[ { "id": "2003-sharma-triggering-the-interferon-antivira", "slug": "2003-sharma-triggering-the-interferon-antivira", "url": "/publications/2003-sharma-triggering-the-interferon-antivira/", "title": "Triggering the Interferon Antiviral Response Through an IKK-Related Pathway", "authors": [ "Sonia Sharma", "Benjamin R. tenOever", "Nathalie Grandvaux", "Guo-Ping Zhou", "Rongtuan Lin", "John Hiscott" ], "author_count": 6, "first_author": "Sonia Sharma", "senior_authors": [ "John Hiscott" ], "corresponding_authors": [ "John Hiscott", "Rongtuan Lin" ], "tenoever_position": 2, "tenoever_role": "middle", "contribution_character": "training period", "year": 2003, "journal": "Science", "volume": "300", "issue": "5622", "pages": "1148-1151", "doi": "10.1126/science.1081315", "doi_url": "https://doi.org/10.1126/science.1081315", "pmid": "12702806", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/12702806/", "pmcid": null, "pmc_url": null, "pmc_pdf_url": null, "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling" ], "themes": [ "ikk-kinases-and-irf-activation" ], "pathogens": [ "Sendai virus", "vesicular stomatitis virus" ], "viral_families": [ "Paramyxoviridae", "Rhabdoviridae" ], "host_species": [ "human", "African green monkey" ], "technologies": [ "in vitro kinase assay", "RNA interference knockdown", "luciferase reporter assay", "electrophoretic mobility shift assay", "GFP fusion localization", "plaque assay", "phosphospecific immunoblotting" ], "biological_systems": [ "HEK293 cells", "A549 cells", "Vero cells", "COS-7 cells" ], "key_concepts": [ "type I interferon induction", "IRF-3 activation", "IRF-7 activation", "virus-activated kinase", "IKK-related kinases", "IKKepsilon", "TBK1", "C-terminal phosphorylation", "innate antiviral state", "transcription factor nuclear translocation" ], "keywords": [ "interferon", "IRF-3", "IRF-7", "IKKepsilon", "TBK1", "innate immunity", "Sendai virus", "vesicular stomatitis virus", "kinase", "antiviral signaling" ], "one_sentence_contribution": "The IKK-related kinases IKKepsilon and TBK1 are identified as components of the virus-activated kinase activity that phosphorylates the C-terminal serine cluster of IRF-3 and IRF-7, linking pathogen sensing to type I interferon gene induction and to establishment of an antiviral state.", "summary_25": "Two related kinases, IKKepsilon and TBK1, phosphorylate the interferon regulatory factors IRF-3 and IRF-7, switching on interferon genes and the cellular antiviral state after virus infection.", "summary_75": "Cells infected by a virus switch on interferon genes, but the enzyme that activates the key transcription factors IRF-3 and IRF-7 had not been identified. Testing the IKK kinase family, the authors found that IKKepsilon and TBK1, and not the classical IKKalpha or IKKbeta, phosphorylate the regulatory tail of both factors, move them into the nucleus and turn on interferon promoters. Silencing the two kinases blocked the response in lung epithelial cells.", "summary_150": "Induction of type I interferon requires phosphorylation of a C-terminal serine and threonine cluster in IRF-3 and IRF-7 by an activity that had been defined only operationally as the virus-activated kinase. Surveying the IKK family, the authors show that IKKepsilon and TBK1, but not IKKalpha or IKKbeta, phosphorylate recombinant IRF-3 and IRF-7 C termini in vitro, with loss of phosphorylation on an alanine-substituted substrate and with a kinase-dead IKKepsilon acting as a block. In cells, IKKepsilon generates Ser396-phosphorylated IRF-3, drives nuclear accumulation of both factors, and produces IRF-containing DNA complexes. Only the two IKK-related kinases activate IFNA4 and IFNB reporters, while all family members tested activate a nuclear factor kappa B reporter, separating the two arms. Silencing IKKepsilon and TBK1 in A549 cells blocks virus-induced IRF-3 phosphorylation and ISG56 induction, and IKKepsilon restricts vesicular stomatitis virus in an IRF-3 dependent manner. The upstream sensing step is not addressed.", "citation": "Sharma S, tenOever BR, Grandvaux N, Zhou G-P, Lin R, Hiscott J. Triggering the Interferon Antiviral Response Through an IKK-Related Pathway. *Science* 2003, volume 300, issue 5622, pages 1148-1151. DOI 10.1126/science.1081315. PMID 12702806.", "sections": { "Citation": "Sharma S, tenOever BR, Grandvaux N, Zhou G-P, Lin R, Hiscott J. Triggering the Interferon Antiviral Response Through an IKK-Related Pathway. *Science* 2003, volume 300, issue 5622, pages 1148-1151.\n\nDOI 10.1126/science.1081315. PMID 12702806.", "One-sentence contribution": "The IKK-related kinases IKKepsilon and TBK1 are identified as components of the virus-activated kinase activity that phosphorylates the C-terminal serine cluster of IRF-3 and IRF-7, linking pathogen sensing to type I interferon gene induction and to establishment of an antiviral state.", "Executive summary": "Rapid induction of type I interferon on virus infection requires the coordinated activation of several transcription factors. By 2002 the routes to nuclear factor kappa B and to ATF-2/c-Jun were reasonably well described, but the kinase activity that phosphorylates the C-terminal serine and threonine cluster of IRF-3 and IRF-7, referred to at the time as the virus-activated kinase, had not been assigned to a defined enzyme. This work asked which member of the IKK family carries that activity. The authors expressed IKKalpha, IKKbeta, IKKepsilon and TBK1 in human cells and tested extracts against glutathione S-transferase fusions carrying the IRF-3 and IRF-7 C termini. Only IKKepsilon and TBK1 phosphorylated the IRF substrates, and a substrate in which the serine and threonine cluster was substituted with alanine was not phosphorylated. IKKepsilon expression produced slower migrating IRF-3 forms recognised by an antibody specific for phosphorylated Ser396, drove nuclear accumulation of IRF-3 and IRF-7 fluorescent fusions, and generated IRF-containing protein-DNA complexes. In reporter assays only IKKepsilon and TBK1, among the kinases tested, activated the IFNA4 and IFNB promoters, while all of them activated a nuclear factor kappa B reporter. Silencing IKKepsilon and TBK1 in A549 lung epithelial cells blocked virus-induced IRF-3 phosphorylation and ISG56 induction, and IKKepsilon expression suppressed vesicular stomatitis virus replication in a manner dependent on functional IRF-3. The work supplies the kinase step that had been missing from the interferon induction pathway.", "Scientific context": "Detection of an invading virus initiates signalling cascades that converge on the interferon beta enhancer, where nuclear factor kappa B, ATF-2/c-Jun and the interferon regulatory factors must act together. The paper states the gap plainly. The pathways activating nuclear factor kappa B and ATF-2/c-Jun had been well characterised, whereas the route to IRF-3 and IRF-7 activation remained undelineated and was described by the authors as a critical missing link. It was already established that IRF-3 and IRF-7 are activated by phosphorylation of a C-terminal domain by an uncharacterised virus-activated kinase, that this modification permits dimerisation, nuclear translocation and interferon gene activation, and that IRF-3 acts early while IRF-7 amplifies expression of interferon genes not induced in the first phase. A two-hybrid screen reported earlier had found an interaction between IRF-3 and the C-terminal domain of IKKalpha, which motivated a survey of IKK family members as candidate virus-activated kinases.", "Central question": "Which kinase or kinases constitute the virus-activated kinase activity that phosphorylates the C-terminal regulatory region of IRF-3 and IRF-7, and is that activity required for virus-induced interferon gene expression and for establishment of the antiviral state?", "Experimental strategy": "The design moves from biochemistry to cell biology to loss of function. First, candidate IKK family kinases were expressed in HEK293 cells and their activity tested directly against recombinant IRF-3 and IRF-7 C-terminal fragments, with an alanine-substituted substrate and a catalytically inactive kinase mutant as specificity controls, and with an IkappaBalpha fragment as a substrate that reports the classical IKK activity. Second, the consequences of kinase expression for the transcription factors themselves were read out at three levels that correspond to the accepted steps of IRF activation, namely site-specific phosphorylation detected with a phosphospecific antibody, subcellular localisation of fluorescent IRF fusions, and sequence-specific DNA binding measured by bandshift with antibody supershift for identity. Third, promoter reporters for IFNA4 and IFNB were used to ask whether these kinases are sufficient to drive interferon transcription, with a nuclear factor kappa B reporter run alongside to separate an IRF-specific effect from general IKK signalling. Finally, RNA interference in A549 lung epithelial cells tested necessity in a physiological infection setting, and plaque assays with vesicular stomatitis virus tested whether the pathway confers protection.", "Key findings": "1. Whole-cell extracts from cells expressing IKKepsilon or TBK1, but not IKKalpha or IKKbeta, phosphorylated a glutathione S-transferase fusion carrying IRF-3 residues 380 to 427 in vitro, and a version of that substrate with alanine substitutions at Ser396, Ser398, Ser402, Thr404 and Ser405 was not phosphorylated. A catalytically inactive IKKepsilon(K38A) blocked the phosphorylation (Figure 1A). The observation is direct evidence that these two kinases act on the known IRF-3 regulatory cluster.\n2. IKKepsilon transcribed and translated in vitro and then immunoprecipitated phosphorylated both the IRF-3 and the IRF-7 C-terminal fragments, while IKKalpha, IKKbeta and IKKepsilon all phosphorylated an IkappaBalpha fragment (Figure 1B). The authors read this as direct action of IKKepsilon on the IRF substrates rather than action through an intermediate kinase in the extract.\n3. Inducible IKKepsilon expression generated slower migrating IRF-3 species detected by an antibody specific for phosphorylation at Ser396, a site previously shown to be critical for physiological IRF-3 activation (Figure 2A).\n4. IRF-3 and IRF-7 fluorescent fusions were predominantly cytoplasmic when expressed alone. Co-expression of IKKepsilon moved approximately 35 percent of IRF-3 and 95 percent of IRF-7 into the nucleus, while IKKepsilon(K38A) left under 5 percent nuclear. TBK1 also drove nuclear localisation (Figure 2B). The quantitative difference between the two factors is the basis for the authors' later suggestion that IRF-7 may be the preferred IKKepsilon substrate.\n5. IKKepsilon expression produced IRF-7 and IRF-3 containing protein-DNA complexes on an IRF-7 binding site and on the ISG15 interferon-stimulated response element, confirmed by antibody supershift (Figure 2C).\n6. Expression of IKKepsilon with IRF-7 stimulated the IFNA4 reporter about 2000-fold and IKKepsilon stimulated the IFNB reporter about 40-fold. Sendai virus infection alone or with IKKepsilon gave about 60-fold IFNB stimulation, and this was blocked by a dominant negative IRF-3 (Figure 3, A and B).\n7. Of IKKalpha, IKKbeta, IKKepsilon, TBK1 and NIK tested against the IFNA4 promoter with IRF-7, only IKKepsilon and TBK1 activated it, whereas every one of them gave 10 to 40-fold stimulation of a nuclear factor kappa B reporter (Figure 3D and supplementary figures). The separation is the paper's argument that the IRF branch is specific to the IKK-related kinases.\n8. RNA interference against TBK1 and IKKepsilon in A549 cells eliminated IKKepsilon protein and reduced TBK1 by 70 to 75 percent. In these cells virus-induced phosphorylation of endogenous IRF-3 was inhibited, ISG56 induction was reduced, and IRF-7-driven IFNA4 reporter activation decreased (Figure 4, A and B). This is the loss-of-function evidence that the two kinases are required, not merely sufficient.\n9. IKKepsilon expression reduced vesicular stomatitis virus titres by four logs to 10^6 plaque-forming units per millilitre, and co-expression of dominant negative IRF-3 restored titres to 10^10, with immunoblotting showing IKKepsilon-induced ISG56 and suppressed viral nucleocapsid protein (Figure 4, C and D). The authors state that an identical experiment with TBK1 gave similar results, citing unpublished data.", "Mechanistic model": "The data support a model in which virus sensing engages IKKepsilon and TBK1, which phosphorylate the C-terminal serine and threonine cluster of IRF-3 and IRF-7, permitting nuclear translocation, sequence-specific DNA binding and transcription of interferon genes, whose products then induce interferon-stimulated genes such as ISG56 and restrict virus replication. The kinase-to-substrate step is demonstrated biochemically and the requirement in infected cells is demonstrated by silencing. What the study does not establish is how virus detection reaches these kinases. The upstream sensor and the adaptor architecture that activates IKKepsilon and TBK1 after infection are outside the scope of the experiments, and the paper does not address them. Nor does the study resolve whether IKKepsilon and TBK1 act on the IRFs within a single complex or in separate complexes, or whether either kinase phosphorylates the IRFs directly in infected cells as opposed to in the reconstituted assays. The authors propose, on the basis of expression patterns and the localisation data, that TBK1 and IRF-3 may serve an early ubiquitous phase of the response while IKKepsilon, for which IRF-7 may be the preferred substrate, mediates amplification, and they further propose a possible functional link to the IKKalpha and IKKbeta complexes through TANK and IKKgamma/NEMO. Both of these are author interpretation rather than demonstrated results in this paper.", "Conceptual or technical advance": "Assigning the virus-activated kinase activity to IKKepsilon and TBK1 converts an operationally defined activity into named enzymes that can be expressed, mutated, silenced and, in principle, inhibited. It also draws a functional line within the IKK family, separating a nuclear factor kappa B arm served by IKKalpha and IKKbeta from an interferon regulatory factor arm served by the IKK-related kinases, with the promoter reporter comparison providing the evidence for that separation. The phosphospecific Ser396 readout and the kinase-dead and alanine-substituted controls together give a tractable assay system for the step, and the demonstration that IKKepsilon-driven restriction of vesicular stomatitis virus depends on functional IRF-3 makes the pathway testable as an antiviral effector axis rather than only a transcriptional one.", "Relationship to the broader research program": "This paper predates the tenOever laboratory and comes from doctoral training in the Hiscott laboratory at the Lady Davis Institute and McGill University, where Benjamin tenOever was one of three authors marked as having contributed equally and was not the senior author. Correspondence was handled by John Hiscott and Rongtuan Lin. Read against the later independent work, the paper sits at the origin of a continuing interest in how virus detection is converted into a transcriptional antiviral program and in how that program is measured. The interferon-stimulated gene readouts and the IRF-3 dependent logic used here recur in the later laboratory's work on innate immune signalling and on transcriptional profiling of infected tissue, though establishing those continuities is category 3 synthesis and requires the later papers to be set alongside this one.", "Related publications": "- Blanco-Melo and colleagues, 2020, conceptual extension. That study reads out the type I interferon response as a transcriptional program during SARS-CoV-2 infection, the same output whose induction step is defined here, although no direct methodological line is asserted from this paper.\n\nThe list is deliberately short. This record was built from one paper read in isolation, and the reference list of this report points to work by other laboratories rather than to the tenOever corpus.", "Limitations and boundaries": "Much of the evidence rests on ectopic expression of kinases and transcription factors in HEK293, Vero and COS-7 cells, together with recombinant fragment substrates, so the results establish sufficiency and direct biochemical capability rather than the stoichiometry or the complex composition that operates in an infected cell. The necessity argument comes from a single loss-of-function setting, RNA interference in A549 lung epithelial cells with partial TBK1 knockdown of 70 to 75 percent, and no genetic knockout is presented here. Two viruses are used, Sendai virus and vesicular stomatitis virus, both RNA viruses assayed over hours in cell culture, so nothing is shown for other virus classes, for primary cells or tissues, or for animal infection. The claim that TBK1 behaves like IKKepsilon in the virus replication experiment is supported by data not shown. The authors themselves flag that the response may be cell-type specific, since TBK1 is ubiquitously expressed while IKKepsilon expression is inducible in lymphoid and other cell types, and they present the division of labour between the two kinases and the possible link to the classical IKK complex as suggestions rather than findings. The pathway upstream of the kinases is not addressed.", "Audience summaries": "### 25 words\n\nTwo related kinases, IKKepsilon and TBK1, phosphorylate the interferon regulatory factors IRF-3 and IRF-7, switching on interferon genes and the cellular antiviral state after virus infection.\n\n### 75 words\n\nCells infected by a virus switch on interferon genes, but the enzyme that activates the key transcription factors IRF-3 and IRF-7 had not been identified. Testing the IKK kinase family, the authors found that IKKepsilon and TBK1, and not the classical IKKalpha or IKKbeta, phosphorylate the regulatory tail of both factors, move them into the nucleus and turn on interferon promoters. Silencing the two kinases blocked the response in lung epithelial cells.\n\n### 150 words\n\nInduction of type I interferon requires phosphorylation of a C-terminal serine and threonine cluster in IRF-3 and IRF-7 by an activity that had been defined only operationally as the virus-activated kinase. Surveying the IKK family, the authors show that IKKepsilon and TBK1, but not IKKalpha or IKKbeta, phosphorylate recombinant IRF-3 and IRF-7 C termini in vitro, with loss of phosphorylation on an alanine-substituted substrate and with a kinase-dead IKKepsilon acting as a block. In cells, IKKepsilon generates Ser396-phosphorylated IRF-3, drives nuclear accumulation of both factors, and produces IRF-containing DNA complexes. Only the two IKK-related kinases activate IFNA4 and IFNB reporters, while all family members tested activate a nuclear factor kappa B reporter, separating the two arms. Silencing IKKepsilon and TBK1 in A549 cells blocks virus-induced IRF-3 phosphorylation and ISG56 induction, and IKKepsilon restricts vesicular stomatitis virus in an IRF-3 dependent manner. The upstream sensing step is not addressed." }, "discoveries": [], "relationships": [ { "from": "2003-sharma-triggering-the-interferon-antivira", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2003-sharma-triggering-the-interferon-antivira" }, { "from": "2007-tenoever-multiple-functions-of-the-ikk-rela", "to": "2003-sharma-triggering-the-interferon-antivira", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2007-tenoever-multiple-functions-of-the-ikk-rela" }, { "from": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "to": "2003-sharma-triggering-the-interferon-antivira", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2020-blanco-melo-imbalanced-host-response-to-sars-c" } ], "canonical_url": "https://tenoeverlab.us/publications/2003-sharma-triggering-the-interferon-antivira/", "controlled_vocabulary": { "pathogens": [ "vsv", "sendai-virus" ], "technologies": [ "plaque-assay", "sirna-knockdown", "luciferase-promoter-reporter", "emsa", "in-vitro-kinase-assay", "phospho-immunoblotting", "fluorescent-fusion-localization" ] } }, { "id": "2007-tenoever-multiple-functions-of-the-ikk-rela", "slug": "2007-tenoever-multiple-functions-of-the-ikk-rela", "url": "/publications/2007-tenoever-multiple-functions-of-the-ikk-rela/", "title": "Multiple Functions of the IKK-Related Kinase IKKε in Interferon-Mediated Antiviral Immunity", "authors": [ "Benjamin R. tenOever", "Sze-Ling Ng", "Mark A. Chua", "Sarah M. McWhirter", "Adolfo García-Sastre", "Tom Maniatis" ], "author_count": 6, "first_author": "Benjamin R. tenOever", "senior_authors": [ "Tom Maniatis" ], "corresponding_authors": [ "Tom Maniatis" ], "tenoever_position": 1, "tenoever_role": "first", "contribution_character": "training period", "year": 2007, "journal": "Science", "volume": "315", "issue": "5816", "pages": "1274-1278", "doi": "10.1126/science.1136567", "doi_url": "https://doi.org/10.1126/science.1136567", "pmid": "17332413", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/17332413/", "pmcid": null, "pmc_url": null, "pmc_pdf_url": null, "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling" ], "themes": [ "ikk-kinases-and-irf-activation", "transcription-factor-selectivity" ], "pathogens": [ "influenza A virus", "influenza A/WSN/33" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "mouse" ], "technologies": [ "gene knockout mice", "Affymetrix microarray", "RT-PCR", "quantitative PCR", "electrophoretic mobility shift assay", "chromatin immunoprecipitation", "in vitro kinase assay", "mass spectrometry", "site-directed mutagenesis", "plaque assay" ], "biological_systems": [ "Ikbke-deficient mice", "primary mouse embryonic fibroblasts", "Stat1-deficient embryonic fibroblasts", "bone marrow-derived macrophages", "mouse lung tissue" ], "key_concepts": [ "IKKε", "type I interferon signalling", "ISGF3", "STAT1 serine phosphorylation", "interferon-stimulated response element", "interferon-stimulated gene subsets", "ADAR1", "promoter selectivity", "antiviral immunity", "IKK-related kinases" ], "keywords": [ "IKKε", "Ikbke", "STAT1", "ISGF3", "ISRE", "interferon-stimulated genes", "ADAR1", "influenza virus", "TBK1", "innate immunity" ], "one_sentence_contribution": "Mice lacking IKKε produce normal interferon-β but fail to induce roughly a third of interferon-stimulated genes, because interferon activates IKKε, which phosphorylates STAT1 at Ser708 and thereby determines whether ISGF3 occupies a subset of response elements.", "summary_25": "Mice lacking IKKε make normal interferon but cannot switch on a third of interferon-response genes, because IKKε phosphorylates STAT1 and directs it to particular promoters.", "summary_75": "IKKε was thought to act only in producing interferon-β. Mice lacking it made interferon normally yet still succumbed to influenza, because a defined subset of interferon-stimulated genes was never induced. The defect persisted when interferon was added to cells directly, placing IKKε inside interferon signalling. Interferon activates IKKε, which phosphorylates STAT1 at serine 708, and that residue is required for the ISGF3 complex to occupy the affected promoters but not others.", "summary_150": "The IKK-related kinases TBK1 and IKKε had been assigned to interferon-β induction, with IKKε treated as redundant. Ikbke-null mice challenged with influenza A/WSN/33 showed higher viral loads and reduced survival at sublethal doses despite normal interferon, cytokine and antibody responses. Transcriptional profiling of infected lung and of primary fibroblasts showed that roughly 30 percent of interferon-stimulated genes, including Adar1, Ifit3 and Ifi203, were poorly induced, and adenosine-to-guanosine editing of influenza matrix mRNA was largely lost in the knockout, giving the ADAR1 defect a functional readout. The defect persisted when recombinant interferon-β was supplied, locating it in signalling. ISGF3 failed to occupy the affected response elements, and rescue required IKKε catalytic activity. Interferon activated IKKε at Thr501, and recombinant IKKε phosphorylated STAT1 at Ser708, a residue required for ISGF3 occupancy at IKKε-dependent but not IKKε-independent elements. The structural basis of that requirement is proposed rather than demonstrated.", "citation": "tenOever BR, Ng SL, Chua MA, McWhirter SM, García-Sastre A, Maniatis T. Multiple Functions of the IKK-Related Kinase IKKε in Interferon-Mediated Antiviral Immunity. Science, 2007, volume 315, issue 5816, pages 1274-1278. DOI 10.1126/science.1136567. PMID 17332413.", "sections": { "Citation": "tenOever BR, Ng SL, Chua MA, McWhirter SM, García-Sastre A, Maniatis T. Multiple Functions of the IKK-Related Kinase IKKε in Interferon-Mediated Antiviral Immunity. Science, 2007, volume 315, issue 5816, pages 1274-1278.\n\nDOI 10.1126/science.1136567. PMID 17332413.", "One-sentence contribution": "Mice lacking IKKε produce normal interferon-β but fail to induce roughly a third of interferon-stimulated genes, because interferon activates IKKε, which phosphorylates STAT1 at Ser708 and thereby determines whether ISGF3 occupies a subset of response elements.", "Executive summary": "Type I interferon induction and type I interferon signalling had been treated as two largely separate arms of the antiviral response, with the IKK-related kinases TBK1 and IKKε assigned to the induction arm as activators of IRF3 and IRF7. This study asked what IKKε actually does in a whole animal. Mice deficient in Ikbke were generated and challenged with influenza A/WSN/33. They were more susceptible to infection at sublethal doses and carried higher pulmonary viral loads, yet their production of interferon-β, other cytokines and virus-specific antibody was not detectably impaired. Transcriptional profiling of infected lung and of primary embryonic fibroblasts instead showed that a defined subset of interferon-stimulated genes, including Adar1, Ifit3 and Ifi203, was poorly induced. Loss of ADAR1 induction had a measurable consequence, since adenosine-to-guanosine editing of influenza matrix mRNA was largely absent in the knockout animals. Treating fibroblasts with recombinant interferon-β reproduced the defect, placing IKKε in the signalling arm rather than the induction arm. Gel shift and chromatin immunoprecipitation assays showed that ISGF3 failed to occupy the affected promoters, and rescue required IKKε kinase activity. Interferon-β activated IKKε, and recombinant IKKε phosphorylated STAT1 at Ser708, Ser744 and Ser747 in vitro. A STAT1 Ser708Ala mutant selectively lost binding at an IKKε-dependent element while retaining binding at an IKKε-independent one. The work reframes IKKε as a determinant of promoter selectivity within the interferon response.", "Scientific context": "Virus infection triggers pattern recognition receptors that activate latent kinase complexes, among them the IKK complex and the IKK-related kinases TBK1 and IKKε, which assemble the interferon-β enhanceosome through ATF2 and cJun, NF-κB, IRF3 and IRF7. Secreted interferon-β then engages its receptor, activates TYK2 and JAK1, and drives tyrosine phosphorylation of STAT1 and STAT2, which associate with IRF9 to form ISGF3 and bind interferon-stimulated response elements across a large family of interferon-stimulated genes. Work in cultured cells before this study had placed virus-inducible IKKε in a role redundant with the ubiquitously expressed TBK1 in activating IRF3 and IRF7, a conclusion the paper attributes to earlier reports from several groups. What IKKε contributes in an intact animal, and whether its only function lies upstream of interferon production, was unresolved.", "Central question": "What is the in vivo function of IKKε in antiviral immunity, and is that function confined to the induction of interferon-β or does it extend into the interferon signalling pathway itself?", "Experimental strategy": "The design begins with loss of function in a whole animal rather than in a cell line, because a kinase reported to be redundant with TBK1 ex vivo may nonetheless carry a non-redundant role in vivo. Ikbke-null mice were generated and challenged intranasally with influenza A/WSN/33 across a dose range, allowing separation of a survival phenotype from a lethality ceiling. Cytokine output and antibody responses were measured to test the expected induction defect, and unbiased microarray profiling of infected lung was used to look for defects the candidate assays would miss. Primary embryonic fibroblasts from the same animals then allowed the phenotype to be moved into a controlled setting, where recombinant interferon-β could be supplied exogenously. That step is the pivot of the design, since a defect that persists when interferon is provided from outside the cell cannot be an interferon induction defect. Promoter occupancy was then interrogated directly by gel shift and chromatin immunoprecipitation, kinase requirement by reconstitution with wild-type and catalytically inactive IKKε, and substrate identity by in vitro phosphorylation with mass spectrometry followed by phosphosite mutants expressed in Stat1-null fibroblasts.", "Key findings": "1. Ikbke-null mice were more susceptible to influenza A/WSN/33 than wild-type littermates at sublethal doses, continuing to lose body mass below 80 percent of starting weight after inoculation with 700 plaque-forming units, whereas doses above 7000 plaque-forming units killed both cohorts (Fig. 1A and 1B). The observation establishes a non-redundant requirement for IKKε in host survival of influenza infection within this dose window.\n\n2. Knockout mice carried elevated pulmonary viral load by quantitative PCR for viral nucleocapsid mRNA and by plaque assay (Fig. 2A). Lung histopathology showed an inflammatory infiltrate of lymphocytes, macrophages and neutrophils.\n\n3. Virus-induced cytokine output was not detectably impaired. Interferon-α, interferon-β, interferon-γ, RANTES and IL2 in lung and serum, and virus-specific antibody amounts, were comparable between genotypes (Fig. 2B). This is the finding that separates the phenotype from the expected interferon induction defect.\n\n4. Microarray profiling of pooled infected lung identified a subset of interferon-stimulated genes, including Ifit3, Ifi203 and Adar1, that were poorly induced without IKKε, confirmed by RT-PCR (Fig. 2B).\n\n5. The failure to induce ADAR1 had a functional readout on the virus. Sequencing of the influenza matrix mRNA stem loop from infected lung showed that more than 30 percent of transcripts carried at least one adenosine-to-guanosine transition in control mice, against less than 5 percent in knockout mice (Fig. 2C, 96 transcripts per cohort). The authors read this as evidence that IKKε is required for induction of a subset of interferon-stimulated genes that includes an effector with antiviral activity in vivo.\n\n6. Primary embryonic fibroblasts reproduced the phenotype. Replication rates were comparable at 2 days post infection but titres continued to rise in knockout cells thereafter (Fig. 2D), with comparable interferon-β induction but reduced induction of the same interferon-stimulated gene subset. Low amounts of virus-inducible Adar1 mRNA were still seen, whereas Ifi203 induction was completely blocked (Fig. 2E).\n\n7. Treating fibroblasts with recombinant interferon-β reproduced the defect. About 30 percent of interferon-inducible genes were poorly induced in knockout cells (Fig. 3A and 3B). Adar1, Ifit3 and Ifi203 were affected while Irf7, Prkra and Stat1 were not. Because interferon was supplied exogenously, this places the lesion in the signalling arm.\n\n8. The affected elements share a consensus resembling characterized ISGF3 binding sites, and gel shift assays showed an interferon-inducible DNA-protein complex that failed to form on IKKε-dependent elements from Oas1b, Mx1 and Adar1 in extracts from knockout cells (Fig. 3C). Antibodies to IRF9, STAT1 and STAT2 each disrupted the complex, identifying it as ISGF3 (Fig. 3D).\n\n9. Reintroducing wild-type IKKε into knockout fibroblasts restored interferon-induced ISGF3 binding, while the catalytically inactive Lys38Ala mutant did not (Fig. 3E). Occupancy therefore depends on IKKε kinase activity and not on the protein as a scaffold.\n\n10. IKKε is activated by interferon itself. A phosphospecific antibody to Thr501 detected rapid IKKε phosphorylation in interferon-treated cells (Fig. 4A). The paper notes that TBK1 is not activated by interferon treatment and lacks the equivalent residue. The suggestion that p38 signalling mediates IKKε activation is presented by the authors as speculation, not as a demonstrated step.\n\n11. Recombinant IKKε phosphorylated STAT1 at Ser708, Ser744 and Ser747 in vitro, mapped by mass spectrometry. Expressed in Stat1-null fibroblasts, the Ser708Ala mutant markedly reduced ISGF3 binding and occupancy at the IKKε-dependent Adar1 element but not at the IKKε-independent Irf7 element, by both gel shift and chromatin immunoprecipitation, whereas a C-terminal deletion removing Ser744 and Ser747 had no effect at either element (Fig. 4D and 4E).\n\n12. Interferon-γ-stimulated gene induction was normal in bone marrow-derived macrophages from knockout mice, and IKKε-independent elements showed normal ISGF3 binding, bounding the defect to a subset of the type I interferon response.", "Mechanistic model": "The study establishes the central link, that IKKε is activated downstream of the interferon receptor and phosphorylates STAT1 at Ser708, and that Ser708 is required for ISGF3 occupancy at a promoter subset. It does not establish the structural basis of that requirement, and the authors state that the structural consequences of the phosphorylation remain to be determined.\n\nThe model the authors propose is that phosphorylation of STAT1 Ser708 favours STAT1 and STAT2 heterodimer formation over STAT1 homodimers, drawing on a published crystal structure from another group rather than on structural data generated here. Because STAT2 is thought to tether STAT1 and IRF9 to the element, heterodimer formation would be required for productive ISGF3 assembly. Computational comparison of IKKε-dependent and IKKε-independent elements identified a purine-rich region upstream of the IKKε-independent elements, which the authors suggest could act as an additional STAT2 contact. In their reading, elements lacking that purine tract would need a more stable STAT1, STAT2 and IRF9 interaction, and it is that stabilization that Ser708 phosphorylation supplies. This account is author interpretation built on sequence analysis and on structures from the literature, and the paper does not test it directly.\n\nA second proposal in the paper, that STAT1 is subject to IKKε-dependent processing or degradation, rests on the observation of discrete STAT1 breakdown products in wild-type but not knockout lung and on higher STAT1 amounts in knockout fibroblasts. The authors explicitly present this as a suggestion that pointed them toward STAT1 as a candidate substrate, not as an established mechanism.", "Conceptual or technical advance": "The work moves IKKε out of a purely redundant position upstream of interferon production and places it inside interferon signalling, where its activity determines which interferon-stimulated genes are transcribed. That makes the interferon-stimulated gene set divisible into IKKε-dependent and IKKε-independent classes distinguishable by their response elements, which gives a handle for asking why a cell would regulate effector genes and signalling-machinery genes separately. The authors advance the interpretation that IKKε-dependent genes act as direct antiviral effectors while IKKε-independent genes serve the signalling machinery that integrates innate and adaptive immunity, and they frame the phenotype as a failure of the local response despite an intact systemwide one. The identification of a serine phosphosite on STAT1 that governs promoter selectivity rather than overall pathway activation also supplies a mechanism by which a transcription factor complex can be tuned rather than switched.", "Relationship to the broader research program": "This paper predates the independent tenOever laboratory and was carried out with the Maniatis laboratory at Harvard together with the García-Sastre laboratory at Mount Sinai, with tenOever as first author. Its recurring concerns carry forward into the later corpus. The use of influenza A virus infection in a defined host genetic background as the assay for an innate immune pathway, and the treatment of the interferon-stimulated gene set as a structured output rather than a single readout, both recur.\n\nCategory 3 synthesis, visible only across papers. ADAR1 enters this paper as an IKKε-dependent effector whose editing activity is measured directly on viral RNA. ADAR1 and the boundary between double-stranded RNA sensing and RNA-directed processes remain active subjects in the laboratory's later work, so this paper marks an early point of contact with that theme. Establishing the continuity requires the later papers and is not supported by this paper alone.", "Related publications": "- McWhirter et al. 2005, Cell, on IKK-related kinase function, methodological foundation for the IKKε reagents and framing used here, and co-authored by a member of this author list.\n- Sharma et al. 2003 and Fitzgerald et al. 2003, predecessor reports assigning TBK1 and IKKε to IRF3 and IRF7 activation, which supply the redundancy model this paper revises.\n- Kim and Maniatis 1996, predecessor, on STAT1 processing and degradation, cited as precedent for the STAT1 turnover observation.\n\nNo relationship to the other papers in this batch is asserted, since none of them cite or extend this work directly.", "Limitations and boundaries": "The in vivo work uses a single virus, influenza A/WSN/33, in mice, and the survival phenotype is confined to a narrow dose window, since doses above 7000 plaque-forming units killed both genotypes. The knockout is germline and constitutive, so cell-intrinsic and systemic contributions are not separated, and no conditional or cell-type-restricted allele was used. Microarray profiling of lung was performed on pooled samples, which limits statistical treatment of the transcriptional differences. The editing readout is based on 96 sequenced matrix transcripts per cohort from pooled material. Phosphorylation of STAT1 by IKKε is shown with recombinant protein in vitro, and the functional test of Ser708 uses ectopic expression of mutant STAT1 in Stat1-null fibroblasts rather than a knock-in animal, so the requirement is established for reconstituted cells rather than for endogenous STAT1 in vivo. The paper does not show that Ser708 phosphorylation of endogenous STAT1 rises after interferon treatment in an IKKε-dependent manner. The structural interpretation of Ser708, the purine tract model for IKKε-independent elements, and the p38 link to IKKε activation are all proposals rather than demonstrations. Interferon-γ responses and IKKε-independent elements are unaffected, so the conclusions do not extend to the type II interferon response or to the interferon-stimulated gene set as a whole.", "Audience summaries": "### 25 words\n\nMice lacking IKKε make normal interferon but cannot switch on a third of interferon-response genes, because IKKε phosphorylates STAT1 and directs it to particular promoters.\n\n### 75 words\n\nIKKε was thought to act only in producing interferon-β. Mice lacking it made interferon normally yet still succumbed to influenza, because a defined subset of interferon-stimulated genes was never induced. The defect persisted when interferon was added to cells directly, placing IKKε inside interferon signalling. Interferon activates IKKε, which phosphorylates STAT1 at serine 708, and that residue is required for the ISGF3 complex to occupy the affected promoters but not others.\n\n### 150 words\n\nThe IKK-related kinases TBK1 and IKKε had been assigned to interferon-β induction, with IKKε treated as redundant. Ikbke-null mice challenged with influenza A/WSN/33 showed higher viral loads and reduced survival at sublethal doses despite normal interferon, cytokine and antibody responses. Transcriptional profiling of infected lung and of primary fibroblasts showed that roughly 30 percent of interferon-stimulated genes, including Adar1, Ifit3 and Ifi203, were poorly induced, and adenosine-to-guanosine editing of influenza matrix mRNA was largely lost in the knockout, giving the ADAR1 defect a functional readout. The defect persisted when recombinant interferon-β was supplied, locating it in signalling. ISGF3 failed to occupy the affected response elements, and rescue required IKKε catalytic activity. Interferon activated IKKε at Thr501, and recombinant IKKε phosphorylated STAT1 at Ser708, a residue required for ISGF3 occupancy at IKKε-dependent but not IKKε-independent elements. The structural basis of that requirement is proposed rather than demonstrated." }, "discoveries": [ "claim-04" ], "relationships": [ { "from": "2007-tenoever-multiple-functions-of-the-ikk-rela", "to": "2003-sharma-triggering-the-interferon-antivira", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2007-tenoever-multiple-functions-of-the-ikk-rela" } ], "canonical_url": "https://tenoeverlab.us/publications/2007-tenoever-multiple-functions-of-the-ikk-rela/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "rt-qpcr", "plaque-assay", "site-directed-mutagenesis", "emsa", "in-vitro-kinase-assay", "mass-spectrometry-proteomics", "chip", "knockout-mice", "microarray" ] } }, { "id": "2009-perez-microrna-mediated-species-specific", "slug": "2009-perez-microrna-mediated-species-specific", "url": "/publications/2009-perez-microrna-mediated-species-specific/", "title": "MicroRNA-mediated species-specific attenuation of influenza A virus", "authors": [ "Jasmine T Perez", "Alissa M Pham", "Maria H Lorini", "Mark A Chua", "John Steel", "Benjamin R tenOever" ], "author_count": 6, "first_author": "Jasmine T Perez", "senior_authors": [ "Benjamin R tenOever" ], "corresponding_authors": [ "Benjamin R tenOever" ], "tenoever_position": 6, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2009, "journal": "Nature Biotechnology", "volume": "27", "issue": "6", "pages": "572-576", "doi": "10.1038/nbt.1542", "doi_url": "https://doi.org/10.1038/nbt.1542", "pmid": "19483680", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/19483680/", "pmcid": null, "pmc_url": null, "pmc_pdf_url": null, "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "programmable-virology" ], "themes": [ "microrna-mediated-viral-attenuation", "molecular-biocontainment" ], "pathogens": [ "influenza A virus", "influenza A virus H1N1 A/Puerto Rico/8/34", "influenza A virus H5N1 A/Vietnam/1203/04" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "human", "mouse", "chicken" ], "technologies": [ "influenza reverse genetics", "site-directed mutagenesis", "northern blot", "luciferase reporter assay", "locked nucleic acid antimiR inhibition", "quantitative RT-PCR", "hemagglutination inhibition assay", "ELISA", "plaque assay" ], "biological_systems": [ "HEK293 cells", "A549 cells", "MDCK cells", "human fibroblasts", "murine lung fibroblasts", "Dicer-deficient murine fibroblasts", "embryonated chicken eggs", "BALB/c mice" ], "key_concepts": [ "microRNA response element", "microRNA-mediated gene silencing", "live attenuated influenza vaccine", "species-specific attenuation", "translational repression", "viral nucleoprotein", "vaccine yield in ovo", "escape mutant resistance", "codon-level engineering of coding sequence" ], "keywords": [ "microRNA", "miR-93", "influenza A virus", "nucleoprotein", "live attenuated vaccine", "reverse genetics", "attenuation", "H5N1", "Dicer", "vaccine safety" ], "one_sentence_contribution": "Engineering microRNA response elements for a mammalian-ubiquitous microRNA directly into the influenza A virus nucleoprotein coding sequence attenuates the virus in mice while leaving growth in embryonated chicken eggs intact, yielding live attenuated vaccine candidates for H1N1 and H5N1.", "summary_25": "Influenza engineered to carry binding sites for a microRNA found in mammals but not chickens is disabled in mice yet grows normally in eggs, producing protective vaccine candidates.", "summary_75": "Live influenza vaccines are grown in chicken eggs but must be weakened in people. The authors exploited a small regulatory RNA, miR-93, that mammals make and chickens do not. Placing binding sites for it inside the nucleoprotein gene left egg growth untouched while cutting lethality in mice by more than a hundredfold. The effect disappeared when the silencing machinery was removed. Vaccinated mice survived lethal H1N1 and H5N1 challenge with broad antibody responses.", "summary_150": "Influenza mRNAs offer almost no untranslated region for inserting regulatory sequence, so the authors engineered near-perfect miR-93 response elements directly into two sites of the nucleoprotein open reading frame, choosing positions where nucleotide changes preserve the class of the encoded amino acid. miR-93 is expressed in mouse and human but not chicken, giving an attenuation signal present in the vaccinee and absent in the egg substrate. A parental virus with the identical amino acid substitutions and disrupted pairing isolated the silencing effect. The doubly targeted virus showed a greater than two log increase in median lethal dose in mice, grew to normal titres in embryonated eggs, and replicated unimpeded in Dicer-deficient fibroblasts. Nucleoprotein mRNA accumulated while protein did not, indicating translational repression. Vaccination gave complete protection against lethal homologous H1N1 and H5N1 challenge. The strategy is orthogonal to temperature-sensitive attenuation and tunable through element number and microRNA choice.", "citation": "Perez JT, Pham AM, Lorini MH, Chua MA, Steel J, tenOever BR. MicroRNA-mediated species-specific attenuation of influenza A virus. Nature Biotechnology. 2009. Volume 27, issue 6, pages 572-576. DOI 10.1038/nbt.1542. PMID 19483680.", "sections": { "Citation": "Perez JT, Pham AM, Lorini MH, Chua MA, Steel J, tenOever BR. MicroRNA-mediated species-specific attenuation of influenza A virus. Nature Biotechnology. 2009. Volume 27, issue 6, pages 572-576.\n\nDOI 10.1038/nbt.1542. PMID 19483680.", "One-sentence contribution": "Engineering microRNA response elements for a mammalian-ubiquitous microRNA directly into the influenza A virus nucleoprotein coding sequence attenuates the virus in mice while leaving growth in embryonated chicken eggs intact, yielding live attenuated vaccine candidates for H1N1 and H5N1.", "Executive summary": "Live attenuated influenza vaccines in use at the time relied on temperature sensitivity for attenuation, a single mechanism with a defined set of excluded recipient groups. The authors asked whether the host microRNA silencing machinery could provide an orthogonal and tunable attenuation mechanism for influenza A virus, one that restricts replication in the vaccinated mammal without restricting yield in the egg substrate used for manufacture. They first established that influenza A virus infection, and the viral NS1 protein on its own, do not block microRNA processing or microRNA-directed silencing in mammalian cells. Because the influenza mRNA has almost no 3' untranslated region available for insertion, response elements were built into the open reading frame of segment five, which encodes nucleoprotein, at two positions where nucleotide changes preserve the hierarchical class of the encoded amino acids. The targeting microRNA, miR-93, was selected because published small RNA profiles show it in mouse and human but not in chicken. A control virus carrying the same three amino acid substitutions with the microRNA pairing disrupted separated silencing effects from protein effects. Viruses carrying both response elements lost more than two logs of lethality in mice, replicated normally in eggs, and replicated normally in Dicer-deficient fibroblasts. Vaccination protected mice against lethal homologous challenge with a broad immunoglobulin response. The approach offers an attenuation mechanism that can be layered onto existing strategies and tuned by the choice and number of elements.", "Scientific context": "Live attenuated influenza vaccines such as FluMist are temperature-sensitive reassortants, and the annual reformulation cycle places a premium on attenuation strategies that transfer easily to new strains. MicroRNA-mediated restriction of viral replication had been demonstrated for lentiviruses, picornaviruses and rhabdoviruses by other laboratories, in each case by placing microRNA target sequences into viral RNA. Extending that idea to influenza A virus faced two specific obstacles. Influenza mRNAs terminate shortly after the stop codon, so the untranslated region used in other systems is essentially unavailable and disruption of that region can cause packaging and replication defects. In addition, it was not settled in the literature whether influenza infection, and particularly the RNA-binding protein NS1, leaves the cellular microRNA pathway functional. Most microRNAs are conserved across vertebrates, but a minority are species restricted, which raises the possibility of an attenuation phenotype that depends on the host species.", "Central question": "Can microRNA response elements placed inside an essential influenza A virus open reading frame attenuate the virus in mammals while preserving replication in the avian system used for vaccine production, and does the resulting virus function as a protective live attenuated vaccine?", "Experimental strategy": "The design has three linked parts. First, a feasibility test in cell culture asking whether influenza infection or NS1 expression perturbs microRNA biogenesis or silencing, assayed by northern blot for precursor and mature forms of an exogenous tissue-restricted microRNA and an exogenous ubiquitous microRNA, and by a luciferase reporter carrying target sites in its 3' untranslated region. Second, a comparative genomics step to choose a microRNA present in mouse and human lung but absent from chicken, using published deep sequencing profiles and confirming mammalian expression by northern blot, RT-PCR and in situ data for the Mcm7 intron that hosts miR-93. Third, engineering and phenotyping. Response elements were introduced by site-directed mutagenesis into two positions of the nucleoprotein coding sequence chosen for amino acid conservation combined with nucleotide plasticity, viruses were rescued by plasmid-based reverse genetics, and the resulting strains were compared with a parental control carrying the identical amino acid substitutions but disrupted microRNA pairing. Attenuation was then attributed to the silencing pathway by three independent controls, replication in Dicer-deficient fibroblasts, rescue of replication by locked nucleic acid inhibition of miR-93, and growth in eggs where the microRNA is absent. Finally the candidate viruses were tested as vaccines in mice, including an H5N1 six-plus-two reassortant, with weight loss, survival after lethal challenge, and serology as readouts.", "Key findings": "1. Influenza A virus infection did not disrupt the microRNA pathway. Exogenous miR-93 and miR-124 were processed to precursor and mature forms in infected HEK293 cells, endogenous miR-93 levels were unchanged over a 36 hour infection time course in human and murine fibroblasts, and miR-124 suppressed a target reporter by about 90 percent whether or not cells were infected or expressed NS1 (Figure 1a-d). The authors read this as establishing that microRNA-based attenuation of influenza is feasible in mammalian cells.\n\n2. Two positions in the nucleoprotein coding sequence, at nucleotides 225 and 818 of A/Puerto Rico/8/34, could be converted into near-perfect miR-93 binding sites with predicted free energies of about minus 28 and minus 37.1 kcal per mole, at the cost of three amino acid substitutions, I63L, S262T and I265L, each preserving the side chain class observed in natural variation at those loci (Figure 2a).\n\n3. The three substitutions were largely tolerated by the protein. In a polymerase reconstitution reporter assay the parental control nucleoprotein retained most activity, with an approximately 20 percent reduction in RNA-dependent RNA polymerase output relative to wild type (Figure 2b). The parental virus behaved like wild type in cell culture but showed some attenuation in vivo, which the authors attribute to that reduced polymerase activity being more consequential under in vivo selective pressure. This attribution is an interpretation and is not separately tested.\n\n4. The doubly targeted virus was attenuated in mice by more than two logs. Median lethal doses were 1.7 x 10^3 plaque forming units for the parental control and 2.15 x 10^5 for the response-element virus (Figure 2d).\n\n5. Attenuation was species restricted. Wild type, parental and all response-element viruses, in both H1N1 and H5N1 six-plus-two configurations, grew to roughly 10^8 plaque forming units per milliliter in ten-day embryonated chicken eggs, indicating no loss of yield in the manufacturing substrate (Figure 2e,f).\n\n6. Attenuation required the silencing machinery and the specific microRNA. In wild type murine fibroblasts the singly targeted viruses showed mild reduction in hemagglutinin and the doubly targeted virus produced none, whereas all strains replicated to similar high levels in Dicer-deficient fibroblasts (Figure 3a). Pretreatment with a locked nucleic acid inhibitor of miR-93 more than doubled doubly targeted virus protein relative to scrambled control (Figure 3b).\n\n7. The restriction acted on translation rather than on transcript abundance. In infected cells, nucleoprotein mRNA was elevated while nucleoprotein protein was very low, a pattern also seen in vivo at 48 hours (Figure 3c,d and Supplementary Figure 5). The authors interpret the elevated mRNA as reflecting the role of unbound nucleoprotein in the switch from transcription to replication, which is a proposal consistent with the data rather than a tested mechanism here.\n\n8. No escape mutants were recovered. Sequencing of nucleoprotein after ten serial passages in A549 cells and after in vivo infection, from more than 25 colonies per cohort, did not identify revertants. The authors read this as suggesting that the flexibility of microRNA targeting combined with the conservation constraint on nucleoprotein limits reversion. Absence of recovered escape over this passage regime does not establish that escape cannot arise.\n\n9. The targeted viruses protected mice. Intranasal vaccination with 10^3 plaque forming units of the H1N1 response-element virus caused less weight loss than the parental control and gave 100 percent survival after lethal challenge, with neutralizing serum and IgM, IgG1, IgG2a and IgG2b responses (Figure 4a). For the H5N1 reassortant, the parental control killed half the animals whereas all animals receiving the response-element virus survived vaccination and subsequent lethal H5N1 challenge with no signs of morbidity, while mock-vaccinated animals all died (Figure 4b).\n\n10. Tropism and the innate response were not obviously altered. Additional in vivo characterization reported no change in viral tropism and normal cytokine and transcriptional responses to infection (Supplementary Figure 6).", "Mechanistic model": "The data support a model in which mature miR-93, present in mouse and human cells and absent in chicken, loads into the silencing complex and engages the two engineered sites within the nucleoprotein open reading frame, suppressing nucleoprotein translation. Because nucleoprotein is required for genome encapsidation and for the transcription to replication switch, loss of the protein caps the amount of infectious progeny and limits pathogenesis in the mammalian host, while the same genome replicates without restriction in the avian egg and in Dicer-deficient cells.\n\nThe translational rather than degradative character of the restriction is directly supported by the divergence between elevated nucleoprotein mRNA and depleted nucleoprotein protein. The specificity of the effect for miR-93 and for the silencing pathway is directly supported by the Dicer-deficient and antimiR experiments. What the study does not establish is the downstream consequence of nucleoprotein limitation in molecular terms. The proposal that elevated nucleoprotein mRNA reflects a biased transcription to replication switch is an interpretation drawn from prior work on nucleoprotein function, not a mechanism demonstrated here. The study also does not resolve which cells in the mouse lung are responsible for the in vivo attenuation, nor whether the residual in vivo attenuation of the parental control shares any mechanism with the microRNA-dependent effect.", "Conceptual or technical advance": "The work makes an attenuation mechanism available that is genetically encoded, orthogonal to temperature sensitivity, and tunable by the number of response elements and the choice of targeting microRNA. Placing the elements inside a conserved open reading frame rather than in an untranslated region solves the specific constraint imposed by influenza mRNA architecture and, by coupling the target site to codons under amino acid conservation, couples escape to a fitness cost. Because the response elements sit on the nucleoprotein segment, the same engineered segment can be moved into reassortants with different surface antigens, which the authors demonstrate by rescuing an H5N1 six-plus-two virus. The result also makes the species distribution of microRNAs a usable engineering parameter, allowing the attenuating signal to be present in the vaccinee and absent in the production substrate.", "Relationship to the broader research program": "The study sits at the intersection of two recurring interests in this corpus, the interaction between RNA viruses and host small RNA pathways, and the deliberate engineering of viruses as tools and vaccines. The demonstration that influenza infection and NS1 leave microRNA biogenesis and silencing intact in mammalian cells is a foundational observation for later work in the corpus that uses virus-encoded or host microRNAs as instruments. The use of a targeted viral genome as a reagent whose tropism is set by the small RNA content of the cell recurs as a design idea.\n\nCategory 3 synthesis is not attempted here beyond that, because this record was built from one paper read in isolation.", "Related publications": "- tenOever BR and colleagues, 2007, Science, on multiple functions of IKKepsilon in interferon-mediated antiviral immunity. Methodological foundation. Cited in this paper as the source of the RT-PCR and immunoblot procedures used.\n- Makeyev, Zhang, Carrasco and Maniatis, 2007, on miR-124 and neuronal differentiation. Methodological foundation from another laboratory, source of the miR-124 reporter constructs and the miR-124 minigene used in the feasibility experiments.\n- Barnes, Kunitomi, Vignuzzi, Saksela and Andino, 2008, and Kelly, Hadac, Greiner and Russell, 2008, and Edge and colleagues, 2008. Predecessor work from other laboratories applying microRNA targeting to picornaviruses, other viruses and vesicular stomatitis virus, which this study extends to a segmented negative-strand virus and to an open reading frame insertion site.", "Limitations and boundaries": "The attenuation and protection data are in BALB/c mice with intranasal inoculation, and no larger animal, ferret or human data are presented. The H1N1 component is based on A/Puerto Rico/8/34, a laboratory-adapted strain, and the authors state plainly that the H1N1 vaccine described does not protect against strains then in circulation. Challenge experiments are homologous or antigenically matched, so nothing here speaks to heterologous protection or to durability beyond the three-week interval tested. Cohorts are small, with three to four mice per dose in the toxicity studies and four per group in the vaccination studies. The species specificity rests on published microRNA profiles plus the egg and Dicer-deficient controls, and miR-93 expression was not surveyed across all relevant avian or mammalian tissues. Absence of escape mutants is bounded by ten serial passages in A549 cells and by sampling of more than 25 clones per in vivo cohort, which does not exclude rarer escape or escape under different selective regimes. Because MDCK cells express miR-93, plaque assays for the targeted viruses were limited, which constrains direct titre comparison for those strains. The translational repression conclusion rests on mRNA and protein measurements rather than on direct ribosome-level assay.", "Audience summaries": "### 25 words\n\nInfluenza engineered to carry binding sites for a microRNA found in mammals but not chickens is disabled in mice yet grows normally in eggs, producing protective vaccine candidates.\n\n### 75 words\n\nLive influenza vaccines are grown in chicken eggs but must be weakened in people. The authors exploited a small regulatory RNA, miR-93, that mammals make and chickens do not. Placing binding sites for it inside the nucleoprotein gene left egg growth untouched while cutting lethality in mice by more than a hundredfold. The effect disappeared when the silencing machinery was removed. Vaccinated mice survived lethal H1N1 and H5N1 challenge with broad antibody responses.\n\n### 150 words\n\nInfluenza mRNAs offer almost no untranslated region for inserting regulatory sequence, so the authors engineered near-perfect miR-93 response elements directly into two sites of the nucleoprotein open reading frame, choosing positions where nucleotide changes preserve the class of the encoded amino acid. miR-93 is expressed in mouse and human but not chicken, giving an attenuation signal present in the vaccinee and absent in the egg substrate. A parental virus with the identical amino acid substitutions and disrupted pairing isolated the silencing effect. The doubly targeted virus showed a greater than two log increase in median lethal dose in mice, grew to normal titres in embryonated eggs, and replicated unimpeded in Dicer-deficient fibroblasts. Nucleoprotein mRNA accumulated while protein did not, indicating translational repression. Vaccination gave complete protection against lethal homologous H1N1 and H5N1 challenge. The strategy is orthogonal to temperature-sensitive attenuation and tunable through element number and microRNA choice." }, "discoveries": [ "claim-10" ], "relationships": [ { "from": "2010-shapiro-noncanonical-cytoplasmic-processin", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2010-shapiro-noncanonical-cytoplasmic-processin" }, { "from": "2012-perez-a-small-rna-enhancer-of-viral-poly", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2012-perez-a-small-rna-enhancer-of-viral-poly" }, { "from": "2012-pham-replication-in-cells-of-hematopoie", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-pham-replication-in-cells-of-hematopoie" }, { "from": "2013-langlois-microrna-based-strategy-to-mitigat", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2013-langlois-microrna-based-strategy-to-mitigat" }, { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2019-tenoever-synthetic-virology-building-viruse", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "application", "evidence": "stated in the Related publications section of 2019-tenoever-synthetic-virology-building-viruse" } ], "canonical_url": "https://tenoeverlab.us/publications/2009-perez-microrna-mediated-species-specific/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "rt-qpcr", "plaque-assay", "reverse-genetics", "luciferase-promoter-reporter", "site-directed-mutagenesis", "northern-blot", "elisa", "lna-antisense-inhibition", "hemagglutination-inhibition" ] } }, { "id": "2010-perez-influenza-a-virus-generated-small-", "slug": "2010-perez-influenza-a-virus-generated-small-", "url": "/publications/2010-perez-influenza-a-virus-generated-small-/", "title": "Influenza A virus-generated small RNAs regulate the switch from transcription to replication", "authors": [ "Jasmine T. Perez", "Andrew Varble", "Ravi Sachidanandam", "Ivan Zlatev", "Muthiah Manoharan", "Adolfo García-Sastre", "Benjamin R. tenOever" ], "author_count": 7, "first_author": "Jasmine T. Perez", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 7, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2010, "journal": "Proceedings of the National Academy of Sciences", "volume": "107", "issue": "25", "pages": "11525-11530", "doi": "10.1073/pnas.1001984107", "doi_url": "https://doi.org/10.1073/pnas.1001984107", "pmid": "20534471", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/20534471/", "pmcid": "PMC2895093", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2895093/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2895093/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "influenza-genome-regulation" ], "themes": [ "small-viral-rnas" ], "pathogens": [ "influenza A virus", "vesicular stomatitis virus" ], "viral_families": [ "Orthomyxoviridae", "Rhabdoviridae" ], "host_species": [ "human", "mouse", "dog", "chicken" ], "technologies": [ "small RNA deep sequencing", "SOLiD sequencing", "northern blot", "primer extension", "quantitative PCR", "immunoprecipitation", "locked nucleic acid antisense inhibition", "bidirectional plasmid reverse genetics" ], "biological_systems": [ "A549 cells", "HEK293 cells", "MDCK cells", "mouse embryonic fibroblasts", "embryonated chicken eggs" ], "key_concepts": [ "small viral RNA", "transcription to replication switch", "viral RNA-dependent RNA polymerase", "viral ribonucleoprotein", "promoter panhandle", "NEP/NS2", "nucleoprotein", "5-prime triphosphate RNA", "segment-specific regulation" ], "keywords": [ "svRNA", "influenza A virus", "RdRp", "replicase", "transcriptase", "vRNA", "cRNA", "NS2", "small RNA sequencing", "LNA antisense" ], "one_sentence_contribution": "Influenza A virus produces a family of 22 to 27 nucleotide small viral RNAs corresponding to the 5 prime end of each genomic segment, which accumulate as the polymerase shifts toward genome synthesis and whose inhibition selectively depletes genomic RNA without comparably affecting messenger or complementary RNA.", "summary_25": "Influenza makes short RNAs copying the start of each genome segment. They appear as the virus begins copying its genome, and blocking them reduces genome production.", "summary_75": "Influenza A virus must switch its single polymerase from making messenger RNA to copying its genome, and how it does so was unclear. Sequencing small RNAs from infected cells revealed a 22 to 27 nucleotide species matching the start of each genome segment. It appears when genome copying begins, binds the assembled polymerase, and requires the polymerase, nucleoprotein and NEP/NS2 to be made. Blocking one segment's small RNA reduces that segment's genome and viral output.", "summary_150": "Influenza A virus uses one RNA-dependent RNA polymerase for primer-dependent messenger RNA synthesis and for primer-independent genome synthesis, and the transition between the two had no established mechanism. Deep sequencing of small RNAs from infected A549 cells identified a discrete 22 to 27 nucleotide species, svRNA, corresponding to the 5 prime end of each of the eight genomic segments. It accumulates from roughly twelve hours after infection, coincident with the measured rise in genomic RNA and after viral protein appears. It is produced across H1N1, H3N2 and H5N1 and across human, canine, murine and avian systems, and is not induced by an unrelated virus or by interferon. Production requires the polymerase subunits, nucleoprotein and segment 8, with NEP/NS2 partially sufficient, and svRNA associates only with the assembled polymerase. Segment-specific antisense inhibition depletes that segment's genomic RNA while sparing messenger and complementary RNA. The authors propose an svRNA-loaded replicase, which the data support but do not establish.", "citation": "Perez JT, Varble A, Sachidanandam R, Zlatev I, Manoharan M, García-Sastre A, tenOever BR. Influenza A virus-generated small RNAs regulate the switch from transcription to replication. Proceedings of the National Academy of Sciences. 2010. Volume 107, issue 25, pages 11525-11530. DOI 10.1073/pnas.1001984107. PMID 20534471. PMCID PMC2895093.", "sections": { "Citation": "Perez JT, Varble A, Sachidanandam R, Zlatev I, Manoharan M, García-Sastre A, tenOever BR. Influenza A virus-generated small RNAs regulate the switch from transcription to replication. Proceedings of the National Academy of Sciences. 2010. Volume 107, issue 25, pages 11525-11530.\n\nDOI 10.1073/pnas.1001984107. PMID 20534471. PMCID PMC2895093.", "One-sentence contribution": "Influenza A virus produces a family of 22 to 27 nucleotide small viral RNAs corresponding to the 5 prime end of each genomic segment, which accumulate as the polymerase shifts toward genome synthesis and whose inhibition selectively depletes genomic RNA without comparably affecting messenger or complementary RNA.", "Executive summary": "Influenza A virus uses a single RNA-dependent RNA polymerase for two incompatible jobs, primer-dependent messenger RNA synthesis early in infection and primer-independent genome synthesis later, and the molecular event that reassigns the enzyme between these modes had not been identified. Transcription requires the polymerase to remain bound in cis to the 5 prime end of the template, which is also what forces the stuttering that generates the polyadenylated tail, so genome synthesis appears to demand a way of reconstituting the double-stranded promoter without that steric constraint. Working from the premise that a small RNA could supply this function, the authors deep sequenced the sub-40 nucleotide RNA fraction of infected lung epithelial cells and found a discrete species, termed svRNA, matching the 5 prime terminus of each of the eight genomic segments. svRNA accumulates around twelve hours after infection, coincident with the measured rise in genomic RNA and after viral protein is already detectable. It is produced by multiple subtypes, in eggs and in cells from three host species, and it is not induced by an unrelated virus or by type I interferon. Reconstitution from plasmids showed a requirement for the three polymerase subunits, nucleoprotein and segment 8, with NEP/NS2 partially restoring production. svRNA co-precipitates only with the assembled polymerase. Locked nucleic acid inhibitors directed at one segment reduced that segment's genomic RNA and viral titres while leaving the other segments intact.", "Scientific context": "The influenza A virus genome consists of eight negative-sense segments whose conserved 5 prime and 3 prime noncoding ends form a panhandle or corkscrew structure recognised by the polymerase. During transcription the polymerase stays associated with the 5 prime end of the template, and that association both primes elongation and produces the steric hindrance that makes the enzyme stutter over the uracil tract to generate a polyadenylated messenger RNA. Genome synthesis proceeds through a full-length complementary RNA intermediate and is primer independent, which requires the polymerase to read through the 5 prime noncoding region rather than remain locked onto it. The paper states the resulting paradox directly, that the virus must keep the genomic ends associated throughout replication while also allowing complete synthesis across those ends, and that current models fail to reconcile this. Previous work had identified factors correlated with the switch, including complementary RNA stability, nucleotide pools and the soluble pools of nucleoprotein and polymerase, but the authors write that a true underlying mechanism remained elusive. Small RNAs were already known to serve viral life cycles in other systems, which motivated looking for one here.", "Central question": "Does influenza A virus generate a small RNA species, and if so does that species participate in the reassignment of the viral polymerase from transcriptase to replicase activity.", "Experimental strategy": "The design moves from unbiased discovery to requirement testing to functional perturbation. Discovery used SOLiD small RNA deep sequencing of the sub-40 nucleotide fraction from A549 cells infected with A/PR/8/34, which makes no assumption about the sequence or the structure of what might be present. Expression was then characterised by northern blot with a pan-specific probe across an infection time course, set against viral protein by western blot and against genomic RNA accumulation by a strand-selective quantitative PCR, so that the timing of svRNA could be placed relative to the transcription to replication transition rather than merely to infection. Generality was tested across three subtypes in embryonated eggs and across human, canine and murine cells, and specificity was tested against vesicular stomatitis virus and type I interferon to exclude an induced host species. Biogenesis requirements were addressed by transfecting the eight bidirectional segment plasmids and withdrawing one at a time, which separates the contribution of individual viral products from infection as a whole, with NS1 and NEP/NS2 supplied back individually to resolve the segment 8 requirement. Physical association was tested by Flag immunoprecipitation of individual polymerase subunits, the reconstituted trimer, nucleoprotein and NS1 against a synthetic 5 prime triphosphorylated svRNA mimetic. Function was interrogated with locked nucleic acid antisense oligonucleotides against individual segment svRNAs, read out by primer extension that resolves messenger, complementary and genomic RNA separately, which is the readout that can distinguish an effect on transcription from an effect on replication.", "Key findings": "1. Deep sequencing of the sub-40 nucleotide RNA from infected A549 cells at twelve hours returned over four million unique reads, more than ninety percent of which were cellular microRNAs. Roughly thirty percent of the influenza-derived sequences in that fraction were enriched at the 5 prime end of the genomic RNA rather than distributed across the genome as breakdown products were. These svRNAs were 22 to 27 nucleotides, unique to each segment beyond position 14 to 16, and terminated three to four bases short of the polyU tract (Figure 1, Tables S1 to S3).\n2. By northern blot with a pan-specific probe, svRNA became detectable around twelve hours after infection and continued to accumulate to thirty-six hours (Figure 2A). NS1 protein was already visible by eight hours, so protein expression preceded svRNA detection (Figure 2B), and strand-selective quantitative PCR placed the rise in genomic RNA at twelve hours, concurrent with svRNA appearance (Figure 2C). The concurrence is a temporal correlation and the paper does not establish which event is upstream.\n3. svRNA was absent from cells treated with vesicular stomatitis virus or type I interferon, indicating it is not a virus-inducible or interferon-inducible host small RNA (Figure 2D). It was produced by H1N1, H3N2 and H5N1 in embryonated eggs, with levels tracking the extent of replication (Figure 2E), and by H1N1 infection of human, canine and murine fibroblasts (Figure 2F).\n4. Transfection of all eight bidirectional segment plasmids produced svRNA in the absence of infection. Withdrawing segment 1, 2, 3 or 8 abolished it, withdrawing segment 5 abolished the mature species while leaving fragments of approximately 32 and 42 nucleotides, withdrawing segment 7 reduced it, and withdrawing segments 4 or 6 had no significant effect (Figure 3A). Supplying NEP/NS2 back to a segment 1 to 7 transfection gave partial rescue while NS1 gave none (Figure 3B). The authors read this as the polymerase, nucleoprotein and NEP/NS2 constituting the minimal machinery for svRNA production.\n5. A synthetic 5 prime triphosphorylated svRNA mimetic co-precipitated with the reconstituted three-subunit polymerase but not with PB1, PB2 or PA individually, nor with nucleoprotein or NS1 (Figure 3C). Phosphatase treatment confirmed that the bound species carried a 5 prime triphosphate, alongside a less abundant species lacking it (Figure 3D).\n6. Despite carrying an exposed 5 prime triphosphate, transfected svRNA did not induce IRF3 phosphorylation or interferon beta, whereas polyinosinic-polycytidylic acid and an NS1 RNA-binding mutant virus did (Figure S3B). The authors take this as consistent with published length and structure requirements for cytosolic helicase recognition rather than as a direct test of receptor engagement.\n7. A locked nucleic acid complementary to segment 4 svRNA had little effect on HA messenger RNA or complementary RNA but substantially reduced HA genomic RNA by primer extension (Figure 4A). Nucleoprotein and NS1 protein were unaffected and HA protein modestly reduced (Figure 4B), while supernatant transferred to MDCK cells produced a marked loss of HA, NP and NS1 signal, which the authors attribute to a packaging defect arising from loss of segment 4 genomic RNA (Figure 4C).\n8. Inhibitors against NA or NS svRNA left HA transcripts unchanged, indicating that the effect of the segment 4 inhibitor is segment restricted (Figure 5A). Prolonged segment 4 inhibition removed HA protein without affecting NP or NS1 (Figure 5B) and reduced infectious titres by approximately eighty percent (Figure 5C).", "Mechanistic model": "The study does not establish a definitive mechanism. It identifies a viral small RNA, places its appearance at the time of the transcription to replication transition, shows that it binds the assembled polymerase, and shows that blocking it selectively depletes genomic RNA. It does not show that svRNA acts on the polymerase to cause the switch, and it does not determine how svRNA is made.\n\nThe model the authors propose is that the replicase form of the polymerase is an NEP/NS2-containing complex loaded with svRNA, while the transcriptase form lacks svRNA and therefore remains bound in cis to the 5 prime template end, which permits the stuttering that generates the polyadenylated tail. Because inhibition acts segment by segment, they note that the model implies eight distinct replicase complexes distinguished by which svRNA is bound, with the bound svRNA acting as a guide that could reconstitute the promoter in trans and free the genomic ends. This is author interpretation and is presented as such in the discussion.\n\nBiogenesis is left open. The authors consider cleavage of the genomic RNA by the polymerase and synthesis from the complementary RNA intermediate, argue against cleavage because it would destroy the template, and note that synthesis from complementary RNA would require a stochastic svRNA-independent round of complementary RNA production. They connect this second possibility to prior work on complementary RNA stability as the trigger for the switch. Neither route is demonstrated here.", "Conceptual or technical advance": "The work makes an RNA species a candidate participant in a step of the influenza life cycle that had been described only in terms of proteins, nucleotide pools and RNA stability, and it supplies the reagents that make that participation testable, namely the pan-specific and segment-specific detection probes, the synthetic 5 prime triphosphorylated mimetics, and the segment-specific locked nucleic acid inhibitors. Because the svRNA sequences derive from the conserved noncoding ends, the observation that the species is produced across subtypes and host species raises the possibility of inhibitors that are not strain restricted, which the authors name as a potential therapeutic direction rather than demonstrate.", "Relationship to the broader research program": "The paper sits at the intersection of two recurring interests in this corpus, the temporal control of influenza gene expression and the use of small RNA biology as both a subject and a tool. The finding that the virus times a transition using an RNA species anticipates later work from the same laboratory on how influenza schedules its own gene expression through the splicing of segment 8, and the deep sequencing and small RNA northern blot methods used here recur across the laboratory's subsequent small RNA work. Reading this alongside later papers in the corpus, the framing of viral noncoding RNA as a regulatory rather than merely incidental product is a thread that continues, which is a category 3 synthesis observation drawn from setting this paper next to Chua and colleagues in 2013 and the laboratory's later small RNA studies rather than something stated here.", "Related publications": "- Chua and colleagues, 2013, follow-up. The later paper on suboptimal splicing of the influenza segment 8 transcript addresses the same question of how the virus times its life cycle, and it cites this work when discussing genomic RNA accumulation and premature ribonucleoprotein export.\n- Robb and colleagues, 2009, predecessor, from another laboratory. Cited here as the report that NEP/NS2 regulates transcription and replication of the influenza genome, which is the basis for interpreting the segment 8 requirement.\n- Vreede and colleagues, 2004 and 2008, predecessor, from another laboratory. Cited as the prior models in which complementary RNA stabilisation and nucleoside triphosphate concentration govern the switch, against which the svRNA model is positioned.", "Limitations and boundaries": "The function of svRNA is inferred from an antisense inhibition experiment, and the segment 4 inhibitor is complementary to a sequence that is also present at the 5 prime terminus of the segment 4 genomic RNA itself, so the reported experiments do not formally separate depletion of the free small RNA from engagement of the template end. No experiment reconstitutes replicase activity with and without svRNA in vitro, so the proposed role in converting the polymerase between activities is not directly tested. The coincidence in timing between svRNA accumulation and the rise in genomic RNA is correlative. The biogenesis route is unresolved and the authors say so. The size of the species is also unresolved, since the sequencing data indicate predominant lengths of 25 and 27 nucleotides while the northern blots indicate approximately 22 and 25 nucleotides, a discrepancy the authors describe as difficult to explain and attribute tentatively to the 5 prime triphosphate. The absence of IRF3 phosphorylation is evidence that transfected svRNA does not trigger this pathway under the conditions used and is not a general claim about innate immune invisibility. All work is in cultured cells and embryonated eggs, with no animal infection model, and the titre reduction of approximately eighty percent was measured in a single-cycle and short growth curve setting rather than in vivo.", "Audience summaries": "### 25 words\n\nInfluenza makes short RNAs copying the start of each genome segment. They appear as the virus begins copying its genome, and blocking them reduces genome production.\n\n### 75 words\n\nInfluenza A virus must switch its single polymerase from making messenger RNA to copying its genome, and how it does so was unclear. Sequencing small RNAs from infected cells revealed a 22 to 27 nucleotide species matching the start of each genome segment. It appears when genome copying begins, binds the assembled polymerase, and requires the polymerase, nucleoprotein and NEP/NS2 to be made. Blocking one segment's small RNA reduces that segment's genome and viral output.\n\n### 150 words\n\nInfluenza A virus uses one RNA-dependent RNA polymerase for primer-dependent messenger RNA synthesis and for primer-independent genome synthesis, and the transition between the two had no established mechanism. Deep sequencing of small RNAs from infected A549 cells identified a discrete 22 to 27 nucleotide species, svRNA, corresponding to the 5 prime end of each of the eight genomic segments. It accumulates from roughly twelve hours after infection, coincident with the measured rise in genomic RNA and after viral protein appears. It is produced across H1N1, H3N2 and H5N1 and across human, canine, murine and avian systems, and is not induced by an unrelated virus or by interferon. Production requires the polymerase subunits, nucleoprotein and segment 8, with NEP/NS2 partially sufficient, and svRNA associates only with the assembled polymerase. Segment-specific antisense inhibition depletes that segment's genomic RNA while sparing messenger and complementary RNA. The authors propose an svRNA-loaded replicase, which the data support but do not establish." }, "discoveries": [ "claim-01" ], "relationships": [ { "from": "2010-perez-influenza-a-virus-generated-small-", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "follow-up", "evidence": "stated in the Related publications section of 2010-perez-influenza-a-virus-generated-small-" } ], "canonical_url": "https://tenoeverlab.us/publications/2010-perez-influenza-a-virus-generated-small-/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "vsv" ], "technologies": [ "rt-qpcr", "reverse-genetics", "small-rna-seq", "northern-blot", "co-ip", "lna-antisense-inhibition", "primer-extension", "short-read-sequencing-platform" ] } }, { "id": "2010-schmid-transcription-factor-redundancy-en", "slug": "2010-schmid-transcription-factor-redundancy-en", "url": "/publications/2010-schmid-transcription-factor-redundancy-en/", "title": "Transcription Factor Redundancy Ensures Induction of the Antiviral State", "authors": [ "Sonja Schmid", "Markus Mordstein", "Georg Kochs", "Adolfo García-Sastre", "Benjamin R. tenOever" ], "author_count": 5, "first_author": "Sonja Schmid", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 5, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2010, "journal": "Journal of Biological Chemistry", "volume": "285", "issue": "53", "pages": "42013-42022", "doi": "10.1074/jbc.m110.165936", "doi_url": "https://doi.org/10.1074/jbc.m110.165936", "pmid": "20943654", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/20943654/", "pmcid": "PMC3009927", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3009927/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3009927/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling" ], "themes": [ "transcription-factor-selectivity" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "human", "mouse" ], "technologies": [ "electrophoretic mobility shift assay", "Affymetrix microarray", "quantitative PCR", "luciferase reporter assay", "lentiviral transduction", "RNA interference knockdown", "knockout mouse infection" ], "biological_systems": [ "HEK293T cells", "2FTGH fibrosarcoma cells", "U3A STAT1-deficient cells", "A549 cells", "primary mouse embryonic fibroblasts", "mouse lung" ], "key_concepts": [ "interferon-stimulated response element", "ISGF3", "IRF7", "IRF3", "transcription factor redundancy", "interferon-stimulated genes", "antiviral state", "type I interferon signaling", "type III interferon signaling", "promoter motif specificity" ], "keywords": [ "ISRE", "IRF7", "ISGF3", "STAT1", "interferon", "influenza A virus", "NS1", "antiviral transcriptome", "EMSA", "gene expression" ], "one_sentence_contribution": "IRF7 and ISGF3 engage overlapping interferon-stimulated response elements and drive largely overlapping antiviral transcriptomes, so that a substantial interferon-like gene program is still induced when type I and type III interferon signaling are both absent.", "summary_25": "Cells lacking interferon receptors still switch on much of the antiviral gene program, because IRF7 reads many of the same promoter elements that the interferon-activated ISGF3 complex uses.", "summary_75": "Antiviral gene induction is usually attributed to interferon signaling through the ISGF3 complex. Mice lacking both type I and type III interferon receptors nonetheless induced many interferon-stimulated genes after influenza A virus infection. Systematic mutagenesis of a model promoter element showed that IRF7 and ISGF3 bind overlapping but distinguishable sequences, and activating IRF7 in interferon-unresponsive human cells reproduced most of that gene set, indicating a redundant route to the antiviral state.", "summary_150": "The antiviral transcriptional response has been framed as a consequence of interferon secretion and ISGF3 assembly. Infection of IFNAR1 and IL28R alpha double knockout mice with NS1-deficient influenza A virus induced a substantial set of interferon-stimulated genes despite the absence of ISGF3 activity. Working from the ISG15 interferon-stimulated response element, the authors mutated the core motif and its flanks and compared binding of reconstituted IRF7 and reconstituted ISGF3 by mobility shift, resolving positions that confer specificity for one complex, for the other, or for both, and deriving candidate consensus sequences for each. Activation of IRF7 in STAT1-deficient U3A cells induced a gene set overlapping about 80 percent with the set induced in infected knockout lungs, and promoter elements from individual genes bound the predicted factors, with MxA restricted to ISGF3 and CXCL10 restricted to IRF7. Endogenous IRF7 was required for CXCL10 and MAP3K8 induction in knockout fibroblasts under translational blockade.", "citation": "Schmid S, Mordstein M, Kochs G, García-Sastre A, tenOever BR. Transcription Factor Redundancy Ensures Induction of the Antiviral State. Journal of Biological Chemistry. 2010. Volume 285, issue 53, pages 42013-42022. DOI 10.1074/jbc.m110.165936. PMID 20943654. PMCID PMC3009927.", "sections": { "Citation": "Schmid S, Mordstein M, Kochs G, García-Sastre A, tenOever BR. Transcription Factor Redundancy Ensures Induction of the Antiviral State. Journal of Biological Chemistry. 2010. Volume 285, issue 53, pages 42013-42022. DOI 10.1074/jbc.m110.165936. PMID 20943654. PMCID PMC3009927.", "One-sentence contribution": "IRF7 and ISGF3 engage overlapping interferon-stimulated response elements and drive largely overlapping antiviral transcriptomes, so that a substantial interferon-like gene program is still induced when type I and type III interferon signaling are both absent.", "Executive summary": "The prevailing account of the antiviral transcriptional response placed interferon secretion and subsequent ISGF3 activation at its center, with interferon regulatory factors acting mainly upstream by inducing interferon itself. Infection of mice lacking both the type I and the type III interferon receptors with an NS1-deficient influenza A virus showed that a large block of genes normally classed as interferon-stimulated genes was still induced, which raised the question of which transcription factor was responsible and how its DNA binding preferences relate to those of ISGF3.\n\nThe study combined biochemical dissection of a single well-characterized element, the ISG15 interferon-stimulated response element, with transcriptional profiling in cells and animals unable to signal through interferon. Systematic mutagenesis of the core motif and its flanking sequence, read out by electrophoretic mobility shift assay against reconstituted IRF7 and reconstituted ISGF3, separated positions that confer IRF7 specificity, ISGF3 specificity, or binding by both. Profiling of STAT1-deficient U3A cells in which IRF7 was activated by IKK epsilon defined an IRF7-driven transcriptome in the absence of interferon signaling, and about 80 percent of the genes induced in that setting were also induced in the infected knockout mouse lung.\n\nThe work reframes the interferon-stimulated response element as a family of motifs with distinguishable factor preferences and supports a model in which IRF7 provides redundancy that secures the antiviral state when interferon signaling is unavailable.", "Scientific context": "Type I and type III interferons signal through distinct receptors that converge on the assembly of ISGF3, a complex of STAT1, STAT2 and IRF9, which binds interferon-stimulated response elements upstream of more than one hundred interferon-stimulated genes. The IRF family binds a partially overlapping element, and IRF3 and IRF7 had been assigned the upstream role of inducing interferon itself. IRF3 is ubiquitously expressed, while basal IRF7 is largely restricted to hematopoietic cells and is induced elsewhere by interferon or tumor necrosis factor alpha. Earlier profiling of IRF3 and of IRF7 activity existed, but no distinction was drawn between an element that binds IRFs, one that binds ISGF3, and one that binds both. The paper states the gap in those terms. A prior report by Mordstein and colleagues on mice lacking both interferon receptors provided the animal model, and the observation of interferon-stimulated gene induction in those animals despite the absence of ISGF3 activity is what motivated the study.", "Central question": "Do IRF7 and ISGF3 act on the same DNA elements, and can IRF7 alone reconstruct an interferon-like transcriptome in the absence of type I and type III interferon signaling, thereby providing redundancy in establishing the antiviral state?", "Experimental strategy": "The design pairs an in vivo demonstration that the phenomenon exists with an in vitro dissection of why it exists. Mice carrying disruptions in IFNAR1 and IL28R alpha were infected with wild-type or NS1-deficient influenza A virus, which removes the viral antagonist of IRF activation, so that virus-driven, interferon-independent transcription could be measured by microarray and quantitative PCR. For biochemistry, IRF7 or the three ISGF3 components were expressed exogenously in fibroblasts and activated with IKK epsilon or interferon beta, giving matched extracts whose binding to a panel of synthetic probes could be compared directly. The ISG15 element was chosen because it carries both a complete ISGF3 consensus and a complete IRF element, so that core positions and flanking positions could be mutated one at a time. STAT1-deficient U3A cells provided a human cellular system in which IRF7 activity can be read transcriptionally with no possibility of interferon feedback, and primary fibroblasts from Irf3, Irf7 and double knockout mice, treated with cycloheximide to block autocrine signaling, provided a genetic test using endogenous factors.", "Key findings": "1. In mice lacking both interferon receptors, NS1-deficient influenza A virus induced interferon beta and interferon lambda 2 transcripts and a set of genes annotated as interferon-stimulated genes, at comparable viral nucleoprotein levels between viral cohorts. Some interferon-stimulated genes, notably Mx1, were not induced (Figure 1). The observation is direct. The inference drawn by the authors, that this residual program reflects IRF3 and IRF7 activity, is interpretation at this stage of the paper.\n\n2. Exogenously expressed IRF7 activated by IKK epsilon, and reconstituted ISGF3 activated by interferon beta or IKK epsilon, both drove an ISG15 element reporter and both bound an ISG15 element probe. Supershift assigned the IRF7 complex to an IRF7 homodimer rather than an IRF3 and IRF7 heterodimer, and confirmed STAT1, STAT2 and IRF9 in the ISGF3 complex. Monomeric IRF9 binding was detectable and was inversely related to ISGF3 assembly (Figure 2).\n\n3. Systematic probe mutagenesis separated the requirements of the two complexes. Minimizing the element to a single core raised ISGF3 and IRF9 binding, randomizing both flanks abolished ISGF3 and IRF9 binding while leaving IRF7 binding intact, and individual core substitutions produced positions that affect only ISGF3, only IRF7, or both (Figure 3). The authors derive a minimal IRF7 motif of AAWNCGAAA, with WWNNGAAANNGAAA also compatible, and an ISGF3 consensus of WBVGGAAANNGAAACT. These consensus statements are the authors' generalization from the probe panel.\n\n4. Favorable bases at the minus 2 and minus 3 positions compensated for an unfavorable base at core position 7 that otherwise abolishes IRF7 binding (Figure 3N). The authors read this as IRF7 making minor groove contacts at those upstream positions.\n\n5. In STAT1-deficient U3A cells, IRF7 with IKK epsilon induced interferon alpha 1 without inducing MxA, and microarray profiling showed induction not only of known IRF3-regulated genes such as IFIT1, IFIT2, IFIT3 and RSAD2 but also of IFIT5, IFIH1, GBP1, OAS2, OASL and IRF9 (Figure 4). About 80 percent of the genes upregulated in this setting overlapped the set induced in the infected knockout mouse lung. The overlap figure is an observation. The further statement that the redundancy is evolutionarily conserved among vertebrates is an extrapolation offered by the authors.\n\n6. In U3A cells stably reconstituted with GFP, IRF7 or STAT1 and challenged with interferon beta or an NS1-mutant influenza A virus, MxA behaved as an ISGF3-dependent gene, OAS1 was induced by both ISGF3 and IRF7, and CXCL10, MAP3K8 and interferon alpha 1 were induced by IRF7 but not by interferon beta treatment (Figure 5). Knockdown of endogenous IRF3 did not abolish these IRF7-dependent inductions.\n\n7. In primary mouse embryonic fibroblasts treated with polyinosinic-polycytidylic acid under cycloheximide, CXCL10 and MAP3K8 were induced in Irf3 knockout cells but not in Irf7 knockout or double knockout cells, placing the requirement on endogenous IRF7 and excluding an autocrine interferon contribution (Figure 5C).\n\n8. Elements taken from the promoters of IRF7-regulated genes sorted into the two predicted motif classes, and direct binding tests assigned the MxA element to ISGF3 only, the OAS1 element to IRF7 and ISGF3, the ISG15 element to IRF3, IRF7 and ISGF3, and the CXCL10 element to IRF7 (Table 1 and Figure 6). In extracts from interferon-treated, polyinosinic-polycytidylic acid stimulated cells, the single inducible CXCL10 element complex was supershifted only by an IRF7 antibody, which ties the ectopic expression model to endogenous activity.", "Mechanistic model": "The mechanism supported here is a DNA-level one and it is established at the level of binding and transcript abundance rather than by structural work. Interferon-stimulated response elements are not a single class. Core positions plus the immediate flanking sequence determine whether an element is read by IRF7, by ISGF3, or by both, with ISGF3 requiring contacts that extend beyond the consensus core, presumably through STAT1 and STAT2, and IRF7 tolerating more variation because favorable upstream minor groove contacts can offset a poor core contact. Because a large fraction of interferon-stimulated gene promoters carry elements of the permissive class, activated IRF7 alone reproduces much of the interferon-stimulated transcriptome without any interferon receptor engagement, and the cellular level of IRF7 therefore sets how much of that program a cell can mount independently of interferon.\n\nThe study does not establish the structural basis of the differential contacts. The major and minor groove assignments are inferred from mutational effects and from published structures of related complexes rather than demonstrated here. Stoichiometry of ISGF3 on DNA is explicitly left open by the authors. The relative contribution of IRF7 homodimers and any residual IRF3 and IRF7 heterodimers in the infected animal is constrained but not settled, and the authors say that heterodimer formation cannot be ruled out.", "Conceptual or technical advance": "The element rather than the cytokine becomes the unit of analysis. Assigning a gene to the interferon-stimulated class no longer implies that its induction requires interferon signaling, and the paper supplies sequence criteria by which a promoter can be sorted into IRF7-responsive, ISGF3-responsive, or universal categories. The authors suggest that the defined requirements should support computational prediction of a gene's inducibility given the levels of IRF3, IRF7 and ISGF3, which is put forward as a prospect rather than demonstrated. Practically, the STAT1-deficient U3A system with inducible IRF7 activation gives a way to read IRF7 transcriptional output with interferon feedback removed.", "Relationship to the broader research program": "The paper sits in the tenOever laboratory's continuing interest in how the cell-intrinsic antiviral transcriptional response is wired and in what the influenza A virus NS1 protein suppresses. The use of NS1-deficient and NS1-mutant influenza A virus as a tool to unmask host transcriptional responses recurs across the corpus. The mapping of which host genes are inducible independently of interferon signaling also informs later work in the corpus on interferon-independent antiviral mechanisms. Category 3 synthesis, visible only when this paper is placed beside Shapiro and colleagues 2014 on Drosha as an interferon-independent antiviral factor, is that the laboratory pursued interferon-independent arms of antiviral defense from two directions, one transcriptional and one post-transcriptional, in the same period.", "Related publications": "- Shapiro and colleagues 2014, Drosha as an interferon-independent antiviral factor. Conceptual extension. Shares the question of what antiviral activity persists when interferon signaling is removed, and shares an author, Sonja Schmid.\n- Mordstein and colleagues 2008, PLoS Pathogens. Methodological foundation. Source of the IFNAR1 and IL28R alpha double knockout mouse used here and of the prior observation that motivated the study.\n- tenOever and colleagues 2007, Science. Predecessor. Cited as the source of the ISGF3 binding model and EMSA conditions used here.\n- Perez and colleagues 2010, PNAS. Methodological foundation. Cited for the quantitative PCR and Western blot procedures used here.", "Limitations and boundaries": "The transcriptomic conclusions rest on Affymetrix arrays from pooled material, three lungs pooled per cohort at each time point with no biological replication at the array level in the animal experiment, with validation by quantitative PCR on a small number of genes. The IRF7 transcriptome is defined under exogenous overexpression of IRF7 together with IKK epsilon in a single transformed human fibrosarcoma line lacking STAT1, which is a strong and non-physiological activation regime, and the endogenous validation is limited to one element, CXCL10, and to a small panel of genes in primary mouse fibroblasts. The binding analysis is built almost entirely on derivatives of one element, the ISG15 element, so the derived consensus sequences are generalizations from a single sequence context rather than from an unbiased selection. Binding was measured with cell extracts on naked oligonucleotides, so chromatin context, cooperativity with other virus-activated factors, and promoter occupancy in cells are not addressed. The infection work uses one virus, influenza A virus, in one mouse background carrying intact Mx1 alleles, and the interferon-independent program was defined with an NS1-deficient or NS1-mutant virus rather than with wild-type virus. The proposal that restricted IRF7 expression exists to avoid unnecessary toxicity is stated by the authors as speculation and is not tested.", "Audience summaries": "### 25 words\n\nCells lacking interferon receptors still switch on much of the antiviral gene program, because IRF7 reads many of the same promoter elements that the interferon-activated ISGF3 complex uses.\n\n### 75 words\n\nAntiviral gene induction is usually attributed to interferon signaling through the ISGF3 complex. Mice lacking both type I and type III interferon receptors nonetheless induced many interferon-stimulated genes after influenza A virus infection. Systematic mutagenesis of a model promoter element showed that IRF7 and ISGF3 bind overlapping but distinguishable sequences, and activating IRF7 in interferon-unresponsive human cells reproduced most of that gene set, indicating a redundant route to the antiviral state.\n\n### 150 words\n\nThe antiviral transcriptional response has been framed as a consequence of interferon secretion and ISGF3 assembly. Infection of IFNAR1 and IL28R alpha double knockout mice with NS1-deficient influenza A virus induced a substantial set of interferon-stimulated genes despite the absence of ISGF3 activity. Working from the ISG15 interferon-stimulated response element, the authors mutated the core motif and its flanks and compared binding of reconstituted IRF7 and reconstituted ISGF3 by mobility shift, resolving positions that confer specificity for one complex, for the other, or for both, and deriving candidate consensus sequences for each. Activation of IRF7 in STAT1-deficient U3A cells induced a gene set overlapping about 80 percent with the set induced in infected knockout lungs, and promoter elements from individual genes bound the predicted factors, with MxA restricted to ISGF3 and CXCL10 restricted to IRF7. Endogenous IRF7 was required for CXCL10 and MAP3K8 induction in knockout fibroblasts under translational blockade." }, "discoveries": [ "claim-04" ], "relationships": [ { "from": "2010-schmid-transcription-factor-redundancy-en", "to": "2014-shapiro-drosha-as-an-interferon-independen", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2010-schmid-transcription-factor-redundancy-en" }, { "from": "2011-ng-i-b-kinase-ikk-regulates-the-balan", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2011-ng-i-b-kinase-ikk-regulates-the-balan" }, { "from": "2012-langlois-hematopoietic-specific-targeting-o", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2012-langlois-hematopoietic-specific-targeting-o" }, { "from": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-shapiro-evidence-for-a-cytoplasmic-micropr" }, { "from": "2014-schmid-mitogen-activated-protein-kinase-m", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-schmid-mitogen-activated-protein-kinase-m" }, { "from": "2014-schmid-mitogen-activated-protein-kinase-m", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "companion", "evidence": "stated in the Related publications section of 2014-schmid-mitogen-activated-protein-kinase-m" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" }, { "from": "2015-benitez-in-vivo-rnai-screening-identifies-", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2015-benitez-in-vivo-rnai-screening-identifies-" }, { "from": "2019-eggenberger-type-i-interferon-response-impairs", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2019-eggenberger-type-i-interferon-response-impairs" }, { "from": "2025-manivasagam-transcriptional-repressor-capicua-", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2025-manivasagam-transcriptional-repressor-capicua-" } ], "canonical_url": "https://tenoeverlab.us/publications/2010-schmid-transcription-factor-redundancy-en/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "rt-qpcr", "sirna-knockdown", "luciferase-promoter-reporter", "emsa", "lentiviral-transduction", "knockout-mice", "microarray" ] } }, { "id": "2010-shapiro-noncanonical-cytoplasmic-processin", "slug": "2010-shapiro-noncanonical-cytoplasmic-processin", "url": "/publications/2010-shapiro-noncanonical-cytoplasmic-processin/", "title": "Noncanonical cytoplasmic processing of viral microRNAs", "authors": [ "Jillian S. Shapiro", "Andrew Varble", "Alissa M. Pham", "Benjamin R. tenOever" ], "author_count": 4, "first_author": "Jillian S. Shapiro", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 4, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2010, "journal": "RNA", "volume": "16", "issue": "11", "pages": "2068-2074", "doi": "10.1261/rna.2303610", "doi_url": "https://doi.org/10.1261/rna.2303610", "pmid": "20841420", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/20841420/", "pmcid": "PMC2957047", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957047/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957047/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense" ], "themes": [ "noncanonical-microrna-biogenesis" ], "pathogens": [ "Sindbis virus" ], "viral_families": [ "Togaviridae" ], "host_species": [ "human", "mouse" ], "technologies": [ "reverse genetics", "recombinant alphavirus engineering", "small RNA Northern blot", "small RNA cloning and sequencing", "siRNA knockdown", "reporter-based post-transcriptional silencing assay", "multicycle growth curve", "confocal immunofluorescence" ], "biological_systems": [ "human fibroblasts", "murine embryonic fibroblasts", "Dicer-deficient fibroblasts", "DGCR8-deficient fibroblasts", "Ifnar1-deficient fibroblasts", "BHK21 cells", "Vero cells", "HEK293 cells" ], "key_concepts": [ "noncanonical microRNA biogenesis", "mirtron-like processing", "virtron", "Dicer dependence", "microprocessor independence", "Exportin-5 independence", "post-transcriptional gene silencing", "antiviral RNA interference in vertebrates", "viral self-targeting", "cytoplasmic hairpin processing" ], "keywords": [ "RNA interference", "microRNA", "Sindbis virus", "Dicer", "DGCR8", "Exportin-5", "miR-124", "viral engineering", "small RNA", "interferon-independent restriction" ], "one_sentence_contribution": "Insertion of a primary microRNA locus into the exclusively cytoplasmic Sindbis virus genome yields mature, functional miR-124 through a Dicer-dependent but microprocessor- and Exportin-5-independent route, defining a cytoplasmic hairpin-processing activity in vertebrate cells that the authors term a virtron.", "summary_25": "A hairpin carried by a virus that stays in the cytoplasm still becomes a working microRNA, needing Dicer but not the nuclear microRNA machinery, and restricting the virus.", "summary_75": "Vertebrate microRNAs are normally cut first in the nucleus and only finished in the cytoplasm. Putting a microRNA gene into Sindbis virus, which never enters the nucleus, still produced mature miR-124. The process required Dicer but not DGCR8, Exportin-5, or interferon signalling. The small RNA silenced a reporter and cut the virus back by about two logs in a Dicer-dependent way, indicating an uncharacterised cytoplasmic route to functional small RNAs.", "summary_150": "Canonical microRNA biogenesis requires nuclear cropping by Drosha and DGCR8 followed by Exportin-5-dependent export and cytoplasmic dicing. Shapiro and colleagues inserted the mmu-miR-124-2 primary locus into an extra subgenomic transcript of Sindbis virus, an alphavirus restricted to the cytoplasm, and recovered abundant precursor and mature miR-124 at levels comparable to plasmid overexpression without measurable replicative cost at high multiplicity. Genetic tests in knockout fibroblasts placed the activity downstream of Dicer and showed no requirement for DGCR8, Exportin-5, or type I interferon signalling, while small RNA sequencing revealed 3 prime heterogeneity comparable to that of known mirtrons. The product was functional, silencing a target-bearing reporter and attenuating the virus by roughly two logs at low multiplicity in a Dicer-dependent, largely interferon-independent manner, with plasmid-supplied miR-124 reducing SV124 protein 5.8-fold. The authors name these products virtrons and state that the responsible processing mechanism remains undefined.", "citation": "Shapiro JS, Varble A, Pham AM, tenOever BR. Noncanonical cytoplasmic processing of viral microRNAs. RNA. 2010. Volume 16, issue 11, pages 2068-2074. DOI 10.1261/rna.2303610. PMID 20841420. PMCID PMC2957047.", "sections": { "Citation": "Shapiro JS, Varble A, Pham AM, tenOever BR. Noncanonical cytoplasmic processing of viral microRNAs. RNA. 2010. Volume 16, issue 11, pages 2068-2074.\n\nDOI 10.1261/rna.2303610. PMID 20841420. PMCID PMC2957047.", "One-sentence contribution": "Insertion of a primary microRNA locus into the exclusively cytoplasmic Sindbis virus genome yields mature, functional miR-124 through a Dicer-dependent but microprocessor- and Exportin-5-independent route, defining a cytoplasmic hairpin-processing activity in vertebrate cells that the authors term a virtron.", "Executive summary": "RNA interference serves as a primary antiviral defence in plants and many invertebrates, while vertebrates are generally understood to rely on interferon-driven innate immunity, with small RNAs confined to microRNA regulation of host transcripts. Canonical microRNA maturation begins in the nucleus with Drosha and DGCR8, requires Exportin-5 for nuclear export, and finishes with Dicer in the cytoplasm. Whether a hairpin that never enters the nucleus can be processed in vertebrate cells, and whether the resulting small RNA can act against the virus that produced it, was unresolved. To test this, the authors grafted the murine mmu-miR-124-2 primary microRNA locus into an additional nonessential subgenomic transcript of Sindbis virus, an alphavirus that replicates only in the cytoplasm. The recombinant virus generated abundant precursor and mature miR-124 at levels comparable to plasmid-based overexpression, and did so without detectable cost to viral protein accumulation or cell infectivity at high multiplicity. Genetic dissection placed the activity downstream of Dicer but showed no requirement for DGCR8, Exportin-5, or type I interferon signalling. The virus-derived miR-124 silenced a sensor transcript bearing complementary target sites and imposed roughly two logs of Dicer-dependent, interferon-independent attenuation on the virus at low multiplicity. The work identifies a cytoplasmic route to functional small RNAs in vertebrate cells and leaves the molecular identity of the processing step open.", "Scientific context": "The field distinguished two established routes to a pre-microRNA. Canonical primary microRNAs are cropped in the nucleus by the Drosha and DGCR8 microprocessor, exported by Exportin-5, and diced in the cytoplasm. Mirtrons bypass Drosha by arising from very short introns that are spliced, debranched, and refolded, but they remain nuclear in origin and Exportin-5 dependent. Separately, vertebrates were thought to have replaced RNA interference as an antiviral system with pattern recognition receptors and type I interferon. Two observations kept the question open. Artificial microRNA target sites engineered into viral genomes restrict replication in vertebrate cells, and deep sequencing had recovered small RNAs mapping to cytoplasmic RNA viruses, raising the possibility that cells can cleave viral RNA structures directly. The paper positions itself against this background by supplying both the hairpin substrate and its target within a single cytoplasmic virus, removing the need for an RNA-dependent RNA polymerase to generate double-stranded RNA.", "Central question": "Can a primary microRNA hairpin delivered by a virus that never enters the nucleus be processed into a mature, functional small RNA in vertebrate cells, and if so, which components of the canonical microRNA machinery does that processing require and can the resulting small RNA restrict the virus that encodes it?", "Experimental strategy": "The design rests on compartmental logic. Sindbis virus clone TE12Q carries a duplicated subgenomic promoter and a unique cloning site, allowing the mmu-miR-124-2 locus to be carried as an extra subgenomic message without disturbing the structural or nonstructural genes. Because Sindbis replication and transcription are strictly cytoplasmic, any mature miR-124 recovered from infected cells must either reflect nuclear trafficking of the viral transcript or a cytoplasmic processing route. miR-124 was chosen because it is neuron-restricted and therefore effectively absent from fibroblasts, giving a clean background, and because its canonical processing and its behaviour in engineered viruses had been characterised previously. Requirement for each biogenesis component was then tested by genetics rather than inhibitors, using fibroblasts lacking Dicer, DGCR8, or IFNAR1, and by pooled siRNA depletion of Exportin-5 in human fibroblasts. Function was assessed with a green fluorescent protein reporter carrying tandem miR-124 target sites in its untranslated region, and antiviral consequence was assessed by low multiplicity multicycle growth curves in the same panel of knockout fibroblasts, so that any attenuation could be assigned to Dicer rather than to interferon or to steric effects on the polymerase.", "Key findings": "1. Recombinant Sindbis virus carrying the pri-miR-124-2 locus in an extra subgenomic transcript, designated SV124, was rescued to stock titres comparable to the parental virus and produced abundant approximately 60-nucleotide precursor and approximately 20-nucleotide mature miR-124 in human fibroblasts from four hours post-infection through thirty-six hours, at levels comparable to a plasmid-based miR-124 expression construct (Figure 1B). Sindbis core protein levels and near-complete cell infectivity were equivalent between the parental and recombinant viruses at high multiplicity (Figures 1B and 1C). The authors read the absence of a replicative defect as indicating that a viral protein prevents formation of the genomic hairpin or that the viral polymerase resolves the structure during replication, and this interpretation is not tested directly.\n\n2. Production of virus-derived miR-124 required Dicer. In Dicer-deficient murine embryonic fibroblasts both the Sindbis-derived miR-124 and the endogenous miR-93 were lost, while wild-type fibroblasts supported robust synthesis (Figure 2A).\n\n3. Depletion of Exportin-5 by pooled siRNA in human fibroblasts, confirmed by loss of Exportin-5 protein, did not change the level of Sindbis-derived miR-124 (Figure 2B). The authors take this as evidence that the small RNA does not pass through a nuclear intermediate.\n\n4. Cloning and sequencing of more than fifty individual small RNA products from SV124-infected cells showed that about half matched endogenous mature miR-124 exactly and about 44 percent carried some degree of 3 prime heterogeneity (Figure 2C). The authors note that this degree of heterogeneity is comparable to that reported for the mirtrons miR-344, miR-668 and miR-702.\n\n5. Synthesis of Sindbis-derived miR-124 proceeded normally in DGCR8-deficient fibroblasts, whereas endogenous miR-93 was lost in those cells, and it also proceeded normally in fibroblasts lacking a functional type I interferon receptor (Figure 3A). Processing is therefore microprocessor-independent and does not depend on interferon signalling.\n\n6. The virus-derived small RNA was functional. A green fluorescent protein reporter bearing tandem miR-124 target sites was silenced by SV124 infection, and the general reduction in host protein synthesis characteristic of alphavirus infection was enhanced in SV124 relative to parental Sindbis (Figure 3B). The authors interpret the reporter result as post-transcriptional gene silencing by virus-produced miR-124.\n\n7. At low multiplicity, SV124 was attenuated by approximately two logs relative to parental Sindbis in wild-type fibroblasts at forty-eight hours (p equals 0.008). The difference was not significant in Dicer-deficient cells (p equals 0.164) and was reduced to roughly one log but still significant in Ifnar1-deficient cells (p equals 0.015) (Figure 4A). The authors read this as Dicer-dependent, largely interferon-independent self-restriction rather than steric hindrance of the polymerase or increased pathogen-associated molecular pattern production.\n\n8. Supplying miR-124 in trans from a plasmid reduced SV124 core protein by 5.8-fold while leaving parental Sindbis core protein unaffected (Figure 4B), showing sequence-specific targeting of the recombinant virus.\n\n9. Sequence and free-energy analysis of the two viral RNA species indicated that the negative-strand genome carries a perfect miR-124 target with a substantially more favourable hybridisation free energy than the corresponding site on the positive-strand genome, which lacks a seed match longer than six nucleotides (Figure 4C). The authors propose on this basis that targeting occurs on the negative-strand genome, which is inference from sequence rather than a demonstrated site of cleavage.", "Mechanistic model": "The study does not establish the molecular mechanism of processing, and the authors say so directly, noting that how a capped and polyadenylated cytoplasmic transcript of roughly 500 nucleotides is converted into a precursor hairpin remains unknown and that future work must define it. What the data constrain is the boundary of the pathway. The activity operates on a substrate that is never nuclear, it requires Dicer, it does not require DGCR8 or Exportin-5, and it does not require type I interferon signalling. The product enters the silencing machinery, because a complementary reporter is repressed and because the virus encoding the hairpin is attenuated in a Dicer-dependent manner. The authors group these features under the term virtron and point out that while Dicer dependence with microprocessor independence resembles mirtrons, the Exportin-5 independence, the overall GC content, and the hairpin end structures do not match previously characterised endogenous mirtrons. What the data do not constrain is the identity of the enzyme or complex that liberates the precursor from the subgenomic transcript, whether an endogenous cellular RNA would be handled the same way, and whether the attenuation reflects cleavage of the negative-strand genome as proposed or another route to reduced replication.", "Conceptual or technical advance": "The work makes a cytoplasmic route to mature microRNA in vertebrate cells experimentally visible and supplies a genetic framework for isolating it, since the substrate can be delivered at will by a cytoplasmic virus and each canonical biogenesis component can be removed independently. It also provides a design principle for engineered viral vectors, showing that a primary microRNA locus can be carried in an alphavirus subgenomic message with little cost to replication at high multiplicity while yielding a functional small RNA, and that the same arrangement produces measurable sequence-specific self-restriction at low multiplicity. The separation of Dicer-dependent restriction from interferon-dependent restriction gives a way to ask how much small RNA activity persists in vertebrate cells independently of the interferon system.", "Relationship to the broader research program": "The paper belongs to a line of work in the laboratory on engineering RNA viruses to carry and produce small RNAs, and on the use of microRNA targeting as a tool for species-specific or tissue-specific attenuation of virus replication. The immediately preceding work from the group established that influenza A virus can be engineered to synthesise microRNAs and that microRNA target insertion attenuates influenza A virus in a species-specific way. The present study extends that framework from a segmented negative-sense virus that replicates in the nucleus to a positive-sense virus that is strictly cytoplasmic, which is what makes the compartmental argument possible. The recurring question the paper feeds is whether small RNA activity in vertebrates retains any antiviral role alongside interferon. The authors are cautious on this point, arguing that because virtrons entail self-targeting they are unlikely to be genuine viral products and would more plausibly be cellular by-products of infection, and stating that establishing whether they constitute an antiviral pathway requires substantially more work.", "Related publications": "- Varble et al. 2010 (PNAS), methodological foundation. Established engineered RNA viral synthesis of microRNAs and provided the pri-miR-124-2 construct and the GFP_124t reporter used here.\n- Perez et al. 2009 (Nature Biotechnology), predecessor. Established microRNA-mediated species-specific attenuation of influenza A virus, the targeting logic that the self-targeting experiments here build on, and supplied constructs cited in the methods.\n- Perez et al. 2010 (PNAS), companion. Influenza A virus-generated small RNAs and their regulatory role, from the same laboratory in the same period, and the source of the small RNA Northern protocol used here.\n- tenOever 2009 (Discovery Medicine), review or synthesis. The author's own framing of microRNA-based manipulation of viral therapeutics, cited in the introduction as the basis for the vestigial-pathway argument.", "Limitations and boundaries": "The findings rest on immortalised fibroblast lines from human and mouse and on a single engineered alphavirus carrying a single microRNA locus, so the generality across cell types, primary cells, tissues, and other cytoplasmic viruses is untested. No animal work is included. The attenuation phenotype appeared only at low multiplicity, and the authors note explicitly that rapid replication at high multiplicity in immortalised fibroblasts may have masked it, which means the magnitude of any effect is regime-dependent. Exportin-5 was removed by pooled siRNA knockdown rather than by genetic deletion, so residual protein cannot be excluded even though the immunoblot showed effective loss. The proposed site of targeting on the negative-strand genome rests on free-energy and seed-match analysis, not on direct mapping of cleavage. The processing enzyme responsible for generating the precursor from the subgenomic transcript is not identified. The system supplies the hairpin artificially, so the study does not show that natural infections generate comparable structures, and the authors themselves frame natural virtrons as hypothetical. Finally, the silencing observed was described as reduced relative to canonical microRNA activity, which bounds how much regulatory weight the pathway can carry.", "Audience summaries": "### 25 words\n\nA hairpin carried by a virus that stays in the cytoplasm still becomes a working microRNA, needing Dicer but not the nuclear microRNA machinery, and restricting the virus.\n\n### 75 words\n\nVertebrate microRNAs are normally cut first in the nucleus and only finished in the cytoplasm. Putting a microRNA gene into Sindbis virus, which never enters the nucleus, still produced mature miR-124. The process required Dicer but not DGCR8, Exportin-5, or interferon signalling. The small RNA silenced a reporter and cut the virus back by about two logs in a Dicer-dependent way, indicating an uncharacterised cytoplasmic route to functional small RNAs.\n\n### 150 words\n\nCanonical microRNA biogenesis requires nuclear cropping by Drosha and DGCR8 followed by Exportin-5-dependent export and cytoplasmic dicing. Shapiro and colleagues inserted the mmu-miR-124-2 primary locus into an extra subgenomic transcript of Sindbis virus, an alphavirus restricted to the cytoplasm, and recovered abundant precursor and mature miR-124 at levels comparable to plasmid overexpression without measurable replicative cost at high multiplicity. Genetic tests in knockout fibroblasts placed the activity downstream of Dicer and showed no requirement for DGCR8, Exportin-5, or type I interferon signalling, while small RNA sequencing revealed 3 prime heterogeneity comparable to that of known mirtrons. The product was functional, silencing a target-bearing reporter and attenuating the virus by roughly two logs at low multiplicity in a Dicer-dependent, largely interferon-independent manner, with plasmid-supplied miR-124 reducing SV124 protein 5.8-fold. The authors name these products virtrons and state that the responsible processing mechanism remains undefined." }, "discoveries": [ "claim-07" ], "relationships": [ { "from": "2010-shapiro-noncanonical-cytoplasmic-processin", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2010-shapiro-noncanonical-cytoplasmic-processin" }, { "from": "2010-shapiro-noncanonical-cytoplasmic-processin", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2010-shapiro-noncanonical-cytoplasmic-processin" }, { "from": "2012-backes-degradation-of-host-micrornas-by-p", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-backes-degradation-of-host-micrornas-by-p" }, { "from": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2012-langlois-in-vivo-delivery-of-cytoplasmic-rn" }, { "from": "2012-perez-a-small-rna-enhancer-of-viral-poly", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-perez-a-small-rna-enhancer-of-viral-poly" }, { "from": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2012-shapiro-evidence-for-a-cytoplasmic-micropr" }, { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" }, { "from": "2013-varble-an-in-vivo-rnai-screening-approach", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-varble-an-in-vivo-rnai-screening-approach" }, { "from": "2014-schmid-a-versatile-rna-vector-for-deliver", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-schmid-a-versatile-rna-vector-for-deliver" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" }, { "from": "2015-aguado-microrna-function-is-limited-to-cy", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-aguado-microrna-function-is-limited-to-cy" }, { "from": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2017-aguado-rnase-iii-nucleases-from-diverse-k" }, { "from": "2017-morales-sars-cov-encoded-small-rnas-contri", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2017-morales-sars-cov-encoded-small-rnas-contri" } ], "canonical_url": "https://tenoeverlab.us/publications/2010-shapiro-noncanonical-cytoplasmic-processin/", "controlled_vocabulary": { "pathogens": [ "sindbis-virus" ], "technologies": [ "reverse-genetics", "immunofluorescence-microscopy", "sirna-knockdown", "small-rna-seq", "small-rna-northern-blot", "growth-curve", "silencing-reporter-assay" ] } }, { "id": "2010-varble-engineered-rna-viral-synthesis-of-", "slug": "2010-varble-engineered-rna-viral-synthesis-of-", "url": "/publications/2010-varble-engineered-rna-viral-synthesis-of-/", "title": "Engineered RNA viral synthesis of microRNAs", "authors": [ "Andrew Varble", "Mark A. Chua", "Jasmine T. Perez", "Balaji Manicassamy", "Adolfo García-Sastre", "Benjamin R. tenOever" ], "author_count": 6, "first_author": "Andrew Varble", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 6, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2010, "journal": "Proceedings of the National Academy of Sciences", "volume": "107", "issue": "25", "pages": "11519-11524", "doi": "10.1073/pnas.1003115107", "doi_url": "https://doi.org/10.1073/pnas.1003115107", "pmid": "20534531", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/20534531/", "pmcid": "PMC2895125", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2895125/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2895125/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "programmable-virology", "small-rna-antiviral-defense" ], "themes": [ "noncanonical-microrna-biogenesis", "rna-vectors-for-delivery" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "human", "mouse", "canine", "chicken" ], "technologies": [ "influenza reverse genetics", "small RNA Northern blotting", "stem-loop quantitative RT-PCR", "5' RACE", "flow cytometry", "confocal immunofluorescence", "multicycle growth curves" ], "biological_systems": [ "MDCK cells", "murine fibroblasts", "Dicer-deficient fibroblasts", "CAD neuronal precursor cells", "HEK293 cells", "embryonated chicken eggs" ], "key_concepts": [ "viral microRNA synthesis", "RNA virus vectors", "Drosha and DGCR8 processing", "intron-encoded microRNA", "post-transcriptional gene silencing", "viral ribonucleoprotein accessibility", "segment 8 engineering", "small RNA delivery" ], "keywords": [ "influenza A virus", "miR-124", "microRNA", "RNA interference", "reverse genetics", "NS segment", "nuclear export protein", "RNA virus vector" ], "one_sentence_contribution": "Influenza A virus can be engineered to encode a cellular pri-microRNA inside an artificial intron of segment 8 and to produce mature, silencing-competent miR-124 during infection without measurable loss of replication or genome stability.", "summary_25": "Influenza virus was rebuilt to carry a human-type microRNA gene in an artificial intron. It made working microRNA, silenced targets, and grew normally, contradicting an assumed limit.", "summary_75": "MicroRNAs made by viruses had been found almost only in DNA viruses, and RNA viruses were thought unable to make them without destroying their own genomes. By splitting two overlapping genes in influenza segment 8, the authors created an intron that carried a cellular microRNA precursor. The virus produced mature, active miR-124 through the normal cellular pathway, silenced a reporter, drove neuron-like differentiation, and replicated as well as wild-type virus.", "summary_150": "Biogenesis of microRNAs requires nuclear cleavage of a hairpin by Drosha, which was argued to prevent RNA viruses from encoding one, since cleavage would fragment the genome and the genome would also be a perfect target of the resulting microRNA. Influenza A virus segment 8 was reengineered to separate the overlapping NS1 and NEP/NS2 reading frames, generating an intron into which the murine miR-124-2 locus was inserted. The recombinant virus produced mature miR-124 within four hours of infection at levels matching abundant cellular microRNAs, in a splicing-dependent, orientation-dependent and Dicer-dependent manner, while protein expression and multicycle growth matched wild type. Complementary RNA levels and 5' RACE gave no evidence of Drosha cleavage of the genome, and target sites on the genomic strand were not silenced while identical sites in messenger RNA were. The authors attribute genome protection to nuclear ribonucleoprotein organization, which the data are consistent with but do not establish.", "citation": "Varble A, Chua MA, Perez JT, Manicassamy B, García-Sastre A, tenOever BR. Engineered RNA viral synthesis of microRNAs. Proceedings of the National Academy of Sciences. 2010. Volume 107, issue 25, pages 11519-11524. DOI 10.1073/pnas.1003115107. PMID 20534531. PMCID PMC2895125.", "sections": { "Citation": "Varble A, Chua MA, Perez JT, Manicassamy B, García-Sastre A, tenOever BR. Engineered RNA viral synthesis of microRNAs. Proceedings of the National Academy of Sciences. 2010. Volume 107, issue 25, pages 11519-11524.\n\nDOI 10.1073/pnas.1003115107. PMID 20534531. PMCID PMC2895125.", "One-sentence contribution": "Influenza A virus can be engineered to encode a cellular pri-microRNA inside an artificial intron of segment 8 and to produce mature, silencing-competent miR-124 during infection without measurable loss of replication or genome stability.", "Executive summary": "Virus-encoded microRNAs had been described almost exclusively in DNA viruses, and the prevailing explanation held that an RNA virus carrying a microRNA hairpin in its genome would be destroyed by RNase III processing of that hairpin or silenced by its own product. The authors tested whether this constraint is absolute by building it into a negative-sense RNA virus. Segment 8 of influenza A/PR/8/34 was reconfigured so that the overlapping NS1 and NEP/NS2 reading frames were split, creating an intergenic region and an extended intron, into which the murine miR-124-2 locus was inserted in forward or reverse orientation alongside a scrambled control. Recombinant viruses were rescued by plasmid-based reverse genetics and characterized for microRNA production, genome integrity and silencing activity. The engineered virus produced mature miR-124 by four hours postinfection at levels comparable to abundant endogenous microRNAs, in an orientation-dependent and splicing-dependent manner, and production required Dicer. Viral protein expression and multicycle growth matched wild type. Complementary DNA levels and 5' RACE gave no evidence that the genomic hairpin was cleaved by Drosha, and target sites placed on the genomic strand were not silenced while the same sites in messenger RNA were. Virus-produced miR-124 silenced a reporter and induced neuron-like morphology in CAD cells. The work removes an assumed barrier and defines influenza as a candidate vector for transient small RNA delivery.", "Scientific context": "MicroRNAs are processed from primary transcripts by the nuclear RNase III complex of Drosha and DGCR8, exported by Exportin 5, cleaved by Dicer and loaded into RISC. At the time of this study, microRNAs of viral origin had been isolated from herpesviruses, polyomaviruses and adenoviruses, all nuclear DNA viruses with large coding capacity. No RNA virus had been shown to make a functional microRNA. Two arguments were advanced for that absence. First, cytoplasmic replication places the genome away from Drosha and DGCR8. Second, and applying even to nuclear RNA viruses such as influenza, excision of a hairpin from the genome by an RNase III enzyme would fragment genomic RNA, and the genome would additionally present a perfect complement to the microRNA it produced and so become a target of its own silencing machinery. Deep sequencing had by then recovered heterogeneous viral small RNA species from several RNA viruses, but their biogenesis and function were unresolved. The paper sets out to determine whether the constraint is intrinsic to RNA genomes or simply unexploited.", "Central question": "Can an RNA virus genome accommodate and express a functional microRNA, or do RNase III processing of the genomic hairpin and self-directed post-transcriptional gene silencing impose an absolute barrier on RNA virus microRNA synthesis.", "Experimental strategy": "The design exploits the one feature of influenza A virus that supplies a nuclear intron. Segments 7 and 8 undergo splicing, and segment 8 is the shorter, so it was chosen as the more tolerant insertion site. The endogenous splice acceptor was disrupted and recreated downstream of the NS1 stop codon, separating the overlapping NS1 and NEP/NS2 reading frames and generating an intergenic region that both extends the NS1 3' untranslated region and lengthens the NEP/NS2 intron. A cellular pri-microRNA placed there is excised in the spliced lariat rather than from the genome, which allows the two proposed obstacles to be separated experimentally. Three inserts were used, a scrambled sequence, the murine miR-124-2 locus in the 5' to 3' orientation, and the same locus reversed, and matched constructs were cloned into a spliced red fluorescent protein plasmid so that plasmid-based and virus-based microRNA output could be compared directly. Viruses were rescued from plasmids and grown in embryonated eggs. Genome integrity was interrogated with a reverse transcription primer specific to the complementary RNA noncoding region, which excludes messenger RNA, and by 5' RACE. Self-targeting was tested with a separate virus pair carrying miR-142 target sites oriented either to the genomic strand or to NS1 messenger RNA, assayed in cells stably expressing miR-142. Function was tested against a reporter carrying miR-124 target sites and against a cellular differentiation readout in CAD cells.", "Key findings": "1. Splitting the NS1 and NEP/NS2 reading frames and inserting scrambled or pri-miR-124 sequence into the resulting intergenic region did not reduce viral protein expression, with nucleoprotein, NS1 and NEP/NS2 all robust, and multicycle growth curves showed no significant titer decrease relative to wild-type A/PR/8/34 (Figure 1C and 1D).\n2. The recombinant virus produced mature miR-124 at levels comparable to plasmid-driven expression and to the abundant endogenous miR-93, detectable by four hours postinfection and sustained through infection (Figures 1B and 2A). Precursor miR-124 was visible at four hours and absent later, which the authors read as evidence that the export machinery was not being saturated, in contrast to a previously reported adenovirus delivery system.\n3. Expression was orientation dependent, occurring only with the locus in its native 5' to 3' configuration, and required splicing of NEP/NS2, since a construct placing the hairpin in the NS1 3' untranslated region without splicing produced no small RNA (Figure 1B and Figure S1).\n4. Stem-loop specific quantitative RT-PCR, which discriminates microRNAs differing by a single nucleotide, reported roughly 25-fold induction, and the authors interpret this as strong evidence that the viral product is an accurate mimetic of endogenous miR-124 (Figure 2B). MicroRNA accumulation tracked viral replication as measured by PB2 (Figure 2C).\n5. MiR-124 was produced in wild-type but not Dicer-deficient fibroblasts, confirmed by loss of endogenous miR-93 in the same cells and corroborated by stem-loop RT-PCR (Figures 2D and 2E). Biogenesis therefore proceeds through the canonical cellular pathway.\n6. Using a reverse transcription primer specific to the complementary RNA noncoding region, 5' and 3' ends of the NS complementary RNA were equally represented in scrambled and miR-124 viruses, and primers over the hairpin confirmed the insert was still present rather than lost to a revertant (Figures 3C, 3D and 3E). Because cleavage would block further genome synthesis, the authors read comparable complementary RNA levels as indicating that viral genomic RNA is not a favorable Drosha substrate.\n7. 5' RACE on complementary RNA recovered, in addition to full-length product, a heterogeneous population of approximately 500 nucleotide species from the miR-124 virus, none of which terminated at the base of the hairpin. The authors attribute these to random replication intermediates or PCR-mediated splice variants rather than to Drosha activity (Figure 3F).\n8. Viruses carrying miR-142 target sites oriented to the genomic strand showed no change in NS1 levels in miR-142 expressing cells, whereas the same sites in NS1 messenger RNA caused pronounced loss of NS1 without affecting nucleoprotein (Figure 4C). The authors conclude that accessibility of genomic RNA to RISC is not sufficient to reduce viral transcript levels, and propose the nuclear localization and ribonucleoprotein organization of the genome as the reason.\n9. Virus-produced miR-124 reduced the number of green fluorescent cells carrying a miR-124 targeted reporter by 47.4 percent relative to the scrambled virus, and infection of CAD cells with the miR-124 virus induced neuron-like morphology comparable to that produced by serum starvation (Figures 5A and 5B). The microRNA is therefore loaded into RISC and active on both an artificial reporter and a cellular differentiation program.", "Mechanistic model": "The data support a specific route of biogenesis. Because microRNA production requires NEP/NS2 splicing, is orientation dependent, and is not accompanied by detectable cleavage of complementary RNA or of the NS1 3' untranslated region, the authors conclude that the sole source of miR-124 is the excised lariat generated during NEP/NS2 splicing, which presents the hairpin to Drosha and DGCR8 as a conventional nuclear pri-microRNA substrate while leaving the genome intact. Dicer dependence places the remainder of the pathway in the canonical route.\n\nFor the second half of the model the evidence is weaker and the paper is explicit about interpretation. Why the genomic strand escapes both Drosha cleavage and RISC-directed silencing is not established here. The authors propose nuclear localization and the molecular organization of the viral ribonucleoprotein complex, in which genomic RNA is coated with nucleoprotein and bound by polymerase, as the reason the negative-sense strand is neither a favorable RNase III substrate nor an accessible target. No structural or biochemical experiment testing ribonucleoprotein occlusion is reported, so this remains an inference from the absence of cleavage and of silencing rather than a demonstrated mechanism. Absence of a detected cleavage product is also, by construction, a negative result, and the assays constrain cleavage to levels below detection rather than excluding it.", "Conceptual or technical advance": "The study removes an assumed prohibition. The argument that RNA viruses cannot make microRNAs because the hairpin would destroy the genome does not hold for at least one nuclear RNA virus, because splicing provides a route by which a hairpin reaches Drosha in a transcript rather than in the genome. That reframes the absence of natural RNA virus microRNAs as a question about selection rather than about physical possibility, and the discussion sets out the selective arguments, including the modest magnitude of microRNA repression, the acute nature of most RNA virus infections, and the evolutionary cost of carrying a hairpin.\n\nTechnically, the split NS1 and NEP/NS2 segment 8 with an intergenic insertion site becomes a reusable cassette. It carries a mammalian pri-microRNA without a replication penalty, produces high transient small RNA levels without saturating export, and does not integrate, which distinguishes it from lentiviral delivery. The authors note that an influenza-based vector would be confined to the respiratory tract and point to the clinical safety record of live attenuated influenza strains, framing the platform as a candidate for respiratory delivery of designed hairpins. That application is proposed, not demonstrated here.", "Relationship to the broader research program": "This paper establishes the engineering foundation for a line of work in which RNA viruses are used as programmable small RNA delivery vehicles and as instruments for asking what small RNAs do during infection. The segment 8 intergenic insertion strategy, the scrambled-insert control design, and the pairing of small RNA Northern blotting with stem-loop RT-PCR recur in later studies from the laboratory that deliver microRNAs or short hairpins from recombinant viruses and that use engineered viruses to interrogate host determinants of replication.\n\nCategory 3 synthesis. Read alongside Langlois and colleagues in 2012, which extends microRNA delivery to a cytoplasmic RNA virus in animals, and Varble and colleagues in 2013, which converts virus-encoded hairpin delivery into an in vivo screening platform, this paper is the point at which the vector concept is validated in cell culture. That trajectory is visible only when the papers are placed side by side and is not claimed by this paper.", "Related publications": "- Perez and colleagues, 2009, MicroRNA-mediated species-specific attenuation of influenza A virus, cited here as reference 38 and sharing an author. Methodological foundation for microRNA target site engineering in the influenza genome.\n- Brown and colleagues, 2007, on exploiting endogenous microRNAs to regulate transgene expression, cited as reference 26 and the source of the miR-142 targeting logic used in Figure 4. Methodological foundation.\n- Makeyev and colleagues, 2007, on miR-124 and neuronal differentiation, cited as reference 23 and the basis for the CAD cell readout. Methodological foundation.\n- Langlois and colleagues, 2012, In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs. Follow-up, extending engineered viral microRNA synthesis beyond a nuclear virus and into animals.\n- Varble and colleagues, 2013, An In Vivo RNAi Screening Approach to Identify Host Determinants of Virus Replication. Application of virus-encoded hairpin delivery.\n- tenOever, 2016, The Evolution of Antiviral Defense Systems. Review or synthesis, treating the relationship between RNA viruses and the small RNA machinery at the conceptual level.", "Limitations and boundaries": "The system is acknowledged by the authors to be artificial. It demonstrates capability, not natural occurrence, and the paper is careful to state that whether nature has produced an RNA virus that makes a microRNA remains unresolved. All conclusions rest on a single virus, influenza A/PR/8/34, and on a single inserted locus, murine miR-124-2, in one insertion site in segment 8, so tolerance of other hairpins, other segments or other strains is untested. The route to Drosha depends on splicing, which most RNA viruses do not perform, so the result does not generalize to cytoplasmic RNA viruses.\n\nEvery experiment reported here is in cell culture or in embryonated eggs. There is no animal infection, no assessment of attenuation or pathogenesis in a host, and no test of whether the insert is retained over serial passage or in vivo, so genome stability is established only over the conditions assayed. Absence of Drosha cleavage and of genome silencing is inferred from unchanged complementary RNA levels, from 5' RACE products that do not map to the hairpin base, and from unchanged NS1 levels with genomically oriented target sites, all of which bound these activities below assay sensitivity rather than excluding them. Silencing of the reporter was partial, at 47.4 percent, consistent with the modest magnitude of microRNA repression the discussion itself invokes. The proposed explanation involving ribonucleoprotein organization is not directly tested. The therapeutic framing for respiratory delivery is an extrapolation offered in the discussion and is not supported by data in this paper.", "Audience summaries": "### 25 words\n\nInfluenza virus was rebuilt to carry a human-type microRNA gene in an artificial intron. It made working microRNA, silenced targets, and grew normally, contradicting an assumed limit.\n\n### 75 words\n\nMicroRNAs made by viruses had been found almost only in DNA viruses, and RNA viruses were thought unable to make them without destroying their own genomes. By splitting two overlapping genes in influenza segment 8, the authors created an intron that carried a cellular microRNA precursor. The virus produced mature, active miR-124 through the normal cellular pathway, silenced a reporter, drove neuron-like differentiation, and replicated as well as wild-type virus.\n\n### 150 words\n\nBiogenesis of microRNAs requires nuclear cleavage of a hairpin by Drosha, which was argued to prevent RNA viruses from encoding one, since cleavage would fragment the genome and the genome would also be a perfect target of the resulting microRNA. Influenza A virus segment 8 was reengineered to separate the overlapping NS1 and NEP/NS2 reading frames, generating an intron into which the murine miR-124-2 locus was inserted. The recombinant virus produced mature miR-124 within four hours of infection at levels matching abundant cellular microRNAs, in a splicing-dependent, orientation-dependent and Dicer-dependent manner, while protein expression and multicycle growth matched wild type. Complementary RNA levels and 5' RACE gave no evidence of Drosha cleavage of the genome, and target sites on the genomic strand were not silenced while identical sites in messenger RNA were. The authors attribute genome protection to nuclear ribonucleoprotein organization, which the data are consistent with but do not establish." }, "discoveries": [ "claim-10" ], "relationships": [ { "from": "2010-shapiro-noncanonical-cytoplasmic-processin", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2010-shapiro-noncanonical-cytoplasmic-processin" }, { "from": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-langlois-in-vivo-delivery-of-cytoplasmic-rn" }, { "from": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "review or synthesis", "evidence": "stated in the Related publications section of 2012-langlois-in-vivo-delivery-of-cytoplasmic-rn" }, { "from": "2012-perez-a-small-rna-enhancer-of-viral-poly", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-perez-a-small-rna-enhancer-of-viral-poly" }, { "from": "2012-pham-replication-in-cells-of-hematopoie", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-pham-replication-in-cells-of-hematopoie" }, { "from": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-shapiro-evidence-for-a-cytoplasmic-micropr" }, { "from": "2013-chua-influenza-a-virus-utilizes-subopti", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-chua-influenza-a-virus-utilizes-subopti" }, { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" }, { "from": "2013-varble-an-in-vivo-rnai-screening-approach", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-varble-an-in-vivo-rnai-screening-approach" }, { "from": "2014-schmid-a-versatile-rna-vector-for-deliver", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-schmid-a-versatile-rna-vector-for-deliver" }, { "from": "2014-varble-influenza-a-virus-transmission-bot", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-varble-influenza-a-virus-transmission-bot" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-in-vivo-rnai-screening-identifies-", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-benitez-in-vivo-rnai-screening-identifies-" }, { "from": "2016-tenoever-the-evolution-of-antiviral-defense", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2016-tenoever-the-evolution-of-antiviral-defense" }, { "from": "2017-morales-sars-cov-encoded-small-rnas-contri", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2017-morales-sars-cov-encoded-small-rnas-contri" }, { "from": "2019-tenoever-synthetic-virology-building-viruse", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2019-tenoever-synthetic-virology-building-viruse" }, { "from": "2023-uhl-adar1-biology-can-hinder-effective", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2023-uhl-adar1-biology-can-hinder-effective" } ], "canonical_url": "https://tenoeverlab.us/publications/2010-varble-engineered-rna-viral-synthesis-of-/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "reverse-genetics", "immunofluorescence-microscopy", "flow-cytometry", "small-rna-northern-blot", "growth-curve", "small-rna-rt-qpcr", "five-prime-race" ] } }, { "id": "2011-ng-i-b-kinase-ikk-regulates-the-balan", "slug": "2011-ng-i-b-kinase-ikk-regulates-the-balan", "url": "/publications/2011-ng-i-b-kinase-ikk-regulates-the-balan/", "title": "IκB kinase ε (IKKε) regulates the balance between type I and type II interferon responses", "authors": [ "Sze-Ling Ng", "Brad A. Friedman", "Sonja Schmid", "Jason Gertz", "Richard M. Myers", "Benjamin R. tenOever", "Tom Maniatis" ], "author_count": 7, "first_author": "Sze-Ling Ng", "senior_authors": [ "Tom Maniatis" ], "corresponding_authors": [ "Tom Maniatis", "Benjamin R. tenOever" ], "tenoever_position": 6, "tenoever_role": "middle", "contribution_character": "co-led", "year": 2011, "journal": "Proceedings of the National Academy of Sciences", "volume": "108", "issue": "52", "pages": "21170-21175", "doi": "10.1073/pnas.1119137109", "doi_url": "https://doi.org/10.1073/pnas.1119137109", "pmid": "22171011", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/22171011/", "pmcid": "PMC3248534", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3248534/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3248534/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling" ], "themes": [ "transcription-factor-selectivity" ], "pathogens": [ "influenza A virus", "influenza A virus H1N1 A/Puerto Rico/8/34" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "mouse", "human" ], "technologies": [ "RNA sequencing", "ChIP sequencing", "electrophoretic mobility shift assay", "coimmunoprecipitation", "size-exclusion chromatography", "recombinant kinase assay", "adenoviral overexpression", "quantitative PCR", "motif discovery" ], "biological_systems": [ "murine embryonic fibroblasts", "bone marrow-derived macrophages", "HeLa cells expressing E1A", "293T cells", "Ikbke knockout mice" ], "key_concepts": [ "IKKepsilon", "STAT1 serine 708 phosphorylation", "ISGF3 assembly", "GAF complex", "interferon-stimulated response element", "gamma-activated sequence", "type I versus type II interferon balance", "STAT1 homodimer interface", "transcription factor complex competition", "interferon-stimulated gene selectivity" ], "keywords": [ "IKKepsilon", "STAT1", "ISGF3", "GAF", "interferon signaling", "JAK-STAT", "ISRE", "GAS", "influenza A virus", "ChIP-seq" ], "one_sentence_contribution": "Phosphorylation of STAT1 serine 708 by IKKε blocks formation of the STAT1 homodimer that constitutes GAF while leaving the STAT1 and STAT2 heterodimer of ISGF3 intact, biasing the shared STAT1 pool and the interferon-stimulated transcriptome toward the type I response.", "summary_25": "A kinase induced during infection phosphorylates STAT1 at a dimer contact point, preventing one interferon complex from forming and channeling the protein into the antiviral alternative.", "summary_75": "Two interferon pathways compete for the same protein, STAT1. Paired with STAT2 it drives the antiviral type I program, paired with itself it drives the type II program. The authors show that IKKε phosphorylates STAT1 at a residue sitting in the self-pairing interface, which blocks self-pairing but not partnering with STAT2. Cells lacking IKKε run the wrong program, bind the wrong promoters, and are more readily infected by influenza A virus.", "summary_150": "STAT1 is shared between ISGF3, the type I interferon complex that acts at interferon-stimulated response elements, and GAF, the STAT1 homodimer that acts at gamma-activated sequences. The authors show that IKKε phosphorylation of STAT1 serine 708, a residue located in the homodimer interface of the published crystal structure, blocks assembly of the activated homodimer while leaving the STAT1 and STAT2 interaction intact. In IKKε-deficient fibroblasts and macrophages, ISGF3 assembly and response element binding fall while GAF assembly and gamma-activated sequence binding rise, transcript and protein signatures shift correspondingly, and influenza A virus replicates earlier. Adenoviral IKKε produces the inverse shift, and recombinant STAT1 phosphorylated by both a JAK kinase and IKKε fails to bind a gamma-activated sequence while still supporting ISGF3. Genome-wide, expression and STAT1 occupancy track a continuous score of relative type I and type II responsiveness. The structural explanation is proposed rather than solved here.", "citation": "Ng SL, Friedman BA, Schmid S, Gertz J, Myers RM, tenOever BR, Maniatis T. IκB kinase ε (IKKε) regulates the balance between type I and type II interferon responses. Proceedings of the National Academy of Sciences. 2011. Volume 108, issue 52, pages 21170-21175. DOI 10.1073/pnas.1119137109. PMID 22171011. PMCID PMC3248534.", "sections": { "Citation": "Ng SL, Friedman BA, Schmid S, Gertz J, Myers RM, tenOever BR, Maniatis T. IκB kinase ε (IKKε) regulates the balance between type I and type II interferon responses. Proceedings of the National Academy of Sciences. 2011. Volume 108, issue 52, pages 21170-21175.\n\nDOI 10.1073/pnas.1119137109. PMID 22171011. PMCID PMC3248534.", "One-sentence contribution": "Phosphorylation of STAT1 serine 708 by IKKε blocks formation of the STAT1 homodimer that constitutes GAF while leaving the STAT1 and STAT2 heterodimer of ISGF3 intact, biasing the shared STAT1 pool and the interferon-stimulated transcriptome toward the type I response.", "Executive summary": "STAT1 is a shared component of two distinct interferon-driven transcription factor complexes. With STAT2 and IRF9 it forms ISGF3, which acts on interferon-stimulated response elements downstream of type I interferon. As a homodimer it forms GAF, which acts on gamma-activated sequences downstream of type II interferon. Because the two complexes draw on the same protein, the allocation of STAT1 between them determines which antiviral program a cell runs. Earlier work from these laboratories had shown that IKKε phosphorylates STAT1 at serine 708 and that this is required for an effective antiviral response, without explaining how. Here the authors ask how that phosphorylation acts. Using IKKε knockout fibroblasts and macrophages, gel shift assays, coimmunoprecipitation with conformation-reporting STAT1 mutants, recombinant kinase reconstitution, RNA sequencing and STAT1 ChIP sequencing, they find that loss of IKKε reduces ISGF3 assembly and DNA binding while increasing GAF assembly and binding, with a reciprocal shift in the gene expression program and increased permissiveness to influenza A virus. In a structural model of the GAF complex, serine 708 sits at the homodimer interface and makes hydrogen bonds there. Recombinant STAT1 phosphorylated by both a JAK kinase and IKKε failed to bind a gamma-activated sequence but still supported ISGF3. The work positions IKKε as a switch that partitions STAT1 between the two complexes.", "Scientific context": "Type I and type II interferons induce overlapping but distinct transcriptomes, and much of the overlap traces to the shared use of STAT1 and to promoters that carry both element classes. The IKK-related kinases IKKε and TBK1 were known to phosphorylate IRF3 and IRF7 during induction of interferon beta. Knockout mouse studies from several laboratories had, however, placed TBK1 rather than IKKε at the center of type I interferon induction, leaving the phenotype of IKKε loss unexplained by an induction defect. The immediately preceding work from these authors, published in 2007, showed that IKKε knockout mice make normal amounts of type I interferon after influenza A virus infection but respond abnormally to it, losing a subset of interferon-stimulated proteins and failing to clear virus, and identified STAT1 serine 708 as an IKKε substrate. That work also distinguished IKKε-dependent from IKKε-independent classes of interferon-stimulated response element. What remained unresolved was the molecular consequence of the serine 708 modification and why it should matter selectively for a subset of genes.", "Central question": "How does IKKε-mediated phosphorylation of STAT1 produce an effective type I interferon response, and specifically does that phosphorylation act by changing which STAT1-containing transcription factor complex assembles?", "Experimental strategy": "The design moves from phenotype to complex to residue and then back out to the genome. Loss-of-function fibroblasts and macrophages from Ikbke knockout and wild type mice provide the comparison throughout, with influenza A virus infection establishing that the signaling defect has a consequence for virus control. Complex-level questions are addressed by gel shift assays against three probe classes, an IKKε-dependent response element from the Adar1 promoter, an IKKε-independent element from the Irf7 promoter, and a gamma-activated sequence from the Irf1 promoter, plus size-exclusion chromatography to read the relative abundance of the two complexes directly. Gain of function is tested by adenoviral IKKε delivery into E1A-expressing HeLa cells, chosen because E1A blocks IRF3-driven interferon induction and so allows interferon input to be set by the experimenter. The residue-level question is approached through the published crystal structure of the phosphotyrosine STAT1 dimer on DNA, through coimmunoprecipitation using STAT1 mutants that cannot form the unstimulated antiparallel dimer and therefore report only on the activated parallel dimer, and through reconstitution with purified recombinant proteins phosphorylated in vitro by a JAK kinase and by IKKε. Finally, RNA sequencing and STAT1 alpha ChIP sequencing in bone marrow-derived macrophages test whether the model holds across the genome, with genes ordered along a beta to gamma mixture score that quantifies the relative response to the two interferons.", "Key findings": "1. IKKε-deficient fibroblasts are more permissive to influenza A virus. Viral protein was detectable by 6 hours after infection in knockout cells against 12 hours in wild type, and interferon beta pretreatment did not significantly reduce viral load in the knockout, which the authors read as locating the defect in type I interferon signaling rather than in interferon production (Figure 1A).\n\n2. The protein-level signature of IKKε loss is reciprocal. IFIT1 and IFIT2, both primarily type I driven, appeared later and at lower levels in knockout cells, while IRF1 and STAT1, both primarily GAF regulated, were not decreased and in the case of IRF1 appeared earlier (Figure 1B).\n\n3. The same reciprocity holds at the transcript level. Ifit2, Mda5 and Viperin were reduced in knockout cells after interferon beta, while Irf1, Irf8 and Icam1 were elevated, and Stat1 message was higher in knockout cells after either interferon, more markedly after interferon gamma (Figure 2A,B).\n\n4. Complex assembly and DNA binding shift in opposite directions. Binding of ISGF3 to the IKKε-dependent element was substantially reduced in knockout extracts while binding to the IKKε-independent element was indistinguishable between genotypes, including in competition titrations (Figure 3A and Figure S1). Binding of GAF to the gamma-activated sequence increased in knockout extracts after either interferon (Figure 3B). Size-exclusion chromatography showed ISGF3 exceeding GAF in wild type extracts with the ratio shifted in the knockout (Figure S2).\n\n5. Adding IKKε produces the mirror-image result. Adenoviral IKKε in E1A-expressing HeLa cells did not reduce ISGF3 binding to the response element but did reduce GAF binding to the gamma-activated sequence, with an inverse relation between IKKε level and binding (Figure S3A-C). At the gene level, IKKε expression induced IFIT2 in response to type II interferon and sharply reduced IRF1 (Figure S4).\n\n6. Serine 708 lies at the homodimer interface. In the published crystal structure of the STAT1 homodimer on DNA, serine 708 sits within the dimerization interface and participates in hydrogen bonding there (Figure 4A,B). The authors use this structure to motivate the hypothesis that phosphorylation at this position is incompatible with the homodimer but tolerated in ISGF3. The structural interpretation is theirs and is not tested by new structural work.\n\n7. IKKε blocks activated STAT1 homodimerization but not the STAT1 and STAT2 interaction. Using STAT1 mutants unable to preassociate in the antiparallel conformation, interferon gamma induced dimerization was disrupted by wild type IKKε but not by GFP or by the catalytically dead K38A mutant (Figure 4C). The STAT1 and STAT2 interaction was not disrupted and in fact appeared stabilised before stimulation by IKKε expression (Figure 4D). A phosphorylation-dependent mobility shift in STAT2 led the authors to suggest that IKKε may also phosphorylate STAT2, which is an observation-based suggestion rather than a demonstrated modification.\n\n8. Purified components reproduce the switch. Recombinant STAT1 phosphorylated by a JAK kinase formed GAF and bound the gamma-activated sequence, whereas STAT1 phosphorylated by both JAK and IKKε failed to bind, while IKKε phosphorylation did not disrupt ISGF3 formation (Figure S5). This is the most direct evidence in the paper that the effect is intrinsic to the modified protein rather than an indirect consequence of the knockout state.\n\n9. Genome-wide expression follows the same axis. Among 538 interferon-stimulated genes in wild type macrophages, genes scoring toward the beta end of the mixture scale were expressed at lower levels in knockout cells after interferon beta, and genes scoring toward the gamma end were expressed at higher levels, with a general elevation of baseline expression for gamma-leaning genes in knockout cells (Figure S6A,B).\n\n10. STAT1 occupancy changes correspondingly. Read density at STAT1 alpha peaks near Tlr9 and Ifit2 was higher in wild type macrophages, while peaks near Nos2, Gbp2 and Irf1 were not reduced in knockout cells and Nos2 was higher (Figure S7). The authors conclude that GAF binding is robust in macrophages and that the primary regulatory action of IKKε is enhancement of ISGF3 formation.\n\n11. Sequence context distinguishes the two peak classes. The gamma-activated sequence motif was enriched in peaks near gamma-leaning genes relative to beta-leaning genes, while the response element consensus did not differ significantly between the two sets. Unbiased motif discovery recovered response element motifs, with the version from gamma-leaning genes carrying additional purine-rich contacts (Figure 5A,B). The authors relate this to their earlier proposal that extra DNA contacts can compensate for the absence of an IKKε-modified ISGF3.", "Mechanistic model": "The model the data support is one of competitive allocation of a shared subunit. STAT1 activated by JAK-mediated tyrosine phosphorylation can either homodimerise into GAF or partner with STAT2 and IRF9 into ISGF3. IKKε, itself induced during infection, phosphorylates STAT1 at serine 708 and thereby renders the homodimer interface unfavourable, so the available STAT1 is driven into ISGF3. The consequence at the genome is more binding and transcription at response elements, less at gamma-activated sequences, and a cell better able to restrict influenza A virus.\n\nSeveral links in this chain are directly demonstrated. The reciprocal behavior of the two complexes in gain and loss of function, the requirement for IKKε catalytic activity in disrupting the activated homodimer, and the reconstitution with purified phosphorylated proteins all support the core claim. The structural explanation, that phosphorylation of serine 708 disrupts hydrogen bonding at the homodimer interface while being accommodated in the trimeric complex, is explicitly framed by the authors as a proposal based on an existing crystal structure of the homodimer. No structure of ISGF3 with or without the modification is presented, and the paper states that the structural differences in ISGF3 assembled with and without IKKε are unknown. The suggestion that a differential requirement for serine 708 explains why minimal response elements are IKKε-dependent while longer elements with additional purine contacts are not is likewise a proposal supported by correlation between motif content and genotype sensitivity, not by direct test.", "Conceptual or technical advance": "The work converts a kinase and a phosphosite into a quantitative control point for the choice between two interferon programs. Rather than treating serine 708 phosphorylation as a general potentiator of STAT1 activity, it identifies a structural target, the homodimer interface, and a consequence, reallocation of a limiting shared subunit. That framing makes several things testable that were not before, including the prediction that the switch should be sensitive to the ratio of activated IKKε to STAT1, which the authors state explicitly, and the prediction that promoter element architecture determines which genes feel the loss of the kinase first. Methodologically, the combination of conformation-reporting STAT1 mutants with reconstitution from purified phosphorylated components provides a way to separate a complex assembly defect from a DNA affinity defect, and pairing RNA sequencing with STAT1 ChIP sequencing across a continuous beta to gamma score gives a genome-scale readout of a balance rather than of a single pathway.", "Relationship to the broader research program": "The paper extends a line begun in the 2007 Science report from tenOever and colleagues, which identified serine 708 as an IKKε substrate and separated IKKε-dependent from IKKε-independent response elements without resolving the mechanism. It also connects to the 2010 work of Schmid, Mordstein, Kochs, García-Sastre and tenOever on transcription factor redundancy in induction of the antiviral state, which is cited here in support of the promoter-architecture argument. Taken together with those two papers the recurring question is how a limited set of shared transcription factors is apportioned so that a cell produces the right antiviral program rather than simply a large one. That framing is a category 3 synthesis across those three papers and is offered as such rather than as a claim made by any one of them.", "Related publications": "- tenOever and colleagues, 2007, Science, on multiple functions of IKKepsilon in interferon-mediated antiviral immunity. Predecessor. Established the phosphorylation of STAT1 serine 708 by IKKε, the antiviral requirement for it, and the distinction between IKKε-dependent and IKKε-independent response elements, all of which this paper builds on directly.\n- Schmid, Mordstein, Kochs, García-Sastre and tenOever, 2010, Journal of Biological Chemistry, on transcription factor redundancy in induction of the antiviral state. Conceptual extension within the same program, cited here in support of the interpretation that promoter element architecture determines IKKε dependence.\n- Chen and colleagues, 1998, on the crystal structure of the tyrosine phosphorylated STAT1 dimer bound to DNA. Methodological foundation from another laboratory, the structural basis for locating serine 708 at the dimerization interface.\n- Mao and colleagues, 2005, and Mertens and colleagues, 2006, on the antiparallel and parallel STAT1 conformations. Methodological foundation from other laboratories, the basis for the mutants used to isolate activated dimers in the coimmunoprecipitation experiments.", "Limitations and boundaries": "The cellular work is in mouse embryonic fibroblasts and mouse bone marrow-derived macrophages, plus human cell lines for the overexpression and coimmunoprecipitation experiments, and the conclusions are not extended to whole animals in this paper. Loss of IKKε is constitutive rather than conditional or acute, so secondary adaptation in the knockout cells cannot be excluded, although the recombinant reconstitution and the gain-of-function experiments mitigate this. The overexpression experiments use adenoviral delivery at levels that are not physiological and depend on E1A to suppress endogenous interferon induction, which is itself a perturbation of the system. The structural claim rests on an existing homodimer structure rather than on structures of the phosphorylated protein or of ISGF3, and the paper says directly that the structural consequences within ISGF3 are unknown. The ChIP sequencing was performed with an antibody against STAT1 alpha and at a single 6 hour time point, so it does not resolve kinetics or distinguish complexes except by inference from the associated gene and motif classes. The suggestion that IKKε also phosphorylates STAT2 rests on a mobility shift and is not pursued. The quantitative relationship the authors propose between activated IKKε and STAT1 levels is a stated expectation rather than a measured parameter. Virus work is confined to one influenza A strain in fibroblasts.", "Audience summaries": "### 25 words\n\nA kinase induced during infection phosphorylates STAT1 at a dimer contact point, preventing one interferon complex from forming and channeling the protein into the antiviral alternative.\n\n### 75 words\n\nTwo interferon pathways compete for the same protein, STAT1. Paired with STAT2 it drives the antiviral type I program, paired with itself it drives the type II program. The authors show that IKKε phosphorylates STAT1 at a residue sitting in the self-pairing interface, which blocks self-pairing but not partnering with STAT2. Cells lacking IKKε run the wrong program, bind the wrong promoters, and are more readily infected by influenza A virus.\n\n### 150 words\n\nSTAT1 is shared between ISGF3, the type I interferon complex that acts at interferon-stimulated response elements, and GAF, the STAT1 homodimer that acts at gamma-activated sequences. The authors show that IKKε phosphorylation of STAT1 serine 708, a residue located in the homodimer interface of the published crystal structure, blocks assembly of the activated homodimer while leaving the STAT1 and STAT2 interaction intact. In IKKε-deficient fibroblasts and macrophages, ISGF3 assembly and response element binding fall while GAF assembly and gamma-activated sequence binding rise, transcript and protein signatures shift correspondingly, and influenza A virus replicates earlier. Adenoviral IKKε produces the inverse shift, and recombinant STAT1 phosphorylated by both a JAK kinase and IKKε fails to bind a gamma-activated sequence while still supporting ISGF3. Genome-wide, expression and STAT1 occupancy track a continuous score of relative type I and type II responsiveness. The structural explanation is proposed rather than solved here." }, "discoveries": [ "claim-04" ], "relationships": [ { "from": "2011-ng-i-b-kinase-ikk-regulates-the-balan", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2011-ng-i-b-kinase-ikk-regulates-the-balan" } ], "canonical_url": "https://tenoeverlab.us/publications/2011-ng-i-b-kinase-ikk-regulates-the-balan/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "emsa", "adenoviral-vector", "co-ip", "in-vitro-kinase-assay", "chip", "motif-analysis", "size-exclusion-chromatography" ] } }, { "id": "2012-backes-degradation-of-host-micrornas-by-p", "slug": "2012-backes-degradation-of-host-micrornas-by-p", "url": "/publications/2012-backes-degradation-of-host-micrornas-by-p/", "title": "Degradation of Host MicroRNAs by Poxvirus Poly(A) Polymerase Reveals Terminal RNA Methylation as a Protective Antiviral Mechanism", "authors": [ "Simone Backes", "Jillian S. Shapiro", "Leah R. Sabin", "Alissa M. Pham", "Ismarc Reyes", "Bernard Moss", "Sara Cherry", "Benjamin R. tenOever" ], "author_count": 8, "first_author": "Simone Backes", "senior_authors": [ "Sara Cherry", "Benjamin R. tenOever" ], "corresponding_authors": [ "Sara Cherry", "Benjamin R. tenOever" ], "tenoever_position": 8, "tenoever_role": "senior", "contribution_character": "co-led", "year": 2012, "journal": "Cell Host & Microbe", "volume": "12", "issue": "2", "pages": "200-210", "doi": "10.1016/j.chom.2012.05.019", "doi_url": "https://doi.org/10.1016/j.chom.2012.05.019", "pmid": "22901540", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/22901540/", "pmcid": "PMC3782087", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3782087/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3782087/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense" ], "themes": [ "does-mammalian-antiviral-rnai-exist", "limits-of-microrna-function" ], "pathogens": [ "vaccinia virus", "Amsacta moorei entomopoxvirus", "Sindbis virus", "vesicular stomatitis virus" ], "viral_families": [ "Poxviridae", "Togaviridae", "Rhabdoviridae" ], "host_species": [ "human", "mouse", "hamster", "Drosophila melanogaster", "Amsacta moorei" ], "technologies": [ "small RNA deep sequencing", "small RNA northern blot", "RNA interference knockdown", "recombinant poxvirus engineering", "argonaute immunoprecipitation", "synthetic modified RNA mimetics", "reporter silencing assay" ], "biological_systems": [ "Drosophila DL1 cells", "Amsacta moorei Ld652 cells", "BHK21 cells", "BSC-1 cells", "murine embryonic fibroblasts" ], "key_concepts": [ "microRNA turnover", "nontemplated 3-prime adenylation", "poly(A) polymerase VP55", "VP39 processivity factor", "2-prime O-methylation", "RNA-induced silencing complex", "strand selection", "small RNA-mediated antiviral restriction", "self versus non-self RNA discrimination" ], "keywords": [ "vaccinia virus", "VP55", "microRNA degradation", "polyadenylation", "2-prime O-methyl", "argonaute", "entomopoxvirus", "miR-124", "esiRNA" ], "one_sentence_contribution": "Poxviruses degrade host microRNAs through the catalytic subunit of their own poly(A) polymerase, VP55, which adds short nontemplated adenosine tails to argonaute-loaded guide strands and thereby marks them for cellular decay, while small RNAs carrying a 3 prime terminal 2 prime O-methyl group are spared.", "summary_25": "Poxviruses carry an enzyme that adds short adenosine tails to host microRNAs, marking them for destruction. Small RNAs with a methylated end escape this fate.", "summary_75": "Poxviruses have never been shown to use host microRNAs, and this work suggests why. In insect and mammalian cells, poxvirus infection adds a few nontemplated adenosines to mature microRNAs, after which the cell's own machinery destroys them. The responsible enzyme is VP55, the catalytic subunit of the viral poly(A) polymerase, which is both necessary and sufficient. Small RNAs carrying a methyl group at their 3 prime end are not modified and survive.", "summary_150": "Poxviruses replicate in the cytoplasm and transcribe genes with long 3 prime untranslated regions, yet none had been reported to encode microRNAs. Sequencing small RNAs during vaccinia virus infection of Drosophila cells, entomopoxvirus infection of its natural moth host, and vaccinia infection of mammalian cells showed that mature microRNAs acquire short nontemplated adenosine tracts and then disappear, while endogenous small interfering RNAs do not. Chemical inhibitors placed the activity in early infection, and knockdown together with reconstitution identified VP55, the catalytic subunit of the viral poly(A) polymerase, as necessary and sufficient. A synthetic microRNA bearing seven adenosines was destroyed in uninfected cells, showing that the nuclease is cellular. Tailed guides remained argonaute associated, placing the modification after strand selection, and a 2 prime O-methylated guide was fully protected. Restoring the dominant microRNAs during infection halved virus production. The authors propose that terminal methylation evolved partly to shield small RNAs from this class of attack.", "citation": "Backes S, Shapiro JS, Sabin LR, Pham AM, Reyes I, Moss B, Cherry S, tenOever BR. Degradation of Host MicroRNAs by Poxvirus Poly(A) Polymerase Reveals Terminal RNA Methylation as a Protective Antiviral Mechanism. Cell Host & Microbe. 2012. Volume 12, issue 2, pages 200-210. DOI 10.1016/j.chom.2012.05.019. PMID 22901540. PMCID PMC3782087.", "sections": { "Citation": "Backes S, Shapiro JS, Sabin LR, Pham AM, Reyes I, Moss B, Cherry S, tenOever BR. Degradation of Host MicroRNAs by Poxvirus Poly(A) Polymerase Reveals Terminal RNA Methylation as a Protective Antiviral Mechanism. Cell Host & Microbe. 2012. Volume 12, issue 2, pages 200-210.\n\nDOI 10.1016/j.chom.2012.05.019. PMID 22901540. PMCID PMC3782087.", "One-sentence contribution": "Poxviruses degrade host microRNAs through the catalytic subunit of their own poly(A) polymerase, VP55, which adds short nontemplated adenosine tails to argonaute-loaded guide strands and thereby marks them for cellular decay, while small RNAs carrying a 3 prime terminal 2 prime O-methyl group are spared.", "Executive summary": "Poxviruses are large double-stranded DNA viruses that replicate entirely in the cytoplasm and transcribe genes with long 3 prime untranslated regions, so they might be expected to exploit host microRNAs the way several nuclear DNA viruses do, yet no poxvirus-encoded microRNA had been reported. The authors approached this puzzle from the small RNA side, sequencing the small RNA population of insect and mammalian cells during poxvirus infection. In Drosophila cells infected with vaccinia virus and in tiger moth cells infected with their natural entomopoxvirus, mature microRNAs acquired nontemplated 3 prime adenosines and were lost, while endogenous small interfering RNAs were not. The same happened in mammalian cells, where a recombinant vaccinia virus expressing miR-124 produced heterogeneous tailed species and simultaneously eliminated endogenous miR-93. Inhibitor experiments placed the responsible activity in the early phase of infection, and knockdown and reconstitution identified VP55, the catalytic subunit of the viral poly(A) polymerase, as both necessary and sufficient. Synthetic microRNA carrying seven adenosines was degraded in uninfected cells, indicating that the decay step is cellular. Tailed microRNAs remained associated with argonaute 2, placing the modification after strand selection, and a guide strand bearing a 3 prime 2 prime O-methyl group escaped tailing entirely. Restoring abundant microRNAs during infection roughly halved virus production, which the authors read as evidence that this degradation benefits the virus.", "Scientific context": "Plants, worms and insects use small interfering RNAs derived from the pathogen to restrict virus infection, while chordates respond largely through nucleic acid sensing and type I interferon. The paper frames its starting question as an anomaly in that landscape. Several nuclear DNA viruses and retroviruses encode their own microRNAs to reshape the host transcriptome, and the generalisation in the field at the time was that this strategy belongs to pathogens with large DNA genomes and sustained replication cycles. Poxviruses fit that description but had not been reported to use the pathway, which could not be explained by cytoplasmic replication because the laboratory and others had already shown that cytoplasmic RNA viruses can be engineered to generate functional microRNAs through noncanonical processing. Separately, 2 prime O-methylation was known to protect small RNAs from 3 prime uridylation-dependent decay and from exonucleolytic trimming, and methylation of the messenger RNA cap had recently been implicated in distinguishing host from viral RNA. These two lines had not been connected to poxvirus biology.", "Central question": "Why do poxviruses not appear to exploit the host microRNA pathway, and does poxvirus infection instead act against host small RNAs.", "Experimental strategy": "The study runs the same question through two host contexts and then reduces it to a single viral protein. Insect cells were used first because they carry both microRNA and small interfering RNA pathways, which allows the two classes to be compared within one sample, and because a genuine insect poxvirus pair exists to control for the artificial nature of infecting Drosophila cells with a mammalian virus. Small RNA deep sequencing at nucleotide resolution was the primary discovery readout, since the modification in question is the addition of a few nontemplated bases and would be invisible to abundance-only methods, and it was paired with northern blot so that mobility shift and abundance loss could be seen together. In mammalian cells the authors built a recombinant vaccinia virus carrying the miR-124 locus, which supplies a microRNA that is absent from the cells used and therefore reports on virus-derived processing and modification separately from the endogenous pool. Temporal placement used cycloheximide and cytosine arabinoside to restrict the infection to distinct gene expression classes. Attribution to VP55 used loss of function by small interfering RNA during infection and gain of function by transfection of the tagged subunits alone, with VP39 as the internal specificity control. Chemically defined mimetics carrying zero, one or seven adenosines separated the tagging step from the degradation step, and a 2 prime O-methylated guide strand tested the protection hypothesis directly. Argonaute 2 immunoprecipitation positioned the modification relative to strand selection, and reconstitution of the dominant microRNAs during infection tested whether the degradation matters for virus yield.", "Key findings": "1. Deep sequencing of small RNAs from vaccinia-infected Drosophila cells showed nontemplated adenosines added to mature microRNAs including bantam, miR-7 and miR-34, with Sindbis virus infection serving as a virus that does not perturb silencing. Endogenous small interfering RNAs were not tailed (Figure 1A and 1B). Northern blot showed the modified microRNAs migrating more slowly and falling in abundance (Figure 1C).\n2. Knockdown of argonaute 1 reduced tailing while knockdown of argonaute 2 did not, and loss of argonaute 2 increased tailing of endogenous small interfering RNAs (Figure 1B). The authors interpret the latter as cross-loading of those small RNAs into an argonaute 1 complex rather than a change in the enzyme's preference.\n3. Amsacta moorei entomopoxvirus infection of its natural tiger moth host cells also removed mature miR-11, miR-34 and miR-184 while leaving U6 intact (Figure 1D), establishing the effect in a genuine insect poxvirus pairing rather than only in an abortive heterologous infection.\n4. In mammalian cells, a recombinant vaccinia virus expressing miR-124 yielded low levels of mature miR-124 in heterogeneous forms from 22 to 35 nucleotides and nearly eliminated endogenous miR-93, whereas plasmid, Sindbis virus and vesicular stomatitis virus delivery of the same microRNA gave clean accumulation (Figure 2A). A kinetic series showed a single 22 nucleotide species at three hours, slower migrating species from six hours with a dominant 30 nucleotide product, and loss of miR-93 coincident with that product. Wild-type vaccinia virus lacking the miR-124 locus also destroyed miR-93 (Figure 2B).\n5. Processing of the virus-derived microRNA did not require DGCR8 but did require Dicer, which the authors take as the noncanonical route already described for cytoplasmic RNA viruses (Figures S2A to S2C).\n6. Cycloheximide, which blocks protein synthesis, prevented both the laddering of miR-124 and the loss of miR-93, while cytosine arabinoside, which permits early gene expression only, did not (Figure 3). The activity therefore depends on an early gene product or a protein delivered in the virion.\n7. Sequencing the 20 to 35 nucleotide fraction from infected mammalian cells showed that more than half of the reads for miR-124 and for the unrelated miR-31 carried seven to nine adenosines, while star strands and non-microRNA species in the same fraction did not (Figures 4A and 4B, Table S2). Northern blot confirmed tailing of miR-124 and miR-31 and no modification of pre-miR-124, miR-124 star, transfer RNA or U6 (Figures 4C and 4D).\n8. Sequencing the 19 to 22 nucleotide fraction from infected fibroblasts showed an approximately thirty-fold reduction across endogenous microRNAs, sparing only the virus-produced miR-124, with the overall small RNA profile otherwise preserved (Figure 5A). The authors read the lack of sequence bias as a general rather than a targeted activity.\n9. A synthetic miR-124 carrying seven adenosines was degraded after transfection into uninfected cells, while the untailed and single-adenosine forms were not (Figure 5B). The nuclease step is therefore supplied by the host and does not require a virus-encoded enzyme.\n10. Knockdown of VP55 during infection abolished both the tailing of virus-derived miR-124 and the degradation of miR-93, and transfection of tagged VP55 alone was sufficient to reproduce both, with VP39 inactive in either assay (Figures 6A to 6C). VP55 expression left miR-124 star, transfer RNA and U6 unmodified, matching the specificity seen in infection.\n11. In a reporter assay, miR-124 silenced a GFP construct carrying four perfect target sites, and co-expression of VP55 restored GFP, which the authors present as evidence that tailing renders the microRNA nonfunctional rather than merely altering its mobility (Figure 6D).\n12. Transfected duplex mimetics of the dominant fibroblast microRNAs, the let-7 family together with miR-21, miR-22 and miR-93, resisted degradation during infection, and their presence reduced total virus at sixty hours by roughly half with a reported p value of 0.0038, with a comparable reduction in extracellular enveloped virus (Figures S5C to S5E). The authors state that this restriction could be direct or indirect.\n13. Tailed and untailed miR-124 species both co-precipitated with argonaute 2, while miR-124 star was recovered only in unadenylated form (Figure 7A). The authors interpret this as tailing occurring after strand selection, acting on the guide within or in association with the loaded complex.\n14. A miR-124 guide strand bearing a 3 prime terminal 2 prime O-methyl group was completely protected from adenylation during infection, while the unmethylated duplex was tailed (Figures 7B and 7C).", "Mechanistic model": "The biochemical chain is supported at each step. VP55, an early and virion-associated protein, adds a short nontemplated poly(A) tract of roughly two to nine adenosines to the guide strand of a microRNA after strand selection, and the tailed guide is then removed by host degradative machinery, since a chemically synthesised tailed microRNA is destroyed in the absence of any viral protein. A 3 prime terminal 2 prime O-methyl group blocks the adenylation step and thereby the entire pathway.\n\nTwo elements are interpretation rather than demonstration. The first is where in the complex the reaction occurs. The argonaute 2 immunoprecipitation shows that tailed species are argonaute associated and that star strands are not tailed, and the authors infer from this that VP55 acts through an interaction with argonaute or another component of the mature silencing complex. No direct interaction between VP55 and argonaute is shown. The second is the purpose of the activity. The authors offer two evolutionary readings, evasion of microRNA-mediated restriction arising from the long 3 prime untranslated regions of poxvirus transcripts or from indirect control of host factors, and evasion of a more general small RNA-based restriction of the kind present in insects. They then propose that 2 prime O-methylation may have evolved, at least in part, as a cellular countermeasure protecting antipathogen small RNAs. This last proposal is explicitly speculative in the paper and rests on comparative arguments about the distribution of the HEN1 methyltransferase rather than on experiment.", "Conceptual or technical advance": "The work supplies a concrete reason why a family of large DNA viruses does not use host microRNAs, namely that it destroys them, which converts an absence in the literature into a testable enzymatic activity with a named subunit. It also assigns a second function to a protein previously characterised for its role in viral messenger RNA maturation, so that the VP55 and VP39 heterodimer is described as carrying both replication functions and evasion functions. By showing that a 3 prime terminal methyl group determines whether a small RNA is a substrate, the study places terminal RNA methylation alongside cap methylation as a mark distinguishing RNA that is protected from RNA that is not, and makes that distinction experimentally accessible using defined synthetic substrates. The engineered poxvirus expressing a heterologous microRNA is itself a reusable reagent for separating virus-derived from host-derived small RNA in infected cells.", "Relationship to the broader research program": "The paper belongs to a sustained line of work in this corpus on what happens to small RNA biology during virus infection and on the use of engineered viruses that express microRNAs as experimental instruments. It cites the laboratory's own earlier demonstrations that cytoplasmic RNA viruses can be built to generate functional microRNAs through noncanonical processing, and the recombinant vaccinia virus here extends that toolkit to a DNA virus. It also connects to the laboratory's interest in why chordates rely on interferon rather than RNA interference, since the finding that a virus spends an enzyme on destroying host microRNAs argues that those small RNAs impose a cost on the virus. Setting this paper beside the laboratory's other small RNA work, the recurring move is to treat small RNAs as both a subject of viral antagonism and a means of interrogating infection, which is a category 3 synthesis drawn from reading these papers together rather than a claim made here.", "Related publications": "- Shapiro and colleagues, 2010 and 2012, methodological foundation, from the same laboratory. Cited as the demonstration that cytoplasmic RNA viruses can be engineered to produce functional microRNAs, which is the premise for building the recombinant vaccinia virus used here.\n- Langlois and colleagues, 2012, methodological foundation and companion, from the same laboratory. Supplies the vesicular stomatitis virus expressing miR-124 used as a comparator and the description of noncanonical cytoplasmic microRNA processing.\n- Perez and colleagues, 2009, and Pham and colleagues, 2012, conceptual extension, from the same laboratory. Cited among the reports that viruses can be engineered to be targeted by host microRNAs, the converse application of the same host and small RNA interface.\n- Ameres and colleagues, 2010, predecessor, from another laboratory. Cited as the description of target-directed trimming and tailing of small RNAs, the host process that the viral activity here resembles and appears to exploit.\n- Daffis and colleagues, 2010, predecessor, from another laboratory. Cited as the demonstration that 2 prime O-methylation of the messenger RNA cap distinguishes host from viral RNA, the precedent for the terminal methylation argument.", "Limitations and boundaries": "The authors state plainly that the physiological requirement for VP55 in microRNA degradation cannot be isolated, because VP55 is also required for viral replication and the two activities cannot be separated with the reagents available. The functional test of whether microRNAs restrict the virus therefore relies on reconstituting a pool of four microRNA families rather than on a virus lacking the tailing activity, and it produced an approximately two-fold effect on virus yield that the authors explicitly describe as possibly direct or indirect. All infection work is in cultured cells, and the inference that a fifty percent loss of extracellular enveloped virus would attenuate the virus in an animal is extrapolation from earlier literature rather than a result reported here. The argonaute 2 immunoprecipitation establishes association, not the site or partner of the enzymatic reaction. The protection experiment tests one 2 prime O-methylated guide strand in one sequence context. The evolutionary claims about the origin of 2 prime O-methylation as an antiviral countermeasure are presented in the discussion as speculation supported by comparative genomic arguments from other laboratories, and no evolutionary experiment is performed. Vaccinia virus infection of Drosophila cells is abortive and arrests before intermediate gene expression, which the authors acknowledge and address by adding the natural entomopoxvirus pairing, but the insect data are otherwise from a non-natural host. Finally, the sequencing-based claim of unbiased degradation rests on the microRNA populations of the specific cell types profiled.", "Audience summaries": "### 25 words\n\nPoxviruses carry an enzyme that adds short adenosine tails to host microRNAs, marking them for destruction. Small RNAs with a methylated end escape this fate.\n\n### 75 words\n\nPoxviruses have never been shown to use host microRNAs, and this work suggests why. In insect and mammalian cells, poxvirus infection adds a few nontemplated adenosines to mature microRNAs, after which the cell's own machinery destroys them. The responsible enzyme is VP55, the catalytic subunit of the viral poly(A) polymerase, which is both necessary and sufficient. Small RNAs carrying a methyl group at their 3 prime end are not modified and survive.\n\n### 150 words\n\nPoxviruses replicate in the cytoplasm and transcribe genes with long 3 prime untranslated regions, yet none had been reported to encode microRNAs. Sequencing small RNAs during vaccinia virus infection of Drosophila cells, entomopoxvirus infection of its natural moth host, and vaccinia infection of mammalian cells showed that mature microRNAs acquire short nontemplated adenosine tracts and then disappear, while endogenous small interfering RNAs do not. Chemical inhibitors placed the activity in early infection, and knockdown together with reconstitution identified VP55, the catalytic subunit of the viral poly(A) polymerase, as necessary and sufficient. A synthetic microRNA bearing seven adenosines was destroyed in uninfected cells, showing that the nuclease is cellular. Tailed guides remained argonaute associated, placing the modification after strand selection, and a 2 prime O-methylated guide was fully protected. Restoring the dominant microRNAs during infection halved virus production. The authors propose that terminal methylation evolved partly to shield small RNAs from this class of attack." }, "discoveries": [ "claim-08" ], "relationships": [ { "from": "2012-backes-degradation-of-host-micrornas-by-p", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-backes-degradation-of-host-micrornas-by-p" }, { "from": "2012-backes-degradation-of-host-micrornas-by-p", "to": "2012-perez-a-small-rna-enhancer-of-viral-poly", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2012-backes-degradation-of-host-micrornas-by-p" }, { "from": "2012-backes-degradation-of-host-micrornas-by-p", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2012-backes-degradation-of-host-micrornas-by-p" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2012-backes-degradation-of-host-micrornas-by-p", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2015-aguado-microrna-function-is-limited-to-cy", "to": "2012-backes-degradation-of-host-micrornas-by-p", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-aguado-microrna-function-is-limited-to-cy" } ], "canonical_url": "https://tenoeverlab.us/publications/2012-backes-degradation-of-host-micrornas-by-p/", "controlled_vocabulary": { "pathogens": [ "vsv", "sindbis-virus", "vaccinia-virus", "amsacta-entomopoxvirus" ], "technologies": [ "reverse-genetics", "sirna-knockdown", "small-rna-seq", "small-rna-northern-blot", "silencing-reporter-assay", "argonaute-ip", "synthetic-rna-mimetics" ] } }, { "id": "2012-langlois-hematopoietic-specific-targeting-o", "slug": "2012-langlois-hematopoietic-specific-targeting-o", "url": "/publications/2012-langlois-hematopoietic-specific-targeting-o/", "title": "Hematopoietic-specific targeting of influenza A virus reveals replication requirements for induction of antiviral immune responses", "authors": [ "Ryan A. Langlois", "Andrew Varble", "Mark A. Chua", "Adolfo García-Sastre", "Benjamin R. tenOever" ], "author_count": 5, "first_author": "Ryan A. Langlois", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 5, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2012, "journal": "Proceedings of the National Academy of Sciences", "volume": "109", "issue": "30", "pages": "12117-12122", "doi": "10.1073/pnas.1206039109", "doi_url": "https://doi.org/10.1073/pnas.1206039109", "pmid": "22778433", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/22778433/", "pmcid": "PMC3409765", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409765/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409765/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling", "programmable-virology" ], "themes": [ "calibration-of-interferon-in-vivo", "cell-type-restriction-as-a-tool" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "mouse", "canine", "human" ], "technologies": [ "influenza reverse genetics", "microRNA target site insertion", "small RNA northern blot", "small RNA deep sequencing", "quantitative RT-PCR", "flow cytometry", "MHC class I tetramer staining", "CD8 T cell hybridoma antigen presentation assay", "plaque assay" ], "biological_systems": [ "MDCK cells", "miR-142-expressing MDCK cells", "bone marrow derived macrophages", "primary lung fibroblasts", "mouse embryonic fibroblasts", "JAWS II dendritic cell line", "mouse lung", "lung draining lymph node" ], "key_concepts": [ "cell-type-restricted viral tropism", "miR-142", "antigen presenting cells", "cross-presentation", "RIG-I", "type I interferon induction", "small viral RNA", "influenza nucleoprotein", "CD8 T cell priming", "innate sensing compartment" ], "keywords": [ "influenza A virus", "microRNA targeting", "miR-142", "dendritic cells", "macrophages", "RIG-I", "type I interferon", "cross-presentation", "CD8 T cells", "viral tropism" ], "one_sentence_contribution": "Influenza A virus engineered to be silenced by the hematopoietic microRNA miR-142 shows that replication inside antigen presenting cells is dispensable for CD8 T cell priming and clearance but required for much of the RIG-I-dependent type I interferon response in vivo.", "summary_25": "An influenza virus silenced only inside immune cells still primed normal CD8 T cells and was cleared normally, but induced far less type I interferon.", "summary_75": "Influenza infects airway epithelium and also immune cells, and separating the two contributions has been difficult. By inserting target sites for a blood-lineage microRNA into an essential viral gene, this work built a virus that cannot replicate in immune cells while growing normally in epithelium. Infected mice cleared the virus and made normal antiviral T cells, but produced substantially less type I interferon, implicating infected immune cells as the main source of that alarm signal.", "summary_150": "Whether influenza A virus needs to replicate inside dendritic cells and macrophages, as opposed to simply supplying them with antigen, bears on both how T cells are primed and where interferon comes from. This study engineered a recombinant virus carrying four target sites for miR-142, a microRNA confined to hematopoietic cells, in a duplicated untranslated region of the nucleoprotein segment. The virus grew normally in epithelial cells and fibroblasts and was silenced completely in macrophages, dendritic cells, and the lung draining lymph node, with no escape mutants detected. Mice infected with it showed unchanged weight loss, lung titers, clearance kinetics, and tetramer-positive CD8 T cell responses, despite a measurable loss of direct antigen presentation in culture, which the authors read as sufficiency of cross-presentation. Interferon beta and IRF-7 induction, however, fell in macrophages and in whole lung, and interferon beta induction required RIG-I in the primary cells tested.", "citation": "Langlois RA, Varble A, Chua MA, García-Sastre A, tenOever BR. Hematopoietic-specific targeting of influenza A virus reveals replication requirements for induction of antiviral immune responses. Proceedings of the National Academy of Sciences. 2012. 109(30), 12117-12122. DOI 10.1073/pnas.1206039109. PMID 22778433. PMCID PMC3409765.", "sections": { "Citation": "Langlois RA, Varble A, Chua MA, García-Sastre A, tenOever BR. Hematopoietic-specific targeting of influenza A virus reveals replication requirements for induction of antiviral immune responses. Proceedings of the National Academy of Sciences. 2012. 109(30), 12117-12122.\n\nDOI 10.1073/pnas.1206039109. PMID 22778433. PMCID PMC3409765.", "One-sentence contribution": "Influenza A virus engineered to be silenced by the hematopoietic microRNA miR-142 shows that replication inside antigen presenting cells is dispensable for CD8 T cell priming and clearance but required for much of the RIG-I-dependent type I interferon response in vivo.", "Executive summary": "Influenza A virus replicates chiefly in respiratory epithelium, yet it also enters dendritic cells and macrophages, and the functional consequence of that second compartment has been hard to isolate. Loss-of-function approaches act on the host and therefore remove the cell type along with its other roles. This study instead alters the pathogen. Four perfect target sites for miR-142, a microRNA expressed in cells of hematopoietic origin, were placed into a duplicated packaging region of the influenza nucleoprotein segment, producing a replication-competent virus (NP142t) whose transcription is destroyed specifically in immune cells while remaining intact in epithelial cells and fibroblasts. Silencing was verified at the level of small viral RNA, viral cRNA and mRNA, nucleoprotein protein, multicycle growth, surface hemagglutinin on lung CD45+CD11c+ cells, and infectious virus recovered from the lung draining lymph node. Mice infected with the targeted virus lost weight, cleared virus, and generated nucleoprotein- and polymerase acidic-specific CD8 T cells indistinguishably from mice given the control virus. What changed was innate signaling. Interferon beta and IRF-7 induction fell in cultured macrophages and in whole lung, and interferon beta induction required RIG-I in both macrophages and fibroblasts. The authors read this as evidence that replication intermediates formed in hematopoietic cells are the dominant in vivo trigger of type I interferon, while cross-presentation suffices for cytotoxic T lymphocyte responses.", "Scientific context": "Antigen presenting cells can acquire influenza antigen by direct infection, by phagocytosis of dying infected epithelial cells, or by membrane exchange that transfers peptide-loaded MHC class I. All three routes had been reported to support presentation by CD103+ and CD8α+ dendritic cell subsets, so which route dominates during a natural infection remained contested. In parallel, RIG-I had been established as the sensor driving interferon induction in infected epithelial cells, but whether Toll-like receptors carry the larger share of interferon induction from hematopoietic cells was unresolved. The paper frames both questions as consequences of one limitation, namely that the usual experimental tools delete host genes or host cells and so cannot ask what a single infected compartment contributes while leaving the rest of the animal intact. Prior work cited by the authors had shown that inserting complementary microRNA target sites into a viral genome converts the host microRNA into an effective silencing agent, including miR-93 targeting of influenza nucleoprotein and miR-142 restriction of dengue virus dissemination.", "Central question": "Is influenza A virus replication within cells of hematopoietic origin required for the induction of type I interferon and for the generation of virus-specific CD8 T cell responses in vivo, or can antigen and pathogen-associated molecular patterns supplied by infected epithelial cells substitute for both?", "Experimental strategy": "The design manipulates the pathogen rather than the host so that one variable, viral replication inside immune cells, can be removed without deleting a cell type, a receptor, or a signaling adaptor. Placing miR-142 target sites in the nucleoprotein segment exploits the restriction of miR-142 expression to hematopoietic lineages, and duplicating the segment five packaging sequence creates a genuine 3' untranslated region so that the inserts do not compromise packaging. A length-matched random insert (NPctrl) provides the control virus, which equates for genome length and for any nonspecific effect of the insertion itself. Small RNA deep sequencing of infected human pulmonary epithelial cells first establishes that infection does not itself reshape the host microRNA profile, which is the assumption the whole approach rests on. Silencing is then verified across an engineered cell line expressing exogenous miR-142, primary cells that do or do not express it endogenously, and the intact animal. With the tool validated, pathogenesis, adaptive immunity, and innate signaling are read out in parallel in the same infection model, and the sensor requirement is tested by repeating the interferon readout in RIG-I deficient and littermate control primary cells.", "Key findings": "1. Deep sequencing of infected human pulmonary epithelial cells showed no significant change in host microRNA levels during infection (Fig. S1). The authors take this as validation that microRNA-based targeting can be used as a tool without the infection itself moving the target microRNA.\n2. In MDCK cells engineered to express miR-142, the NP142t virus lost small viral RNA accumulation (Fig. 1A), viral mRNA and cRNA (Fig. S2C), and detectable nucleoprotein (Fig. 1B), while parental MDCK cells supported both viruses. The loss of both mRNA and cRNA is interpreted as rapid targeting of the incoming genome.\n3. Multicycle growth curves showed no difference between NP142t and NPctrl in the absence of miR-142 and more than three logs of attenuation in miR-142-expressing cells (Fig. 1C), indicating the insert imposes no intrinsic fitness cost.\n4. Replication of NP142t was abolished in murine bone marrow derived macrophages, which express endogenous miR-142 (Fig. 1D), and was unchanged in primary lung fibroblasts, which do not (Fig. 1E).\n5. In infected mice, surface hemagglutinin was lost on lung CD45+CD11c+ dendritic cells during NP142t infection and was unchanged on CD45- cells (Fig. 2A), and infectious virus was reduced in the lung draining lymph node while lung titers were unchanged (Fig. 2B). Because only hematopoietic cells traffic to the node during infection, the authors use the node as an independent in vivo measure of silencing.\n6. Weight loss, recovery, and the kinetics of lung viral titer and clearance were indistinguishable between the two viruses (Fig. 2C and 2D), and sequencing confirmed that the absence of a phenotype was not caused by escape mutants (Fig. S3).\n7. Direct antigen presentation was lost. JAWS II antigen presenting cells, which express miR-142 (Fig. 3A), activated nucleoprotein-specific CD8 T cell hybridomas after NPctrl infection but only to the level of naive or ultraviolet-inactivated virus controls after NP142t infection (Fig. 3B).\n8. Despite that loss, the frequency and number of nucleoprotein- and polymerase acidic-specific tetramer positive CD8 T cells were not significantly different between viruses in C57BL/6 mice at 9 days (Fig. 3C and 3D) or in BALB/c mice at 10 days (Fig. S4), and interferon gamma production after peptide restimulation showed a downward trend that did not reach significance (Fig. 3E and 3F). The authors interpret the combination as evidence that cross-presentation or cross-dressing is sufficient for protective cytotoxic T lymphocyte responses.\n9. Interferon beta and IRF-7 induction were reduced in bone marrow derived macrophages infected with NP142t and were unaffected in primary lung fibroblasts (Fig. 4A and 4B), that is, the innate defect tracked with miR-142 expression rather than with the virus itself.\n10. Whole lung interferon beta and IRF-7 messenger RNA were significantly reduced during NP142t infection (Fig. 4C and 4D) even though lung tissue is predominantly nonhematopoietic. This is the observation the authors treat as the central result, and they read it as evidence that the hematopoietic compartment contributes disproportionately to the in vivo interferon response.\n11. Interferon beta induction was abolished in RIG-I deficient macrophages and fibroblasts for both viruses (Fig. 4E and 4F), while transfected double-stranded RNA still induced interferon beta in those cells (Fig. S5), showing the cells retain the capacity to produce interferon.", "Mechanistic model": "The data support a model in which the accumulation of influenza replication intermediates inside hematopoietic cells, rather than uptake of material from infected epithelium, is the dominant in vivo trigger of RIG-I-dependent type I interferon, while antigen for CD8 T cell priming reaches the presenting cell through exogenous routes that do not require the presenting cell to be productively infected. Two of the links in this model are shown directly. Silencing removes replication in hematopoietic cells, and whole lung interferon falls. Interferon beta induction by either virus requires RIG-I in the cells tested. The connection between those two observations is inferred rather than demonstrated, because the RIG-I requirement was established in cultured primary cells and not in a hematopoietic-restricted knockout animal, so the study does not establish that the interferon lost in the lung is produced by hematopoietic cells through RIG-I. The specific replication intermediate that RIG-I engages is not identified here. The authors also note that they cannot explain, from these data, why blocking replication in a numerically minor compartment produces so large a deficit in total interferon, and they raise the possibility that NS1-mediated suppression of sensing in epithelial cells biases the response toward the immune compartment. That possibility is offered as a proposal and is not tested.", "Conceptual or technical advance": "The work makes viral tropism an experimental variable that can be set independently of host genotype. Because a tissue-restricted microRNA is used to silence an essential viral gene, the same animal carries both a permissive and a nonpermissive compartment, and the contribution of one compartment can be read out against an otherwise identical infection. That turns questions previously approachable only by gene deletion or cell depletion, both of which remove functions beyond infection, into gain-of-specificity questions. The validation chain also matters practically, since silencing is verified at the level of small viral RNA, mRNA, cRNA, protein, growth, surface antigen, and organ titer, and escape mutants are excluded by sequencing, which sets the standard for what a microRNA-restricted virus has to demonstrate before its phenotypes can be interpreted.", "Relationship to the broader research program": "Several threads that recur across this corpus intersect here. The use of small RNA biology as an engineering tool rather than only as an object of study appears in the microRNA targeting strategy, and the reference list points to earlier laboratory work using miR-93 to attenuate influenza in a species-specific manner and miR-142 to restrict dengue virus dissemination. Small viral RNA, a product characterized in earlier laboratory work on the influenza transcription to replication switch, is repurposed here as a sensitive readout of ongoing polymerase activity. The question of which cells produce interferon during infection, and which sensor drives it, is continuous with laboratory work on the interferon enhanceosome and on transcription factor redundancy in establishing the antiviral state, both of which are cited. Placing this paper beside later corpus entries that engineer recombinant viruses to deliver or restrict RNA cargo would be category 3 synthesis, and it should be assembled only when those records exist, since it cannot be read off this paper alone.", "Related publications": "- Perez et al. 2009, microRNA-mediated species-specific attenuation of influenza A virus. Methodological foundation, cited as the prior demonstration that microRNA target sites in nucleoprotein render influenza replication inversely proportional to the cognate microRNA.\n- Perez et al. 2010, influenza A virus-generated small RNAs regulate the switch from transcription to replication. Methodological foundation, the source of the small viral RNA readout and of the northern blot protocol used here.\n- Pham, Langlois and tenOever 2012, replication in cells of hematopoietic origin is necessary for dengue virus dissemination. Companion, the same miR-142 restriction strategy applied by the same group to a different virus and a different question.\n- Schmid et al. 2010, transcription factor redundancy ensures induction of the antiviral state. Predecessor, laboratory work on interferon induction cited in the discussion of redundant sensing.\n- tenOever et al. 2007, multiple functions of the IKK-related kinase IKKepsilon in interferon-mediated antiviral immunity. Predecessor, laboratory work on the interferon induction pathway cited for the enhanceosome step.", "Limitations and boundaries": "The findings are established in mice infected intranasally with a single laboratory influenza A strain, and the adaptive immune readouts cover two mouse backgrounds at single late time points rather than a full kinetic series. Silencing removes productive replication in hematopoietic cells, so the model cannot distinguish a requirement for viral replication from a requirement for the downstream products of replication, and it cannot address any contribution of abortive infection that still generates sensed RNA below the detection thresholds used. The RIG-I requirement was tested in cultured primary macrophages and fibroblasts, not in animals with a hematopoietic-restricted deletion, so the in vivo sensor attribution is inferred. Because miR-142 is expressed across hematopoietic lineages, the approach silences replication in dendritic cells, macrophages, and other immune cells together and cannot assign the interferon deficit to a single subset. Interferon was measured as messenger RNA for interferon beta and IRF-7 rather than as secreted protein, and the interferon gamma restimulation difference was a nonsignificant trend that the authors explicitly decline to interpret as a functional defect. Finally, the authors note that the magnitude of the interferon deficit relative to the small size of the infected hematopoietic compartment is not explained by the data presented.", "Audience summaries": "### 25 words\n\nAn influenza virus silenced only inside immune cells still primed normal CD8 T cells and was cleared normally, but induced far less type I interferon.\n\n### 75 words\n\nInfluenza infects airway epithelium and also immune cells, and separating the two contributions has been difficult. By inserting target sites for a blood-lineage microRNA into an essential viral gene, this work built a virus that cannot replicate in immune cells while growing normally in epithelium. Infected mice cleared the virus and made normal antiviral T cells, but produced substantially less type I interferon, implicating infected immune cells as the main source of that alarm signal.\n\n### 150 words\n\nWhether influenza A virus needs to replicate inside dendritic cells and macrophages, as opposed to simply supplying them with antigen, bears on both how T cells are primed and where interferon comes from. This study engineered a recombinant virus carrying four target sites for miR-142, a microRNA confined to hematopoietic cells, in a duplicated untranslated region of the nucleoprotein segment. The virus grew normally in epithelial cells and fibroblasts and was silenced completely in macrophages, dendritic cells, and the lung draining lymph node, with no escape mutants detected. Mice infected with it showed unchanged weight loss, lung titers, clearance kinetics, and tetramer-positive CD8 T cell responses, despite a measurable loss of direct antigen presentation in culture, which the authors read as sufficiency of cross-presentation. Interferon beta and IRF-7 induction, however, fell in macrophages and in whole lung, and interferon beta induction required RIG-I in the primary cells tested." }, "discoveries": [ "claim-10" ], "relationships": [ { "from": "2012-langlois-hematopoietic-specific-targeting-o", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "companion", "evidence": "stated in the Related publications section of 2012-langlois-hematopoietic-specific-targeting-o" }, { "from": "2012-langlois-hematopoietic-specific-targeting-o", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2012-langlois-hematopoietic-specific-targeting-o" } ], "canonical_url": "https://tenoeverlab.us/publications/2012-langlois-hematopoietic-specific-targeting-o/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "rt-qpcr", "plaque-assay", "reverse-genetics", "small-rna-seq", "flow-cytometry", "small-rna-northern-blot", "mirna-target-site-insertion", "tetramer-staining", "antigen-presentation-assay" ] } }, { "id": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "slug": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "url": "/publications/2012-langlois-in-vivo-delivery-of-cytoplasmic-rn/", "title": "In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs", "authors": [ "Ryan A Langlois", "Jillian S Shapiro", "Alissa M Pham", "Benjamin R tenOever" ], "author_count": 4, "first_author": "Ryan A Langlois", "senior_authors": [ "Benjamin R tenOever" ], "corresponding_authors": [ "Benjamin R tenOever" ], "tenoever_position": 4, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2012, "journal": "Molecular Therapy", "volume": "20", "issue": "2", "pages": "367-375", "doi": "10.1038/mt.2011.244", "doi_url": "https://doi.org/10.1038/mt.2011.244", "pmid": "22086233", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/22086233/", "pmcid": "PMC3277236", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277236/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277236/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "programmable-virology", "small-rna-antiviral-defense" ], "themes": [ "noncanonical-microrna-biogenesis", "rna-vectors-for-delivery" ], "pathogens": [ "vesicular stomatitis virus", "Sindbis virus", "influenza A virus" ], "viral_families": [ "Rhabdoviridae", "Togaviridae", "Orthomyxoviridae" ], "host_species": [ "mouse", "human", "hamster" ], "technologies": [ "reverse genetics", "small RNA Northern blotting", "small RNA deep sequencing", "Argonaute 2 immunoprecipitation", "luciferase reporter assays", "flow cytometry", "quantitative RT-PCR", "intranasal and intravenous infection of mice" ], "biological_systems": [ "murine embryonic fibroblasts", "Dicer-deficient fibroblasts", "baby hamster kidney cells", "293T cells", "mouse lung", "mouse spleen", "mouse liver", "mouse kidney", "mouse heart" ], "key_concepts": [ "virtrons", "cytoplasmic microRNA biogenesis", "noncanonical small RNA processing", "Dicer dependence", "star strand accumulation", "RISC loading", "post-transcriptional silencing", "in vivo small RNA delivery", "vector tropism" ], "keywords": [ "vesicular stomatitis virus", "Sindbis virus", "miR-124", "microRNA delivery", "RNA interference", "Ptbp1", "Ifnar1", "Argonaute 2" ], "one_sentence_contribution": "A negative-sense cytoplasmic RNA virus, vesicular stomatitis virus, can be engineered to produce mature Dicer-dependent miR-124 that loads into Argonaute 2, silences targets, reaches many tissues in mice, and persists after the vector itself is cleared.", "summary_25": "A virus that never enters the nucleus was engineered to make a working microRNA, delivering it to many mouse tissues and leaving it behind after clearance.", "summary_75": "MicroRNA production was thought to begin in the nucleus. Vesicular stomatitis virus, which replicates only in the cytoplasm, was engineered to carry a microRNA precursor. It produced abundant mature miR-124 that required Dicer, loaded into Argonaute 2, and silenced targets. In mice the vector delivered the microRNA to lung, spleen, liver, kidney and heart, and the microRNA remained after the virus was cleared, reducing induction of a known target gene.", "summary_150": "Whether microRNAs can be generated outside the nucleus was contested, since the two reported examples came from positive-sense cytoplasmic viruses in transformed cells. Vesicular stomatitis virus, a cytoplasmic virus of negative polarity, was engineered to express the murine pri-miR-124 locus as an independent transcript and compared with Sindbis and influenza vectors carrying the same locus. Mature miR-124 was produced by all three, was lost in Dicer-deficient cells without a change in viral transcription, reached an estimated 25,000 to 35,000 copies per cell, associated with Argonaute 2, and repressed target reporters by roughly 60 to 90 percent without impairing an unrelated microRNA. The cytoplasmic vectors also produced substantial star strand, which repressed its own reporter and represents a genuine off-target liability. In mice the vector reached several organs and the microRNA outlasted the infection, reducing induction of Ptbp1. The processing enzyme responsible for the cytoplasmic step is not identified.", "citation": "Langlois RA, Shapiro JS, Pham AM, tenOever BR. In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs. Molecular Therapy. 2012. Volume 20, issue 2, pages 367-375. DOI 10.1038/mt.2011.244. PMID 22086233. PMCID PMC3277236.", "sections": { "Citation": "Langlois RA, Shapiro JS, Pham AM, tenOever BR. In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs. Molecular Therapy. 2012. Volume 20, issue 2, pages 367-375.\n\nDOI 10.1038/mt.2011.244. PMID 22086233. PMCID PMC3277236.", "One-sentence contribution": "A negative-sense cytoplasmic RNA virus, vesicular stomatitis virus, can be engineered to produce mature Dicer-dependent miR-124 that loads into Argonaute 2, silences targets, reaches many tissues in mice, and persists after the vector itself is cleared.", "Executive summary": "MicroRNA biogenesis is understood to begin in the nucleus, where Drosha and DGCR8 cleave the primary transcript. Reports that positive-sense cytoplasmic RNA viruses could yield mature microRNAs, termed virtrons, raised the possibility of a nuclear-independent route, but that claim had been challenged as an artifact of rapidly dividing transformed cells whose transient nuclear envelope breakdown might give viral transcripts access to the microprocessor. This study addresses both a biological question and a delivery problem. Vesicular stomatitis virus, a cytoplasmic virus of negative polarity, was engineered to express the murine pri-miR-124 locus as an independent transcript inserted between the glycoprotein and polymerase genes, and was compared directly with Sindbis virus and influenza A virus carrying the same locus. The recombinant vesicular stomatitis virus produced mature miR-124 that was abolished in Dicer-deficient fibroblasts, accumulated to an estimated 25,000 to 35,000 copies per cell, associated with Argonaute 2, and repressed reporters carrying miR-124 target sites by roughly 60 to 90 percent without impairing an unrelated microRNA. In mice, the vector delivered miR-124 to lung, spleen, liver, kidney and heart, and in immunocompetent animals the microRNA persisted after viral leader RNA had disappeared, with a measurable reduction in induction of the miR-124 target Ptbp1. Cytoplasmic microRNA production therefore occurs in animals and supplies a transient delivery platform.", "Scientific context": "Canonical microRNA biogenesis requires nuclear cleavage of the primary transcript by Drosha and DGCR8, export by Exportin 5, and cytoplasmic cleavage by Dicer with TRBP, after which the duplex is loaded into an Argonaute protein and one strand is selected as the guide while the other, the star strand, is normally degraded. Bona fide virus-encoded microRNAs had been described only in large DNA viruses. Two reports, one from another group working with a flavivirus replicon and one from this laboratory working with Sindbis virus, had recovered mature microRNAs from positive-sense cytoplasmic RNA viruses, but the mechanism was undefined and an alternative explanation had been raised in the literature, that transformed cells dividing rapidly allow viral transcripts to reach the nuclear microprocessor. Separately, delivery remains the principal obstacle to therapeutic RNA interference. DNA virus vectors had been reported to cause toxicity through saturation of nuclear export and of Argonaute, and lentiviral delivery integrates. The paper therefore sets out to test whether cytoplasmic microRNA production is a genuine process by demonstrating it in animals and with a virus of the opposite polarity.", "Central question": "Can a cytoplasmic RNA virus of negative polarity be engineered to generate a functional microRNA, and does cytoplasmic microRNA production occur in animal tissue rather than only in transformed cells in culture.", "Experimental strategy": "The design turns on polarity, replication compartment and organism. Vesicular stomatitis virus replicates exclusively in the cytoplasm and is of negative polarity, so a microRNA recovered from it cannot be attributed to the positive-strand replication strategy of Sindbis virus, and its transcripts carry a 5' cap and a poly(A) tail like cellular messenger RNA, which makes nuclear import improbable. The murine pri-miR-124 locus was inserted as an independent transcription unit between the glycoprotein and polymerase genes with flanking polymerase recognition signals. A scrambled noncoding insert provided the matched control. Sindbis virus and influenza A virus carrying the same locus, both previously generated in the laboratory, allow the three replication strategies to be compared side by side in a single experiment, with influenza serving as the nuclear reference case. Dicer-deficient fibroblasts test the cytoplasmic half of the pathway. Small RNA deep sequencing quantifies abundance, strand composition and the frequency of the product relative to the whole microRNA pool. Argonaute 2 immunoprecipitation tests RISC loading, and reporters bearing perfect target sites for either the guide or the star strand separate intended from unintended silencing. Interferon alpha receptor knockout mice remove the antiviral response so that different vectors can be compared at matched replication levels and so that systemic spread reveals the accessible tissue range, while wild-type mice test whether the approach works in an immunocompetent host and whether the payload outlives the vector.", "Key findings": "1. Insertion of the pri-miR-124 hairpin did not prevent rescue of recombinant vesicular stomatitis virus, and infection produced a small RNA matching plasmid-derived miR-124 in size, with no change to endogenous miR-93 and no induction of endogenous miR-124 by the scrambled control virus at comparable replication levels (Figures 1b and 1c).\n2. All three engineered viruses produced miR-124 in wild-type fibroblasts, and production of both viral miR-124 and endogenous miR-93 was abolished in Dicer-deficient fibroblasts while viral transcript levels were unchanged, establishing that biogenesis of the viral product requires Dicer and that its loss is not secondary to reduced replication (Figures 2a and 2b).\n3. Deep sequencing recovered approximately 80,000 to 400,000 reads mapping to the 583 nucleotide viral pri-miR-124, with mature miR-124 reaching as much as 2 percent of all profiled microRNAs and an estimated 25,000 to 35,000 copies per cell, against fewer than 15 copies per cell in mock-infected fibroblasts (Figure 3a).\n4. Star strand accumulation was pronounced and restricted to the cytoplasmic vectors, reaching as much as 40 percent of the reads mapping to pri-miR-124, and was not seen for endogenous miR-93 or miR-20a (Figures 3a and 3b). Plasmid-driven miR-124 also produced detectable star strand but at significantly lower levels than the cytoplasmic viruses, which the authors interpret as evidence that overexpression alone does not account for the effect and that star strand accumulation may be a characteristic of cytoplasmic processing.\n5. Argonaute 2 immunoprecipitation recovered virus-derived miR-124 regardless of which virus produced it, with no reduction in Argonaute-associated miR-93, and overexpression of miR-124 from any of the three vectors did not impair repression of a miR-142 reporter by cotransfected miR-142 (Figures 4a and 4b). The authors read this as indicating that the vectors are unlikely to add toxicity through competition for the silencing machinery.\n6. All three viruses repressed a luciferase reporter bearing perfect miR-124 target sites by roughly 60 to 90 percent, and also repressed a reporter carrying the Scp1 3' untranslated region with its endogenous response elements. The star strand reporter was also significantly repressed by the cytoplasmic vectors, though at substantially lower levels, which the authors present as a real off-target risk that could be reduced by altering duplex thermodynamics or the star strand sequence (Figure 4c and Supplementary Figure S3).\n7. In interferon alpha receptor knockout mice, intranasal infection with each miR-124 virus gave high lung levels of the microRNA within one to two days, and intravenous delivery of the vesicular stomatitis vector produced detectable miR-124 in lung, spleen, liver, kidney and heart by two days, tracking viral transcript levels (Figures 5a and 5b).\n8. In wild-type mice, viral leader RNA appeared by one day and fell sharply after four days, while miR-124 remained at high relative levels at five days, which the authors take as evidence that the delivered small RNA persists through clearance of the vector (Figure 6a).\n9. Infection with the control virus raised the miR-124 target transcript Ptbp1 roughly 30-fold over naive lung, whereas the miR-124 virus gave less than a fivefold increase, while induction of the unrelated control transcript Tnfa did not differ significantly between the two viruses (Figure 6b). Because whole lung includes uninfected cells and microRNA repression is modest, the authors interpret the reduced induction as strong evidence of functional silencing in vivo rather than as a measurement of knockdown magnitude in infected cells.", "Mechanistic model": "The study does not establish the mechanism of cytoplasmic microRNA biogenesis, and the discussion opens by saying that it remains unclear. What the data constrain is the following. The cytoplasmic step requires Dicer, since the product is lost in Dicer-deficient cells while viral transcription is unaffected. The product enters Argonaute 2 and silences targets, so it completes the functional pathway. Because the vesicular stomatitis virus pri-miR-124 transcript carries a cap and a poly(A) tail and so resembles a cellular messenger RNA, the authors argue that return of that transcript to the nucleus of primary cells is highly improbable, and they infer from this that either the cytoplasm contains components capable of the first processing step or hairpin formation recruits canonical machinery out of the nucleus. Neither alternative is tested here, and the enzyme performing the pri to pre conversion is not identified.\n\nTwo features are advanced as interpretive links rather than demonstrated mechanism. First, the shared properties of virtrons and mirtrons, namely star strand accumulation and independence from DGCR8 reported in the laboratory's earlier work, are read as suggestive of noncanonical biogenesis, with the alternative explanation of RISC saturation from overexpression argued against by the lower star strand levels seen with plasmid expression. Second, the persistence of miR-124 in lung after loss of viral leader RNA is attributed to the long half-life of microRNAs, which is cited rather than measured here.", "Conceptual or technical advance": "Two things become testable. The demonstration in animals, and in a negative-sense cytoplasmic virus, argues against the proposal that cytoplasmic microRNA production is an artifact of rapidly dividing transformed cells, and so makes the search for a nuclear-independent processing activity, and for endogenous small RNAs made by that route, a defined question rather than a disputed observation.\n\nPractically, the work delivers a vector class with a distinct profile. A cytoplasmic RNA virus does not integrate, does not depend on nuclear export for the primary processing step, produces a high per cell copy number, distributes to many tissues from an intravenous route, and leaves a payload that outlasts the infection. The discussion sets out the resulting design space, including replacement of the natural hairpin with artificial microRNAs engineered to release perfectly complementary small interfering RNAs, combination with microRNA target site technology to restrict tropism, use in oncolytic settings, and, notably, the proposal that a virus could deliver a library of artificial microRNAs only to infected cells so that selection identifies host restriction factors. All of these are proposed applications, not results of this paper. The vectors are framed as suited to conditions with acute phenotypes lasting less than about a week.", "Relationship to the broader research program": "This paper sits between the demonstration that a nuclear RNA virus can be made to produce a microRNA and the later use of engineered viruses as screening instruments. It extends the platform from influenza to two cytoplasmic viruses, moves it from cell culture into mice, and adds quantification by deep sequencing, Argonaute association and in vivo target measurement to the characterization toolkit.\n\nCategory 3 synthesis. Read with Varble and colleagues in 2010, which established the influenza vector, and Varble and colleagues in 2013, which delivers artificial small RNA libraries from a virus and selects on them in animals, the discussion here contains the explicit statement of the screening idea that the 2013 paper implements. That trajectory across three papers is visible when they are placed side by side and is not a claim made by any one of them. The interest in whether RNA viruses interact productively with the host small RNA machinery, and in what that implies about antiviral RNA interference in mammals, recurs across the corpus and is treated conceptually in the 2016 perspective.", "Related publications": "- Varble and colleagues, 2010, Engineered RNA viral synthesis of microRNAs, cited as reference 14 and supplying the influenza vector used here. Predecessor and methodological foundation.\n- Shapiro and colleagues, 2010, Noncanonical cytoplasmic processing of viral microRNAs, cited as reference 10, from the same laboratory and sharing an author, supplying the Sindbis vector and the prior evidence of DGCR8 independence. Predecessor.\n- Perez and colleagues, 2009, MicroRNA-mediated species-specific attenuation of influenza A virus, cited as reference 23. Methodological foundation for microRNA target site engineering.\n- Varble and tenOever, 2011, Implications of RNA virus-produced miRNAs, cited as reference 43. Review or synthesis from the same laboratory.\n- Stojdl and colleagues, 2003, on vesicular stomatitis virus strains defective in innate immune shutdown, cited as reference 49 and the source of the rescue procedure, with tenOever as an author. Methodological foundation.\n- Varble and colleagues, 2013, An In Vivo RNAi Screening Approach to Identify Host Determinants of Virus Replication. Conceptual extension, implementing the library screening idea raised in this discussion.\n- tenOever, 2016, The Evolution of Antiviral Defense Systems. Review or synthesis addressing the relationship between RNA viruses and small RNA pathways.", "Limitations and boundaries": "The mechanism of cytoplasmic pri-microRNA processing is explicitly unresolved, and the argument that the transcript does not reach the nucleus rests on its resemblance to a cellular messenger RNA rather than on a localization experiment. Only one microRNA locus, murine miR-124-2, was tested, in one insertion position per vector, so generality across hairpins and insertion sites is untested, as is the behavior of the artificial microRNAs the discussion proposes for therapeutic use.\n\nMuch of the in vivo work uses interferon alpha receptor knockout mice, chosen deliberately so that vectors could be compared without the confound of differential replication, which means that the tissue distribution seen after intravenous delivery is obtained in the absence of type I interferon signaling and does not establish the range accessible in an immunocompetent animal. In wild-type mice only vesicular stomatitis virus and only the lung were examined for function. The functional in vivo readout is a reduction in virus-induced Ptbp1 induction measured in whole lung, which contains uninfected cells, so it demonstrates activity without quantifying silencing in infected cells, and it is a transcript measurement rather than a protein measurement. Persistence of miR-124 beyond clearance is inferred from loss of viral leader RNA rather than from a direct measure of infectious virus. The star strand is loaded and does repress its target, so off-target silencing is a demonstrated property of these vectors rather than a hypothetical one, and the proposed fixes are untested here. No toxicity, pathogenesis or dose ranging study is reported, and the therapeutic framing throughout the discussion is extrapolation.", "Audience summaries": "### 25 words\n\nA virus that never enters the nucleus was engineered to make a working microRNA, delivering it to many mouse tissues and leaving it behind after clearance.\n\n### 75 words\n\nMicroRNA production was thought to begin in the nucleus. Vesicular stomatitis virus, which replicates only in the cytoplasm, was engineered to carry a microRNA precursor. It produced abundant mature miR-124 that required Dicer, loaded into Argonaute 2, and silenced targets. In mice the vector delivered the microRNA to lung, spleen, liver, kidney and heart, and the microRNA remained after the virus was cleared, reducing induction of a known target gene.\n\n### 150 words\n\nWhether microRNAs can be generated outside the nucleus was contested, since the two reported examples came from positive-sense cytoplasmic viruses in transformed cells. Vesicular stomatitis virus, a cytoplasmic virus of negative polarity, was engineered to express the murine pri-miR-124 locus as an independent transcript and compared with Sindbis and influenza vectors carrying the same locus. Mature miR-124 was produced by all three, was lost in Dicer-deficient cells without a change in viral transcription, reached an estimated 25,000 to 35,000 copies per cell, associated with Argonaute 2, and repressed target reporters by roughly 60 to 90 percent without impairing an unrelated microRNA. The cytoplasmic vectors also produced substantial star strand, which repressed its own reporter and represents a genuine off-target liability. In mice the vector reached several organs and the microRNA outlasted the infection, reducing induction of Ptbp1. The processing enzyme responsible for the cytoplasmic step is not identified." }, "discoveries": [ "claim-10" ], "relationships": [ { "from": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-langlois-in-vivo-delivery-of-cytoplasmic-rn" }, { "from": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2012-langlois-in-vivo-delivery-of-cytoplasmic-rn" }, { "from": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "review or synthesis", "evidence": "stated in the Related publications section of 2012-langlois-in-vivo-delivery-of-cytoplasmic-rn" }, { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" } ], "canonical_url": "https://tenoeverlab.us/publications/2012-langlois-in-vivo-delivery-of-cytoplasmic-rn/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "vsv", "sindbis-virus" ], "technologies": [ "rt-qpcr", "reverse-genetics", "small-rna-seq", "flow-cytometry", "small-rna-northern-blot", "luciferase-promoter-reporter", "in-vivo-infection-route", "argonaute-ip" ] } }, { "id": "2012-perez-a-small-rna-enhancer-of-viral-poly", "slug": "2012-perez-a-small-rna-enhancer-of-viral-poly", "url": "/publications/2012-perez-a-small-rna-enhancer-of-viral-poly/", "title": "A Small-RNA Enhancer of Viral Polymerase Activity", "authors": [ "Jasmine T. Perez", "Ivan Zlatev", "Shilpa Aggarwal", "Sailakshmi Subramanian", "Ravi Sachidanandam", "Baek Kim", "Muthiah Manoharan", "Benjamin R. tenOever" ], "author_count": 8, "first_author": "Jasmine T. Perez", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 8, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2012, "journal": "Journal of Virology", "volume": "86", "issue": "24", "pages": "13475-13485", "doi": "10.1128/jvi.02295-12", "doi_url": "https://doi.org/10.1128/jvi.02295-12", "pmid": "23035211", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/23035211/", "pmcid": "PMC3503082", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503082/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503082/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "influenza-genome-regulation" ], "themes": [ "small-viral-rnas" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "human" ], "technologies": [ "Northern blotting", "primer extension", "small RNA deep sequencing", "RNA immunoprecipitation", "influenza reverse genetics", "polymerase reconstitution assay", "in vitro RNA polymerase assay", "site-directed mutagenesis", "subcellular fractionation", "synthetic 5-prime triphosphate RNA chemistry" ], "biological_systems": [ "A549 cells", "HEK293 cells", "purified recombinant influenza A virus polymerase", "cell-free in vitro polymerase reactions" ], "key_concepts": [ "small viral RNA", "svRNA", "RNA-dependent RNA polymerase", "transcription to replication switch", "allosteric enhancer RNA", "PA RNA binding cleft", "cRNA intermediate", "nuclear export protein NEP", "segment-specific regulation", "genome segment stoichiometry" ], "keywords": [ "influenza A virus", "svRNA", "viral polymerase", "PA subunit", "NEP", "cRNA", "vRNA synthesis", "genome replication", "segment balance", "regulatory RNA" ], "one_sentence_contribution": "Influenza A virus small viral RNAs are shown to be synthesized from the complementary RNA intermediate, to load into the RNA binding cleft of the polymerase PA subunit, and to act there as segment-specific allosteric enhancers of full-length genome synthesis.", "summary_25": "Influenza makes short RNAs from its own genome copies. These dock in a pocket of the viral polymerase and switch it toward copying that specific segment.", "summary_75": "Influenza A virus must switch its polymerase from making messenger RNA to copying its eight genome segments, and it must copy them in balanced amounts. This study shows that short viral RNAs, made from the positive-sense copy of the genome, bind a basic cleft in the polymerase PA subunit and boost full-length genome synthesis without acting as primers. Removing this small RNA from one segment blocks genome synthesis for that segment alone.", "summary_150": "The influenza A virus polymerase transcribes and replicates the same eight templates, and the control of that switch has been attributed variously to new polymerase acting in trans, to nucleoprotein or nucleotide concentrations, and to stabilization of the complementary RNA intermediate. Working in human cell lines, in reconstituted polymerase assays and with purified enzyme, this study shows that the small viral RNAs previously found at the 5-prime ends of the segments are templated from the complementary RNA intermediate, remain nuclear and are largely excluded from virions, and bind the polymerase through the PB1 and PA heterodimer, with the PA basic residue R566 most important. Synthetic small viral RNA promotes full-length genome synthesis from a complementary RNA template even when its 3-prime hydroxyl is blocked, which excludes priming and is read as allosteric enhancement. A recombinant virus lacking small viral RNA from the neuraminidase segment loses genome synthesis for that segment alone.", "citation": "Perez JT, Zlatev I, Aggarwal S, Subramanian S, Sachidanandam R, Kim B, Manoharan M, tenOever BR. A Small-RNA Enhancer of Viral Polymerase Activity. Journal of Virology. 2012. Volume 86, Issue 24, pages 13475-13485. DOI 10.1128/jvi.02295-12. PMID 23035211. PMCID PMC3503082.", "sections": { "Citation": "Perez JT, Zlatev I, Aggarwal S, Subramanian S, Sachidanandam R, Kim B, Manoharan M, tenOever BR. A Small-RNA Enhancer of Viral Polymerase Activity. Journal of Virology. 2012. Volume 86, Issue 24, pages 13475-13485.\n\nDOI 10.1128/jvi.02295-12. PMID 23035211. PMCID PMC3503082.", "One-sentence contribution": "Influenza A virus small viral RNAs are shown to be synthesized from the complementary RNA intermediate, to load into the RNA binding cleft of the polymerase PA subunit, and to act there as segment-specific allosteric enhancers of full-length genome synthesis.", "Executive summary": "Influenza A virus carries a heterotrimeric RNA-dependent RNA polymerase that must perform two different jobs on the same eight templates, first transcribing capped and polyadenylated messenger RNA and later replicating full-length genome segments. How the enzyme switches between these modes, and how it keeps the eight segments in balance so that complete genomes can be packaged, had remained unsettled. Earlier work from this laboratory had identified small viral RNAs of roughly 22 nucleotides derived from the 5-prime ends of the genomic segments and had associated them with the transcription-to-replication switch, but their origin, their location, their binding site on the polymerase and their mode of action were unknown.\n\nThe study combines a plasmid-based polymerase reconstitution system, subcellular fractionation of infected human alveolar epithelial cells, deep sequencing of purified virions, immunoprecipitation of tagged polymerase subunits with synthetic 5-prime triphosphate small viral RNAs, structure-guided mutagenesis of the PA subunit, cell-free polymerase assays with purified recombinant enzyme, and a recombinant virus engineered to lose small viral RNA from one segment.\n\nThe small RNAs are shown to be templated from the complementary RNA intermediate, to remain nuclear rather than being packaged, to require the PB1 and PA heterodimer and in particular the basic residue R566 of PA for binding, and to enhance full-length genome synthesis in vitro without needing a free 3-prime hydroxyl. A virus unable to make small viral RNA from one segment loses genome synthesis for that segment alone and retains normal messenger RNA production.", "Scientific context": "The influenza A virus polymerase enters the nucleus tethered to the partially complementary 3-prime and 5-prime ends of each negative-sense segment, which fold into the panhandle or corkscrew promoter. From that template it first synthesizes capped viral messenger RNA using host-derived cap primers and stuttering at a poly-uridine track to add a poly-adenosine tail, and it later shifts to synthesizing full-length complementary RNA and progeny genomic RNA. The paper states that models for this switch have generally invoked external factors such as newly made polymerase acting in trans, changing concentrations of nucleoprotein, nucleotides or substrates, or simple stabilization of the complementary RNA intermediate, and that the precise contribution of each remains unknown. It further states that little was understood about how a replicase-competent polymerase maintains stoichiometric balance across the eight segments.\n\nPrior work from the same laboratory reported influenza-derived small viral RNAs mapping to the 5-prime ends of each genomic segment and implicated them in the transcription-to-replication switch, and independent work reported that the relative concentrations of template sources bias the polymerase toward replicase mode. What those small RNAs bound, where they acted, and by what mechanism they acted were open questions entering this study.", "Central question": "By what molecular mechanism do influenza A virus small viral RNAs modulate the activity of the viral polymerase, and does that mechanism account both for the transition to genome replication and for the maintenance of balanced amounts of the eight genomic segments?", "Experimental strategy": "The design separates the question into origin, location, binding site and function, and answers each with a system in which the relevant variables can be set independently.\n\nOrigin and cofactor requirements are approached with a bidirectional eight-plasmid expression system in which individual segments can be omitted, and then with a reduced polymerase reconstitution assay containing a single template of defined polarity. Supplying a genomic-sense template or a complementary-sense template directly is what allows the nuclear export protein requirement to be placed upstream or downstream of complementary RNA synthesis. Truncations of the nuclear export protein separate its nuclear export function from its effect on RNA synthesis.\n\nLocation is approached by nuclear and cytoplasmic fractionation of infected A549 cells across a time course, and independently by deep sequencing small RNAs from sucrose-purified virions against small RNAs from infected cells, which tests packaging rather than steady-state distribution.\n\nThe binding site is approached with immunoprecipitation of individually tagged polymerase subunits in the presence of chemically synthesized 5-prime triphosphate small viral RNA, then with subunit truncations to find the minimal binding unit, then with point mutation of the conserved basic residues lining a reported cleft in the PA crystal structure.\n\nFunction is approached in a cell-free reaction with purified trimeric polymerase, an in vitro transcribed complementary RNA minireplicon that cannot itself generate small viral RNA, and synthetic small viral RNA variants including a 13-nucleotide form and forms blocked at the 3-prime hydroxyl, which distinguishes a primer from an allosteric effector. Finally, a recombinant virus carrying a poly-uridine track substitution in the neuraminidase segment tests the contribution during infection while leaving the other seven segments as internal controls.", "Key findings": "1. Omitting individual segments from the bidirectional plasmid system showed that loss of the polymerase subunits, of nucleoprotein, or of segment 8 abolished small viral RNA production, whereas loss of hemagglutinin, neuraminidase or the matrix proteins did not, and complementation identified the nuclear export protein rather than nonstructural protein 1 as the segment 8 component required (Figure 1A). The authors interpret this as a requirement for a fully elongation-competent polymerase together with the nuclear export protein.\n\n2. In the polymerase reconstitution assay, increasing nuclear export protein enhanced complementary RNA synthesis from a genomic-sense template in a dose-dependent manner, and small viral RNA levels tracked with it (Figure 1B). Supplying a complementary-sense template directly made small viral RNA production independent of the nuclear export protein (Figure 1C). The observation is the loss of dependence on the alternate template. The interpretation offered is that the complementary RNA serves as the template for small viral RNA and that the nuclear export protein requirement reflects only its role in generating enough of that intermediate.\n\n3. Nuclear export protein truncations that lacked the nuclear export sequence still drove complementary RNA synthesis and small viral RNA production, whereas truncations retaining the nuclear export sequence but lacking an intact C-terminal domain did neither (Figure 2). The effect on RNA synthesis is therefore separable from nuclear export activity. The authors note this corroborates earlier findings of Robb and colleagues.\n\n4. Subcellular fractionation of infected A549 cells detected small viral RNA predominantly in the nuclear fraction from 8 hours post-infection onward and throughout infection, while nucleoprotein genomic RNA accumulated in the cytoplasm and peaked by 16 hours (Figure 3A). Modest cross-fraction contamination was visible on the blot and is acknowledged in the text.\n\n5. Deep sequencing of sucrose-purified virions found that small RNA reads accounted for about 1.8 percent of segment 1 reads from virions against about 28 percent from infected cells (Figure 3B). The interpretation is that small viral RNAs are largely not packaged, supporting a nuclear site of action.\n\n6. Immunoprecipitation of tagged subunits with synthetic 5-prime triphosphate small viral RNA showed association with the intact trimer and with the PB1 and PA heterodimer, and no association with the green fluorescent protein control (Figure 4A). Truncation mapping identified the PB1 interaction domain of PA, residues 155 to 716, and the catalytic domain of PB1, residues 1 to 500, as the minimal requirement, while the PB2 interaction domain of PB1 and the PA endonuclease domain were dispensable (Figure 4B).\n\n7. Point mutation of R566 in the reported basic cleft of PA strongly reduced small viral RNA binding, K328 and K539 had minimal effects individually, and double and triple combinations substantially reduced binding (Figure 4C). The authors additionally report, as data not shown, that cleft mutants were incapable of both transcription and replication in reconstitution assays, which they read as evidence that the cleft matters for overall polymerase activity and not only for this interaction.\n\n8. In cell-free reactions with purified trimeric polymerase and a complementary RNA minireplicon, little full-length product was made with buffer alone or with increasing scrambled RNA, whereas equimolar synthetic 22-nucleotide small viral RNA produced full-length product (Figure 5A). The first 13 nucleotides were sufficient, and small viral RNAs blocked at the 3-prime hydroxyl still supported full-length synthesis while abolishing aberrant 30 to 40 nucleotide products (Figure 5B). Because extension from the oligonucleotide is chemically prevented, the enhancement cannot be primer-mediated, and the authors conclude the effect is allosteric and is carried by the conserved 5-prime region.\n\n9. Replacing the poly-adenosine track with a poly-uridine track in a complementary RNA template abolished small viral RNA production at 24 and 48 hours in the reconstitution assay (Figure 6A and 6B), indicating that the canonical track is needed for small viral RNA generation from the promoter.\n\n10. A recombinant virus carrying the poly-uridine neuraminidase segment made less total small viral RNA (Figure 6C), retained normal neuraminidase messenger RNA (Figure 6B and 6D), and failed to synthesize neuraminidase genomic RNA robustly while hemagglutinin, nucleoprotein and nonstructural segment genomic RNA levels were unchanged relative to wild type (Figure 6E). The observation is a segment-restricted genome synthesis defect with messenger RNA intact. The interpretation is that small viral RNA acts specifically on replication of its own segment and thereby contributes to segment balance.", "Mechanistic model": "The model the authors advance, shown schematically in Figure 7, runs as follows. The incoming polymerase, bound to the genomic promoter and not yet occupied by small viral RNA, works predominantly as a transcriptase using host caps as primers. As nuclear export protein accumulates from viral transcripts, complementary RNA is synthesized and stabilized. Nascent complementary RNA then serves as the template for small viral RNA. The small viral RNA loads into the basic RNA binding cleft of PA, and the loaded polymerase becomes competent to engage its cognate complementary RNA template and synthesize full-length genomic RNA. Because the 3-prime complementarity of each small viral RNA differs, loading would create segment-specific replication-competent polymerase populations, which the authors propose is how stoichiometric balance across the eight segments is maintained.\n\nSeveral parts of this model are directly demonstrated and others are proposed. Directly demonstrated are the binding of small viral RNA to the PB1 and PA heterodimer, the dependence of that binding on PA basic residues including R566, the sufficiency of synthetic small viral RNA to promote full-length synthesis from a complementary RNA template with purified enzyme, the fact that a blocked 3-prime hydroxyl does not remove that activity, and the segment-restricted genome synthesis defect in the engineered virus. The allosteric character of the effect is an inference from the blocked 3-prime hydroxyl experiment and from the sufficiency of 13 nucleotides, which excludes priming but does not itself show a conformational change. No structure of a small viral RNA bound to the polymerase is presented, and the cleft is identified from a previously reported crystal structure of a PA and PB1 fragment rather than from a complex with this RNA. The proposal that one polymerase complex is dedicated to one segment, and the claim that this is how segment balance is set, is author interpretation consistent with the single-segment defect but not established by a direct measurement of polymerase occupancy. The conclusion that the nuclear export protein requirement reflects nothing more than complementary RNA availability is also an interpretation, supported by the template-swap and truncation experiments.", "Conceptual or technical advance": "The work places a small RNA inside the catalytic machinery of a viral RNA-dependent RNA polymerase and assigns it a defined binding surface and a non-priming mode of action. That makes the transcription-to-replication switch addressable as a ligand-occupancy problem rather than only as a question of protein and nucleotide concentrations. It also supplies a reagent-level toolkit for testing the idea further, namely chemically synthesized 5-prime triphosphate small viral RNAs including 3-prime blocked forms, a purified-enzyme reaction in which de novo small RNA production is suppressed, PA cleft point mutants, and a recombinant virus in which small viral RNA can be removed from a single segment while leaving the others intact. The single-segment virus in particular turns segment balance into something that can be perturbed one segment at a time.\n\nThe paper states in its discussion that this finding is the first example of a small RNA capable of controlling RNA-dependent RNA polymerase activity. That is a priority claim made by the authors.", "Relationship to the broader research program": "The study extends a line of work in this laboratory on small RNAs generated during virus infection and on what those RNAs do to the infected cell and to the virus itself. It builds directly on the earlier identification of influenza small viral RNAs by Perez and colleagues and connects to the laboratory's broader interest in whether small RNA species encountered during infection are regulatory or incidental. Reading this alongside the laboratory's work on mammalian small RNA silencing during infection is category 3 synthesis, and it would need the other records in the corpus to support it, so it is flagged here rather than asserted.\n\nThe engineered poly-uridine virus also belongs to a recurring methodological thread in which recombinant influenza is used as an instrument, with a defined genetic change installed to remove one regulatory element while leaving the rest of the genome as an internal control.", "Related publications": "- Perez and colleagues 2010, Proceedings of the National Academy of Sciences, identification of influenza A virus-generated small RNAs regulating the switch from transcription to replication. Relationship predecessor. Cited in this paper as the source of the original identification and of the methods for small viral RNA detection.\n- Shapiro, Varble, Pham and tenOever 2010, RNA, noncanonical cytoplasmic processing of viral microRNAs. Relationship methodological foundation. Cited for the small RNA library preparation used here.\n- Perez and colleagues 2009, Nature Biotechnology, microRNA-mediated species-specific attenuation of influenza A virus. Relationship predecessor. Cited for the infection procedures used here.\n- Robb, Smith, Vreede and Fodor 2009, Journal of General Virology, on regulation of transcription and replication by the nuclear export protein. Relationship predecessor from another laboratory. The truncation results here are explicitly described as corroborating it.", "Limitations and boundaries": "The cell-based work is confined to two human cell lines, A549 and HEK293, and to influenza A/Puerto Rico/8/34. The in vitro polymerase reactions use a purified trimer from an avian isolate, A/chicken/Nanchang/3-120/01, so the cell-free and infection systems are not matched for strain. No animal work is included, and no claim is made about pathogenesis or transmission.\n\nThe cell-free system lacks nucleoprotein, which the authors note prevents de novo small viral RNA production and also means the reaction does not reproduce the ribonucleoprotein context in which the polymerase normally works. The authors themselves caution that the ability of a short synthetic small viral RNA to promote full-length synthesis in vitro may not reflect a system the virus would use in a cell, since such promiscuity could be disadvantageous.\n\nThe allosteric mechanism is inferred rather than observed. There is no structure of the complex, no direct measurement of a conformational change, and no binding affinity determination. The PA cleft mutants are reported to be dead for both transcription and replication, which means the cleft mutations cannot cleanly separate small viral RNA function from general polymerase function in a functional assay, and this control is presented as data not shown.\n\nThe segment-specificity conclusion rests on one engineered segment, neuraminidase, altered by a poly-uridine track substitution that is known from prior work to affect messenger RNA export as well, so the manipulation is not exclusive to small viral RNA production even though messenger RNA levels for that segment were intact here. The fractionation result showed acknowledged cross-contamination between fractions, and the packaging conclusion rests on relative read proportions from purified virus stocks rather than on a direct assay of virion content.", "Audience summaries": "### 25 words\n\nInfluenza makes short RNAs from its own genome copies. These dock in a pocket of the viral polymerase and switch it toward copying that specific segment.\n\n### 75 words\n\nInfluenza A virus must switch its polymerase from making messenger RNA to copying its eight genome segments, and it must copy them in balanced amounts. This study shows that short viral RNAs, made from the positive-sense copy of the genome, bind a basic cleft in the polymerase PA subunit and boost full-length genome synthesis without acting as primers. Removing this small RNA from one segment blocks genome synthesis for that segment alone.\n\n### 150 words\n\nThe influenza A virus polymerase transcribes and replicates the same eight templates, and the control of that switch has been attributed variously to new polymerase acting in trans, to nucleoprotein or nucleotide concentrations, and to stabilization of the complementary RNA intermediate. Working in human cell lines, in reconstituted polymerase assays and with purified enzyme, this study shows that the small viral RNAs previously found at the 5-prime ends of the segments are templated from the complementary RNA intermediate, remain nuclear and are largely excluded from virions, and bind the polymerase through the PB1 and PA heterodimer, with the PA basic residue R566 most important. Synthetic small viral RNA promotes full-length genome synthesis from a complementary RNA template even when its 3-prime hydroxyl is blocked, which excludes priming and is read as allosteric enhancement. A recombinant virus lacking small viral RNA from the neuraminidase segment loses genome synthesis for that segment alone." }, "discoveries": [ "claim-01" ], "relationships": [ { "from": "2012-backes-degradation-of-host-micrornas-by-p", "to": "2012-perez-a-small-rna-enhancer-of-viral-poly", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2012-backes-degradation-of-host-micrornas-by-p" }, { "from": "2012-perez-a-small-rna-enhancer-of-viral-poly", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-perez-a-small-rna-enhancer-of-viral-poly" }, { "from": "2012-perez-a-small-rna-enhancer-of-viral-poly", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-perez-a-small-rna-enhancer-of-viral-poly" }, { "from": "2012-perez-a-small-rna-enhancer-of-viral-poly", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2012-perez-a-small-rna-enhancer-of-viral-poly" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2012-perez-a-small-rna-enhancer-of-viral-poly", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" }, { "from": "2021-nilsson-payant-reduced-nucleoprotein-availability", "to": "2012-perez-a-small-rna-enhancer-of-viral-poly", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-nilsson-payant-reduced-nucleoprotein-availability" }, { "from": "2022-nilsson-payant-the-host-factor-anp32a-is-required", "to": "2012-perez-a-small-rna-enhancer-of-viral-poly", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2022-nilsson-payant-the-host-factor-anp32a-is-required" } ], "canonical_url": "https://tenoeverlab.us/publications/2012-perez-a-small-rna-enhancer-of-viral-poly/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "reverse-genetics", "small-rna-seq", "site-directed-mutagenesis", "northern-blot", "in-vitro-reconstitution", "primer-extension", "rna-immunoprecipitation", "subcellular-fractionation", "synthetic-rna-mimetics" ] } }, { "id": "2012-pham-replication-in-cells-of-hematopoie", "slug": "2012-pham-replication-in-cells-of-hematopoie", "url": "/publications/2012-pham-replication-in-cells-of-hematopoie/", "title": "Replication in Cells of Hematopoietic Origin Is Necessary for Dengue Virus Dissemination", "authors": [ "Alissa M. Pham", "Ryan A. Langlois", "Benjamin R. tenOever" ], "author_count": 3, "first_author": "Alissa M. Pham", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 3, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2012, "journal": "PLoS Pathogens", "volume": "8", "issue": "1", "pages": "e1002465", "doi": "10.1371/journal.ppat.1002465", "doi_url": "https://doi.org/10.1371/journal.ppat.1002465", "pmid": "22241991", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/22241991/", "pmcid": "PMC3252368", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3252368/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3252368/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "programmable-virology" ], "themes": [ "microrna-mediated-viral-attenuation", "cell-type-restriction-as-a-tool" ], "pathogens": [ "dengue virus serotype 2" ], "viral_families": [ "Flaviviridae" ], "host_species": [ "human", "mouse", "hamster", "mosquito" ], "technologies": [ "microRNA target site insertion", "flavivirus infectious cDNA clone and virus rescue", "in vitro transcription and electroporation", "small RNA Northern blotting", "quantitative RT-PCR", "plaque assay", "magnetic-activated cell sorting", "fluorescence-activated cell sorting", "escape mutant sequencing" ], "biological_systems": [ "C6/36 Aedes albopictus cells", "BHK cells", "HEK293 cells", "human fibroblasts", "Raji B cells", "murine bone marrow derived macrophages", "mouse spleen", "mouse liver", "Ifnar1 and Il28r double knockout mice" ], "key_concepts": [ "viral tropism", "microRNA-mediated attenuation", "miR-142", "hematopoietic cells", "virus dissemination", "post-transcriptional silencing", "RNA-induced silencing complex", "escape mutants", "cell-type restriction of replication", "dengue pathogenesis models" ], "keywords": [ "dengue virus", "miR-142", "tropism", "macrophages", "dendritic cells", "hematopoietic", "dissemination", "microRNA targeting", "escape mutants", "mouse model" ], "one_sentence_contribution": "Inserting hematopoietic-specific miR-142 target sites into the dengue virus 3-prime untranslated region excludes replication from macrophages and dendritic cells in vivo and abolishes spread to other tissues, identifying hematopoietic cells as the dominant amplification compartment.", "summary_25": "Dengue virus was engineered so immune cells silence it. Without replication in those cells the virus stopped spreading, and every surviving genome had deleted the silencing sites.", "summary_75": "Dengue virus infects dendritic cells and macrophages, but whether other cells sustain infection has been unclear. Researchers inserted target sites for a microRNA found only in blood-lineage cells into the dengue genome, so the virus was silenced in those cells alone. In mice the modified virus lost replication in macrophages and dendritic cells and failed to spread to spleen and liver. All virus recovered from animals had deleted the inserted sites.", "summary_150": "Determining which cells actually support dengue virus replication in a host is difficult because viral antigen can reflect entry or uptake rather than productive infection. This study takes a genetic approach instead, inserting four tandem target sites for the hematopoietic-restricted microRNA miR-142 into the variable region of the dengue virus 3-prime untranslated region. The resulting virus replicates normally in cells lacking that microRNA and is silenced in cells expressing it. In interferon receptor deficient mice the targeted virus showed reduced replication in sorted CD11b positive, CD11c positive and CD45 positive cells, and reduced spleen and liver titers by intraperitoneal, intravenous and subcutaneous routes. Sequencing the residual virus recovered from spleen found no intact targeted genomes, only variants that had excised the whole cassette. The authors read the completeness of that conversion as evidence that hematopoietic cells are the predominant site of amplification and are required for dissemination.", "citation": "Pham AM, Langlois RA, tenOever BR. Replication in Cells of Hematopoietic Origin Is Necessary for Dengue Virus Dissemination. PLoS Pathogens. 2012. Volume 8, Issue 1, article e1002465. DOI 10.1371/journal.ppat.1002465. PMID 22241991. PMCID PMC3252368.", "sections": { "Citation": "Pham AM, Langlois RA, tenOever BR. Replication in Cells of Hematopoietic Origin Is Necessary for Dengue Virus Dissemination. PLoS Pathogens. 2012. Volume 8, Issue 1, article e1002465.\n\nDOI 10.1371/journal.ppat.1002465. PMID 22241991. PMCID PMC3252368.", "One-sentence contribution": "Inserting hematopoietic-specific miR-142 target sites into the dengue virus 3-prime untranslated region excludes replication from macrophages and dendritic cells in vivo and abolishes spread to other tissues, identifying hematopoietic cells as the dominant amplification compartment.", "Executive summary": "Dengue virus is known to replicate in dendritic cells and macrophages, but whether other cell types serve as productive reservoirs during infection has been hard to settle. The difficulty is a detection problem. Viral antigen or RNA found in a tissue can reflect active replication, or entry without replication, or uptake of virus or infected debris by that cell. Studies reporting endothelial cell infection have been questioned on exactly these grounds.\n\nThe study takes a genetic rather than a detection-based approach. Four tandem target sites for miR-142, a microRNA abundant in hematopoietic cells and absent elsewhere, were inserted into the variable region of the dengue virus 3-prime untranslated region downstream of the NS5 coding frame. The resulting virus should be silenced wherever miR-142 is present and should replicate normally everywhere else, so the hematopoietic compartment can be subtracted from an otherwise intact infection.\n\nThe targeted virus was attenuated in miR-142-expressing cells and unaffected in cells lacking the microRNA. In interferon receptor deficient mice it showed reduced replication in sorted CD11b positive, CD11c positive and CD45 positive cells, and reduced titers in spleen and liver by three routes of inoculation. Sequencing of the residual virus recovered from spleen showed that every recoverable genome had excised the entire targeting cassette, which the authors read as evidence that the hematopoietic compartment is where the virus is predominantly amplified.", "Scientific context": "Dengue virus is a mosquito-borne flavivirus with four serotypes and a large global burden, and at the time of the study no vaccine or specific therapeutic was available. Infection is thought to begin in resident cutaneous Langerhans dendritic cells at the site of mosquito inoculation, followed by migration through the lymphatic system and recruitment of monocytes and macrophages, which are themselves targets. The paper states that although hematopoietic cells are understood to be major sites of replication, it remains uncertain whether non-hematopoietic cells are permissive during natural infection.\n\nThe paper frames the unresolved point as an interpretive one. A wide range of cell lines from several host species are permissive in culture, and the virus engages many candidate receptors including heparan sulfate, DC-SIGN, CD14 and heat shock proteins 70 and 90, which has led some to propose endothelial cells as targets in vivo. The paper notes that this interpretation has been questioned because viral antigen in endothelial cells could reflect entry rather than replication, and cites work finding undetectable viral RNA in endothelial cells.\n\nSeparately, inserting fully complementary microRNA target sites into a viral genome had already been used to restrict tropism for influenza A virus, poliovirus, vesicular stomatitis virus and measles virus. For dengue specifically, the paper notes that the approach had previously been applied only to a replication-incompetent replicon or to a chimera carrying tick-borne encephalitis virus structural proteins, so a fully replication-competent targeted dengue virus had not been available.", "Central question": "If dengue virus replication is selectively excluded from hematopoietic cells in an otherwise intact animal, can the virus still be amplified and disseminated from a non-hematopoietic compartment?", "Experimental strategy": "The strategy converts a tropism question into a genetic subtraction. Rather than detecting virus in candidate cell types and arguing about whether the signal means replication, the design removes one compartment from the infection and asks what remains.\n\nThe tool is the endogenous microRNA machinery. Perfectly complementary target sites for a cell-type-restricted microRNA make that microRNA function as a virus-specific silencing guide, so the virus is attenuated only where the microRNA is expressed. The choice of miR-142 is what confers the desired restriction, since it is among the most abundant hematopoietic-specific microRNAs and is absent from the fibroblast, hamster kidney and mosquito cells used as permissive controls.\n\nPlacement matters because the flavivirus 3-prime untranslated region carries structures required for translation and RNA synthesis. The insert was therefore placed in the variable region just downstream of the NS5 open reading frame, a position previously reported to tolerate nucleotide insertion. A control virus carrying the same 157 nucleotide cassette in reverse orientation controls for the insertion itself rather than for its sequence, and an unmodified wild-type clone controls for the presence of any insert.\n\nBefore using the tool, the study checks that dengue infection does not itself disable microRNA biogenesis or silencing, since the entire approach depends on that machinery functioning during infection.\n\nIn vivo, mice lacking receptors for type I and type III interferon were used because human dengue isolates lack an adequate immunocompetent animal model. Three inoculation routes address whether the result is an artifact of delivery. Magnetic sorting of splenocytes into CD11b positive, CD11c positive, CD45 positive and CD45 negative fractions separates the targeted compartment from the untargeted one within the same animal. Finally, sequencing the 3-prime untranslated region of virus recovered from infected animals asks whether residual virus represents a genuine non-hematopoietic reservoir or escape from targeting, a distinction that quantitative RT-PCR for NS5 cannot make.", "Key findings": "1. In human fibroblasts carrying a miR-142-targeted green fluorescent protein reporter, supplying miR-142 reduced fluorescence by about 75 percent, and dengue virus infection at a multiplicity of one did not alter that silencing despite confirmed high-level replication (Figure 1). Northern blots for an exogenous microRNA and an endogenous microRNA likewise showed no change during infection (Figure S1). The interpretation offered is that dengue does not interfere with microRNA biogenesis or silencing, which licenses the rest of the approach.\n\n2. The targeted and reverse-orientation control viruses grew to comparable levels in mosquito C6/36 cells and in BHK cells, neither of which express miR-142, while unmodified wild-type virus exceeded both by roughly one log by quantitative RT-PCR and by plaque assay (Figure 2C, Figure 2D, Figure S3). The observation is a fitness cost attributable to the 157 nucleotide insertion itself and independent of its sequence, since the reverse-orientation control behaves the same way.\n\n3. Electroporation of in vitro transcribed genomes into BHK cells expressing miR-142 abolished NS5 protein from the targeted construct alone and left wild-type and control constructs unaffected (Figure 3B). Infection of the same cells gave the same result (Figure 3C), with residual NS5 for the targeted strain attributed by the authors to cells not successfully transfected with the miR-142 plasmid.\n\n4. A variant with the nucleotides complementary to positions 3 and 10 of miR-142 mismatched, intended to destroy seed pairing and the Argonaute 2 cleavage site, was nonetheless still repressed in a miR-142-dependent manner at the protein level (Figure 3D), while repression at the RNA level was significantly weaker than for the fully complementary virus with a reported p value of 0.0178 (Figure 3E). The observation is a dissociation between protein-level and RNA-level repression. The authors interpret it as a shift away from cleavage toward some form of translational repression or steric interference, and they state explicitly that the underlying molecular biology is unclear and that future studies will be required.\n\n5. Infection of two hematopoietic lineages, a human B cell line and murine bone marrow derived macrophages, showed selective attenuation of the targeted virus, whereas HEK293 cells showed no difference between targeted and control virus (Figure 3F). This is the endogenous counterpart of the ectopic experiments and establishes cell-type-specific attenuation without supplied miR-142.\n\n6. In interferon alpha receptor and interleukin 28 receptor double knockout mice inoculated intravenously, CD11b positive and CD11c positive splenocytes showed minimal NS5 transcript from the targeted virus and a significant decrease relative to control virus (Figure 4A).\n\n7. Sorting splenocytes into CD45 positive and CD45 negative fractions showed reduced targeted virus in the hematopoietic fraction as expected, and also reduced growth in the non-hematopoietic fraction, while the ratio of non-hematopoietic to hematopoietic signal was higher for the targeted virus (Figure 4B, Figure S4A). The authors interpret this as showing that the targeted virus is not intrinsically attenuated in non-hematopoietic cells and that its lower titer there is a consequence of the loss of hematopoietic amplification.\n\n8. Across intraperitoneal, intravenous and subcutaneous routes, spleen viral transcript was reduced for the targeted virus, with about a 1.5 log reduction after intraperitoneal delivery and greater attenuation by the intravenous and subcutaneous routes that the authors describe as better simulating natural inoculation (Figure 4C). Titers from spleen and liver showed approximately a three log reduction (Figure 4D, Figure S4B, Figure S4C). Heart, lung and brain titers were undetectable, identifying spleen and liver as the primary replication sites in this model.\n\n9. Reduced targeted virus was also seen in liver, where the paper notes miR-142 expression is lower than in spleen. The authors read this as evidence that liver virus resides predominantly in resident macrophages and dendritic cells, and they raise as a possibility that these cells are needed to sustain basal replication in non-hematopoietic cells. The second part is an interpretation rather than a measurement.\n\n10. Sequencing the 3-prime untranslated region of virus recovered from infected spleens found a complete absence of intact targeted genomes. Every recovered species had lost all four target sites, either by complete excision of the cassette or by replacement with a small host RNA fragment (Figure 5). In cell culture, by contrast, the mutation frequency was unbiased and comparable between cohorts, consistent with polymerase error rather than selection (Figure S5B). The authors note this explains the low residual signal in the NS5 quantitative RT-PCR, which cannot distinguish escape mutants from intact targeted virus, and they argue it makes a pre-existing quasispecies in the stock an unlikely explanation.", "Mechanistic model": "The study does not establish a mechanism for dengue tissue tropism at the molecular level, and it does not resolve the mechanism of the silencing it uses as a tool. What it establishes is a compartment requirement.\n\nThe model supported by the data is that hematopoietic cells, specifically macrophages and dendritic cells, are the compartment in which dengue virus is predominantly amplified in this animal model, and that replication in non-hematopoietic cells is insufficient to sustain dissemination on its own. The strongest evidence for the compartment claim is the escape mutant result. Under conditions where the hematopoietic compartment is closed to the virus, the entire recoverable population has reverted by excising the targeting cassette, which is difficult to reconcile with a productive alternative reservoir in which the targeted virus could have replicated unmodified. The authors state this conclusion as an inference from the completeness of the conversion.\n\nFor the silencing mechanism itself the paper is explicit that the picture is incomplete. Attenuation of the fully complementary virus is presumed by the authors to involve Argonaute 2 cleavage, and they describe the repression of the seed-mismatched variant as an enigma. They offer two alternative hypotheses, that residual extensive complementarity supports non-canonical silencing, or that loaded RISC binding provides steric hindrance that disrupts the long-range 5-prime to 3-prime base pairing required for flavivirus genome cyclization. They note that the second is supported by the nature of the escape mutants, since the only escapes recovered were complete cassette excisions rather than point mutations in seed or central positions. This remains an interpretation, not a demonstration.", "Conceptual or technical advance": "The work supplies a replication-competent dengue virus whose tropism can be edited genetically, which turns a question about which cells matter into an experiment that can be run in an intact animal. Where prior microRNA targeting of dengue had been limited to a replicon or to a chimeric virus, this construct replicates and disseminates, so compartment subtraction can be tested against dissemination rather than only against single-cycle gene expression.\n\nThe design also shows why sequencing the recovered population is necessary rather than optional. Quantitative RT-PCR for a viral gene registers escape mutants and intact targeted virus alike, and in this study that difference was the difference between apparent residual replication and no surviving targeted genomes at all. The authors present the approach as generalizable to other viruses and other cell subsets.", "Relationship to the broader research program": "The study sits in a line of work from this laboratory on engineering RNA viruses to interact with the host small RNA machinery, following directly from the use of microRNA target sites to attenuate influenza A virus in a species-specific manner and from work on engineered RNA viral synthesis of microRNAs, both cited here and both from the same group. The recurring idea is that the host small RNA system can be recruited as a programmable constraint on where a virus is permitted to replicate.\n\nIt also connects to a broader question that runs through the corpus, namely what small RNA pathways actually do during mammalian virus infection as opposed to what they can be made to do. This paper uses the machinery instrumentally and reports that dengue infection leaves it intact. Placing that observation beside the laboratory's other work on small RNA silencing during infection is category 3 synthesis and would need those records to support it.", "Related publications": "- Perez, Pham, Lorini, Chua, Steel and colleagues 2009, Nature Biotechnology, microRNA-mediated species-specific attenuation of influenza A virus. Relationship methodological foundation. Cited here as the precedent for microRNA-mediated viral attenuation and as the source of the extraction and Northern blot protocols used.\n- Varble, Chua, Perez, Manicassamy, Garcia-Sastre and colleagues 2010, Proceedings of the National Academy of Sciences, engineered RNA viral synthesis of microRNAs. Relationship methodological foundation. Cited as the source of the miR-142 target sites used in the construct.\n- Kinney and colleagues 1997, Virology, construction of infectious cDNA clones for dengue 2 virus. Relationship methodological foundation from another laboratory. The pD2/IC-30P-A clone was the backbone for all recombinant viruses here.\n- Mordstein and colleagues 2008, Journal of Virology, on lambda interferon and epithelial resistance. Relationship methodological foundation from another laboratory. Source of the interferon receptor double knockout mice used for the in vivo work.", "Limitations and boundaries": "The animal model lacks receptors for both type I and type III interferon, which the authors chose because human dengue isolates have no adequate immunocompetent model. Interferon signaling shapes which cells support replication and how far a virus spreads, so conclusions about compartment requirements are bounded by that genetic background and should not be transferred directly to immunocompetent hosts or to human infection.\n\nOnly one serotype and one strain were used, dengue virus 2 strain 16681, so serotype-specific differences in tropism are not addressed.\n\nThe insertion itself costs fitness. Both the targeted and the reverse-orientation control virus grew about one log below unmodified virus in the permissive cell lines, so all comparisons are between two insertion-bearing viruses rather than against an unmodified baseline.\n\nThe in vivo analysis rests on spleen and liver, with heart, lung and brain below detection. Skin, the natural site of mosquito inoculation, and the Langerhans cells the introduction identifies as the first target were not sampled. The cell sorting used CD11b, CD11c and CD45 markers on splenocytes, which does not enumerate every hematopoietic subset and does not identify which non-hematopoietic cell types are represented in the CD45 negative fraction.\n\nThe claim that non-hematopoietic cells are not productively infected is stated by the authors as one of two possibilities, the other being that macrophages and dendritic cells are required for spread. The data constrain dissemination, not intrinsic permissiveness of every other cell type, and the paper phrases the closing conclusion in that conditional form.\n\nThe mechanism of attenuation for the seed-mismatched variant is unresolved and the authors say so. The steric interference model for disruption of genome cyclization is a hypothesis supported indirectly by the character of the escape mutants.\n\nEscape mutant sequencing used fifteen randomly selected clones per condition after PCR amplification, so the estimate of population composition has the depth limits of clonal sequencing rather than deep sequencing.", "Audience summaries": "### 25 words\n\nDengue virus was engineered so immune cells silence it. Without replication in those cells the virus stopped spreading, and every surviving genome had deleted the silencing sites.\n\n### 75 words\n\nDengue virus infects dendritic cells and macrophages, but whether other cells sustain infection has been unclear. Researchers inserted target sites for a microRNA found only in blood-lineage cells into the dengue genome, so the virus was silenced in those cells alone. In mice the modified virus lost replication in macrophages and dendritic cells and failed to spread to spleen and liver. All virus recovered from animals had deleted the inserted sites.\n\n### 150 words\n\nDetermining which cells actually support dengue virus replication in a host is difficult because viral antigen can reflect entry or uptake rather than productive infection. This study takes a genetic approach instead, inserting four tandem target sites for the hematopoietic-restricted microRNA miR-142 into the variable region of the dengue virus 3-prime untranslated region. The resulting virus replicates normally in cells lacking that microRNA and is silenced in cells expressing it. In interferon receptor deficient mice the targeted virus showed reduced replication in sorted CD11b positive, CD11c positive and CD45 positive cells, and reduced spleen and liver titers by intraperitoneal, intravenous and subcutaneous routes. Sequencing the residual virus recovered from spleen found no intact targeted genomes, only variants that had excised the whole cassette. The authors read the completeness of that conversion as evidence that hematopoietic cells are the predominant site of amplification and are required for dissemination." }, "discoveries": [ "claim-09", "claim-10" ], "relationships": [ { "from": "2012-backes-degradation-of-host-micrornas-by-p", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2012-backes-degradation-of-host-micrornas-by-p" }, { "from": "2012-langlois-hematopoietic-specific-targeting-o", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "companion", "evidence": "stated in the Related publications section of 2012-langlois-hematopoietic-specific-targeting-o" }, { "from": "2012-pham-replication-in-cells-of-hematopoie", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-pham-replication-in-cells-of-hematopoie" }, { "from": "2012-pham-replication-in-cells-of-hematopoie", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-pham-replication-in-cells-of-hematopoie" }, { "from": "2013-langlois-microrna-based-strategy-to-mitigat", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "application", "evidence": "stated in the Related publications section of 2013-langlois-microrna-based-strategy-to-mitigat" }, { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "application", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2018-aguado-homologous-recombination-is-an-int", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "application", "evidence": "stated in the Related publications section of 2018-aguado-homologous-recombination-is-an-int" } ], "canonical_url": "https://tenoeverlab.us/publications/2012-pham-replication-in-cells-of-hematopoie/", "controlled_vocabulary": { "pathogens": [ "dengue-virus" ], "technologies": [ "rt-qpcr", "plaque-assay", "reverse-genetics", "small-rna-northern-blot", "mirna-target-site-insertion", "viral-population-deep-sequencing", "cell-sorting" ] } }, { "id": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "slug": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "url": "/publications/2012-shapiro-evidence-for-a-cytoplasmic-micropr/", "title": "Evidence for a cytoplasmic microprocessor of pri-miRNAs", "authors": [ "Jillian S. Shapiro", "Ryan A. Langlois", "Alissa M. Pham", "Benjamin R. tenOever" ], "author_count": 4, "first_author": "Jillian S. Shapiro", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 4, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2012, "journal": "RNA", "volume": "18", "issue": "7", "pages": "1338-1346", "doi": "10.1261/rna.032268.112", "doi_url": "https://doi.org/10.1261/rna.032268.112", "pmid": "22635403", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/22635403/", "pmcid": "PMC3383965", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383965/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383965/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense" ], "themes": [ "noncanonical-microrna-biogenesis" ], "pathogens": [ "Sindbis virus", "influenza A virus" ], "viral_families": [ "Togaviridae", "Orthomyxoviridae" ], "host_species": [ "mouse", "golden hamster" ], "technologies": [ "recombinant Sindbis virus", "small RNA Northern blot", "small RNA deep sequencing", "RNA in situ hybridization", "immunofluorescence microscopy", "Argonaute immunoprecipitation", "luciferase reporter silencing assay", "conditional gene knockout", "CFSE cell division tracking" ], "biological_systems": [ "BHK cells", "mouse embryonic fibroblasts", "conditional knockout fibroblast lines" ], "key_concepts": [ "noncanonical microRNA biogenesis", "cytoplasmic pri-miRNA", "microprocessor", "Drosha relocalization", "DGCR8 dependence", "Dicer dependence", "RNA-induced silencing complex loading", "virus-encoded microRNA", "endogenous miRNA landscape stability" ], "keywords": [ "Drosha", "DGCR8", "Dicer", "microRNA biogenesis", "Sindbis virus", "Argonaute", "cytoplasmic processing", "small RNA sequencing", "miR-124", "miR-122" ], "one_sentence_contribution": "Primary microRNA transcripts generated in the cytoplasm by a recombinant Sindbis virus are cleaved without any nuclear involvement yet still require Drosha, which relocalises from nucleus to cytoplasm on infection while the endogenous microRNA profile of the cell remains largely unchanged.", "summary_25": "Microbes that never enter the nucleus can still make microRNAs, because virus infection moves the nuclear enzyme Drosha into the cytoplasm, where it cleaves viral hairpin transcripts.", "summary_75": "MicroRNAs are normally cut first in the nucleus by Drosha and then in the cytoplasm by Dicer. Using a Sindbis virus engineered to carry microRNA hairpins, the authors show that processing happens entirely in the cytoplasm, does not need cell division, and yet still depends absolutely on Drosha. Infection itself moves Drosha out of the nucleus. Despite this relocation, the cell's own microRNA population is essentially unchanged, and the viral microRNA silences targets normally.", "summary_150": "Recombinant Sindbis viruses carrying miR-124, miR-122 or both were used to generate primary microRNA transcripts confined to the cytoplasm. Mature products formed from all constructs, loaded into Argonaute 2 and silenced a target reporter. Serum starvation that fully blocked division did not alter processing efficiency, and in situ hybridisation placed the viral transcript in the cytoplasm, in contrast to the nuclear signal from an influenza A virus expressing the same hairpin. Conditional knockout fibroblasts showed an absolute requirement for Drosha, a requirement for Dicer only at the precursor to mature step, no requirement for TRBP2, PACT or AGO2, and a DGCR8 effect limited to mature microRNA accumulation that the authors leave unresolved. Endogenous Drosha redistributed to the cytoplasm after infection with either parental or microRNA-expressing virus. Small RNA sequencing showed the host microRNA profile largely unchanged even as virus-derived miR-124 reached several percent of total cellular microRNA.", "citation": "Shapiro JS, Langlois RA, Pham AM, tenOever BR. Evidence for a cytoplasmic microprocessor of pri-miRNAs. *RNA* 2012, volume 18, issue 7, pages 1338-1346. DOI 10.1261/rna.032268.112. PMID 22635403. PMCID PMC3383965.", "sections": { "Citation": "Shapiro JS, Langlois RA, Pham AM, tenOever BR. Evidence for a cytoplasmic microprocessor of pri-miRNAs. *RNA* 2012, volume 18, issue 7, pages 1338-1346.\n\nDOI 10.1261/rna.032268.112. PMID 22635403. PMCID PMC3383965.", "One-sentence contribution": "Primary microRNA transcripts generated in the cytoplasm by a recombinant Sindbis virus are cleaved without any nuclear involvement yet still require Drosha, which relocalises from nucleus to cytoplasm on infection while the endogenous microRNA profile of the cell remains largely unchanged.", "Executive summary": "Canonical microRNA biogenesis is compartmentalised. A primary transcript is cleaved in the nucleus by the microprocessor, made up of the RNase III enzyme Drosha and the double-stranded RNA binding protein DGCR8, and the resulting precursor is exported and cleaved again by Dicer in the cytoplasm. Cytoplasmic RNA viruses can be engineered to produce functional microRNAs, which raises the question of how a primary transcript that never enters the nucleus is processed. Using recombinant Sindbis viruses encoding miR-124, miR-122 or both in tandem, the authors show that mature microRNAs are produced from a range of hairpin sequences and transcript positions, load into Argonaute 2 and silence a reporter carrying target sites. Blocking cell division by serum starvation, which removes the possibility that nuclear envelope breakdown grants access to the nuclear microprocessor, did not change the conversion of cytoplasmic primary transcript into mature microRNA, and in situ hybridisation showed the viral transcript confined to the cytoplasm, unlike the nuclear signal from an influenza A virus engineered to express the same hairpin. Genetic tests across conditional knockout fibroblasts showed a complete requirement for Drosha, a requirement for Dicer only at the final maturation step, no requirement for TRBP2, PACT or AGO2, and a partial effect of DGCR8 loss confined to mature microRNA accumulation. Immunofluorescence showed endogenous Drosha redistributed to the cytoplasm after Sindbis virus infection. Small RNA sequencing showed the endogenous microRNA profile essentially unchanged despite this redistribution and despite virus-derived miR-124 reaching several percent of cellular microRNA content.", "Scientific context": "At the time of writing, canonical biogenesis was well described as a stepwise nuclear then cytoplasmic pathway, and a set of noncanonical routes had been catalogued, most of which bypass the microprocessor while retaining Dicer. Mirtrons arise by splicing and debranching, and miR-451 is processed by the catalytic activity of Argonaute 2 rather than by Dicer. A separate line of work from several laboratories, including earlier reports from this group and from Rouha and colleagues, had shown that cytoplasmic RNA viruses can be engineered to yield functional microRNAs, and initial characterisation had suggested those transcripts were processed independently of the canonical microprocessor while still requiring Dicer. The paper states that the mechanism and characteristics of cytoplasmic biogenesis had not been thoroughly elucidated, and that is the gap it sets out to close.", "Central question": "What cellular machinery cleaves a primary microRNA transcript that is generated and retained in the cytoplasm, and does the use of that machinery perturb normal microRNA biogenesis in the infected cell?", "Experimental strategy": "The system is a recombinant Sindbis virus, an alphavirus whose entire replication cycle is cytoplasmic, carrying a microRNA locus so that the primary transcript is produced where the nuclear microprocessor is not. Varying the payload across a second hairpin and a tandem arrangement tests whether processing depends on the particular sequence, structure or position within the transcript. A matched comparison with an influenza A virus engineered to produce the same hairpin provides a nuclear-replicating control for the in situ hybridisation, so that cytoplasmic confinement of the viral transcript is demonstrated rather than assumed. Serum starvation with CFSE dilution as a division readout removes mitotic nuclear envelope breakdown as a route of access to nuclear components. The genetic requirements are then addressed directly with a panel of fibroblast lines lacking individual biogenesis factors, using conditional deletion with adenoviral Cre for the essential factors Drosha and DGCR8 and confirming deletion by loss of the abundant endogenous miR-93. Function is read out with Argonaute 2 immunoprecipitation and a Gaussia luciferase reporter bearing perfect target sites. Finally, small RNA deep sequencing asks whether any of this disturbs the cell's own microRNA population.", "Key findings": "1. A recombinant Sindbis virus expressing miR-122 produced abundant mature 22-nucleotide microRNA at 16 hours, comparable to the earlier miR-124 virus, and a tandem construct carrying both loci in a single transcript yielded both mature species (Figure 1A). Observation aside, the authors read this as evidence that cytoplasmic processing is not restricted to one hairpin sequence or structure.\n2. In the tandem virus, miR-124 accumulated to lower levels than in the single-hairpin virus, which the authors attribute to its position in the transcript rather than to any demonstrated positional mechanism. No intermediate cleavage products were detectable, which is interpreted as rapid processing or degradation of the cytoplasmic primary transcript.\n3. Argonaute 2 immunoprecipitation recovered both miR-122 and miR-124 regardless of transcript source, while a GFP control precipitate recovered neither, and the lower miR-124 yield from the tandem virus was mirrored in Argonaute association and in silencing of a luciferase reporter carrying four perfect miR-122 sites (Figure 1, B and C).\n4. Serum starvation produced a complete block in division as judged by sustained CFSE signal at 24 and 48 hours. Infection of arrested cells gave reduced viral replication but the ratio of cytoplasmic primary transcript to mature miR-124 was unchanged at roughly one to one and a quarter (Figure 2, A and B). The authors state this strongly suggests cell division is not required for cytoplasmic microRNA synthesis.\n5. RNA in situ hybridisation with a probe against the primary miR-124 transcript showed nuclear accumulation in cells infected with the influenza A virus construct and an absence of nuclear signal with cytoplasmic abundance in cells infected with the Sindbis construct (Figure 2D).\n6. Conditional deletion of Drosha abolished both precursor and mature virus-derived miR-124 (Figure 3). This is the central genetic result and it is a direct demonstration that a nominally nuclear enzyme is required for cleavage of a transcript that never reaches the nucleus.\n7. Loss of DGCR8 reduced mature miR-124 and led to accumulation of the precursor without preventing conversion of primary transcript to precursor (Figure 3). The authors explicitly present two competing readings, that DGCR8 is dispensable for cleavage but needed for its accuracy, or that the cytoplasmic activity is DGCR8-independent and the phenotype is an indirect consequence of losing endogenous microRNAs, and they state that future work is needed to distinguish them.\n8. Cells lacking Dicer accumulated both the cytoplasmic primary transcript and a precursor of about 60 nucleotides at levels and size comparable to wild-type cells, with only the mature species absent (Figure 3). This places Dicer at the second cleavage only and argues it is not involved in the first.\n9. Loss of TRBP2 or of PACT left all three RNA species unchanged, and loss of AGO2 left precursor and mature miR-124 unchanged (Figure 3). The authors note that the two double-stranded RNA binding proteins share partial homology and may therefore be redundant rather than simply dispensable.\n10. Immunofluorescence for endogenous Drosha showed nuclear staining in mock-treated fibroblasts and a pronounced redistribution to the cytoplasm after infection with either the parental or the microRNA-expressing Sindbis virus (Figure 4A). The relocalisation is therefore a response to infection and not to the presence of a cytoplasmic hairpin substrate.\n11. Small RNA deep sequencing of mock-treated and infected fibroblasts showed the annotated microRNA profile largely unchanged by infection and by expression of a cytoplasmic primary transcript, while virus-derived miR-124 reached 4.3 percent of total cellular microRNA at 24 hours, with estimated concentrations up to about 55,000 copies per cell (Figure 4, B and C). The authors suggest the stability of the endogenous profile reflects microRNA half-lives longer than 24 hours, which is an interpretation and not a measurement made here.", "Mechanistic model": "The data constrain the pathway but do not establish a complete mechanism, and the authors say as much for the steps that remain open. What is shown is that a cytoplasmic primary transcript is cleaved into a precursor in a Drosha-dependent and Dicer-independent step, that the precursor is then converted to a mature microRNA in a Dicer-dependent step, that the product loads into Argonaute 2 and silences targets, and that Drosha itself moves out of the nucleus on infection. The model the authors propose is that virus infection redistributes Drosha to the cytoplasm where it acts on highly structured cytoplasmic RNAs, giving the enzyme a second function alongside its canonical microprocessor role. What is not established is the composition of the cytoplasmic cleaving activity. The paper cannot say whether DGCR8 is a genuine partner in the cytoplasm, and it raises the alternative that an uncharacterised and possibly virus-inducible double-stranded RNA binding protein substitutes for it. Nor is the trigger or the mechanism of Drosha relocalisation determined, although the authors note prior work that Drosha nuclear localisation is phosphorylation-dependent. The speculation that Drosha relocalisation reflects a role as a virus restriction factor independent of microRNA biogenesis, and the evolutionary framing that follows it, are explicitly offered as speculation.", "Conceptual or technical advance": "The work converts an operational observation, that engineered cytoplasmic viruses yield functional microRNAs, into a defined set of genetic requirements, and in doing so reassigns Drosha from a strictly nuclear enzyme to one whose localisation is conditional on infection. It also revises the earlier reading of these transcripts as microprocessor-independent, since Drosha proves to be strictly required. Practically, the demonstration that a cytoplasmic RNA virus can deliver a microRNA to high copy number without disturbing the host microRNA profile matters for the use of such viruses as small RNA delivery vehicles, and the panel of biogenesis knockout fibroblasts combined with recombinant Sindbis viruses provides a general assay for dissecting noncanonical processing.", "Relationship to the broader research program": "This paper sits within a sustained line of work in the laboratory on engineering RNA viruses to express small RNAs and on what that engineering reveals about host RNA biology. Its own reference list points to earlier reports from the group on noncanonical cytoplasmic processing of viral microRNAs, on engineered RNA viral synthesis of microRNAs, on influenza A virus-generated small RNAs, on microRNA-mediated species-specific attenuation of influenza A virus, and on in vivo delivery of cytoplasmic virus-derived microRNAs. Setting this paper beside the later laboratory work on RNA interference and virus interactions would be category 3 synthesis and is not attempted from this paper alone.", "Related publications": "- Shapiro and colleagues, 2010, predecessor. Cited here as the report of noncanonical cytoplasmic processing of viral microRNAs that this study sets out to characterise mechanistically, and as the source of the recombinant Sindbis virus cloning strategy.\n- Varble and colleagues, 2010, methodological foundation. Cited as the origin of the engineered RNA viral microRNA approach and specifically as the source of the influenza A virus expressing miR-124 used here as the nuclear-replicating comparator.\n- Langlois and colleagues, 2012, companion. Cited as the accompanying demonstration that cytoplasmic virus-derived microRNAs can be delivered in vivo.\n- Perez and colleagues, 2009 and 2010, predecessor. Cited as earlier work from the group on influenza A virus-generated small RNAs and on microRNA-mediated attenuation.\n- Schmid and colleagues, 2010, methodological foundation. Cited for immunoblotting procedure and part of the same laboratory's output.", "Limitations and boundaries": "The primary transcripts studied are virus-derived and engineered, so the findings describe what happens to a structured, capped and polyadenylated hairpin-bearing transcript produced at high abundance by an alphavirus, not a natural cytoplasmic pri-microRNA. All experiments are in cultured cells, chiefly BHK cells and mouse embryonic fibroblasts, at multiplicities of infection from one to ten and over roughly 16 to 24 hours, so nothing is established for primary tissue, for animals or for longer timescales. Two viruses are used and one of them, influenza A virus, only as a localisation comparator. The DGCR8 result is not resolved and the authors present two incompatible explanations for it. The negative results for TRBP2, PACT and AGO2 come from single knockout lines and cannot exclude redundancy, a point the authors make for the two double-stranded RNA binding proteins. The conditional knockouts require six days of Cre expression before infection, during which endogenous microRNAs are depleted, so indirect effects on the small RNA machinery cannot be excluded for any of the deletion phenotypes. The stability of the endogenous microRNA profile is measured at a single 24-hour timepoint by sequencing and is consistent with, but does not demonstrate, the proposed explanation based on microRNA half-life. The mechanism and the functional consequence of Drosha relocalisation are not determined.", "Audience summaries": "### 25 words\n\nMicrobes that never enter the nucleus can still make microRNAs, because virus infection moves the nuclear enzyme Drosha into the cytoplasm, where it cleaves viral hairpin transcripts.\n\n### 75 words\n\nMicroRNAs are normally cut first in the nucleus by Drosha and then in the cytoplasm by Dicer. Using a Sindbis virus engineered to carry microRNA hairpins, the authors show that processing happens entirely in the cytoplasm, does not need cell division, and yet still depends absolutely on Drosha. Infection itself moves Drosha out of the nucleus. Despite this relocation, the cell's own microRNA population is essentially unchanged, and the viral microRNA silences targets normally.\n\n### 150 words\n\nRecombinant Sindbis viruses carrying miR-124, miR-122 or both were used to generate primary microRNA transcripts confined to the cytoplasm. Mature products formed from all constructs, loaded into Argonaute 2 and silenced a target reporter. Serum starvation that fully blocked division did not alter processing efficiency, and in situ hybridisation placed the viral transcript in the cytoplasm, in contrast to the nuclear signal from an influenza A virus expressing the same hairpin. Conditional knockout fibroblasts showed an absolute requirement for Drosha, a requirement for Dicer only at the precursor to mature step, no requirement for TRBP2, PACT or AGO2, and a DGCR8 effect limited to mature microRNA accumulation that the authors leave unresolved. Endogenous Drosha redistributed to the cytoplasm after infection with either parental or microRNA-expressing virus. Small RNA sequencing showed the host microRNA profile largely unchanged even as virus-derived miR-124 reached several percent of total cellular microRNA." }, "discoveries": [ "claim-07" ], "relationships": [ { "from": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2012-shapiro-evidence-for-a-cytoplasmic-micropr" }, { "from": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-shapiro-evidence-for-a-cytoplasmic-micropr" }, { "from": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2012-shapiro-evidence-for-a-cytoplasmic-micropr" }, { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" }, { "from": "2015-benitez-in-vivo-rnai-screening-identifies-", "to": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-in-vivo-rnai-screening-identifies-" }, { "from": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "to": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2017-aguado-rnase-iii-nucleases-from-diverse-k" } ], "canonical_url": "https://tenoeverlab.us/publications/2012-shapiro-evidence-for-a-cytoplasmic-micropr/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sindbis-virus" ], "technologies": [ "reverse-genetics", "immunofluorescence-microscopy", "small-rna-seq", "small-rna-northern-blot", "silencing-reporter-assay", "rna-in-situ-hybridization", "argonaute-ip", "conditional-knockout", "cell-division-tracking" ] } }, { "id": "2013-chua-influenza-a-virus-utilizes-subopti", "slug": "2013-chua-influenza-a-virus-utilizes-subopti", "url": "/publications/2013-chua-influenza-a-virus-utilizes-subopti/", "title": "Influenza A Virus Utilizes Suboptimal Splicing to Coordinate the Timing of Infection", "authors": [ "Mark A. Chua", "Sonja Schmid", "Jasmine T. Perez", "Ryan A. Langlois", "Benjamin R. tenOever" ], "author_count": 5, "first_author": "Mark A. Chua", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 5, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2013, "journal": "Cell Reports", "volume": "3", "issue": "1", "pages": "23-29", "doi": "10.1016/j.celrep.2012.12.010", "doi_url": "https://doi.org/10.1016/j.celrep.2012.12.010", "pmid": "23333274", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/23333274/", "pmcid": "PMC3563938", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563938/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563938/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "influenza-genome-regulation" ], "themes": [ "splicing-and-temporal-control", "ns1-and-interferon-antagonism" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "human", "mouse", "dog" ], "technologies": [ "reverse genetics", "microRNA-mediated attenuation", "small interfering RNA knockdown", "replication-incompetent virus-like vectors", "2A ribosome recoding", "site-directed splice site mutagenesis", "multicycle growth curves", "immunofluorescence microscopy", "intranasal mouse infection" ], "biological_systems": [ "A549 cells", "Jurkat T cells", "murine embryonic fibroblasts", "Dicer1-deficient fibroblasts", "primary lung fibroblasts", "bone marrow-derived macrophages", "MDCK cells", "mouse lung" ], "key_concepts": [ "suboptimal 5-prime splice site", "molecular timer", "nuclear export protein NEP", "NS1 interferon antagonism", "viral ribonucleoprotein export", "bicistronic segment 8", "gene expression stoichiometry", "temporal coordination of the viral life cycle" ], "keywords": [ "influenza A virus", "NS segment", "NEP", "NS2", "NS1", "splicing", "vRNP export", "miR-142", "miR-20", "molecular timer" ], "one_sentence_contribution": "The inefficient 5 prime splice site of influenza A virus segment 8 functions as a timing device, causing the nuclear export protein to accumulate slowly as a minor product of abundant NS1 transcription, with both raising and lowering that rate attenuating the virus through mistimed ribonucleoprotein export.", "summary_25": "Influenza splices one of its genes badly on purpose. The resulting slow build-up of an export protein times the infection, and correcting the splicing cripples the virus.", "summary_75": "Influenza A virus segment 8 makes the interferon antagonist NS1 as its main product and the nuclear export protein NEP through a weak splice site. Silencing NS1 by over ninety percent barely affected the virus, in cells or in mice. Changing NEP in either direction crippled it. Making the splice site efficient raised NEP, sent genome complexes to the cytoplasm hours early, and cost two logs of growth. The poor splice site is a timer.", "summary_150": "Influenza A virus has eight segments with comparable promoters and no obvious way to schedule its life cycle. Segment 8 encodes the interferon antagonist NS1 as the unspliced transcript and the nuclear export protein NEP through a 5 prime splice site used only ten to fifteen percent of the time. Inserting host microRNA target sites allowed NS1 to be reduced by more than ninety percent without touching NEP, and this had little effect on replication or on interferon-regulated gene induction in cell lines, primary cells or mice. Reducing NEP by a 2A recoding site or by small interfering RNA lowered titres, and raising NEP by an extra gene copy or by optimising the splice site lowered them by about two logs in culture and nearly abolished replication in mice. Nucleoprotein reached the cytoplasm hours early when NEP was abundant. The authors propose that inefficient splicing serves as a molecular timer pacing NEP accumulation.", "citation": "Chua MA, Schmid S, Perez JT, Langlois RA, tenOever BR. Influenza A Virus Utilizes Suboptimal Splicing to Coordinate the Timing of Infection. Cell Reports. 2013. Volume 3, issue 1, pages 23-29. DOI 10.1016/j.celrep.2012.12.010. PMID 23333274. PMCID PMC3563938.", "sections": { "Citation": "Chua MA, Schmid S, Perez JT, Langlois RA, tenOever BR. Influenza A Virus Utilizes Suboptimal Splicing to Coordinate the Timing of Infection. Cell Reports. 2013. Volume 3, issue 1, pages 23-29.\n\nDOI 10.1016/j.celrep.2012.12.010. PMID 23333274. PMCID PMC3563938.", "One-sentence contribution": "The inefficient 5 prime splice site of influenza A virus segment 8 functions as a timing device, causing the nuclear export protein to accumulate slowly as a minor product of abundant NS1 transcription, with both raising and lowering that rate attenuating the virus through mistimed ribonucleoprotein export.", "Executive summary": "Influenza A virus carries only ten major proteins on eight segments, each driven by a comparable promoter, yet it must order nuclear entry, genome replication, nuclear export and assembly in time. Segment 8 is bicistronic, producing the interferon antagonist NS1 as the unspliced transcript and the nuclear export protein NEP through a weak 5 prime splice site that captures only ten to fifteen percent of segment-derived messenger RNA. The authors asked whether that inefficiency is functional. Using microRNA target sites inserted into the segment, they silenced NS1 by more than ninety percent without touching NEP and found little effect on replication or on host antiviral gene induction in cell lines, in primary cells or in mice. Manipulating NEP in either direction told a different story. Lowering NEP, either by decoupling it from splicing with a 2A recoding site or by small interfering RNA, reduced titres. Raising NEP, either by supplying an extra copy from a replication-incompetent vector or by optimising the splice site so that NEP became the dominant product, reduced titres by about two orders of magnitude in culture and almost abolished replication in mice. Imaging showed premature cytoplasmic appearance of nucleoprotein when NEP accumulated early and delayed export when NEP was scarce.", "Scientific context": "Segment 8 of influenza A virus is one of two segments that undergo splicing and encodes both NS1 and NEP. NS1 is described in the prior literature as the dominant antagonist of the cellular response to infection, and viruses lacking it are attenuated by more than four logs in animals while retaining wild-type virulence in hosts without antiviral signalling. NEP is a 121 amino acid protein required for production of replication-competent virus, with its best-characterised role in exporting nucleoprotein-associated genomic RNA from the nucleus through association with the matrix protein M1, and disruption of its nuclear export signal abolishes replication. NEP had also been implicated by earlier work in controlling complementary RNA levels, in host tropism and in the synthesis of influenza small viral RNA. What had not been resolved was why the virus makes NEP through a splice site that works poorly, producing only ten to fifteen percent of segment 8 messenger RNA as the spliced form, and whether the relative amounts of the two segment 8 products matter for coordinating the infection.", "Central question": "Is the balance between NS1 and NEP, set by the efficiency of the segment 8 splice site, a requirement for coordinating the influenza A virus life cycle, or is the inefficiency of that splice site incidental.", "Experimental strategy": "The design isolates each segment 8 product and moves its abundance up and down by independent means, so that a shared phenotype cannot be attributed to any one manipulation. NS1 was reduced without disturbing NEP by inserting tandem perfect target sites for host microRNAs into the intergenic region of a modified NS vector, which places silencing under the control of the cell's own microRNA content rather than requiring a mutation in the coding sequence. Two microRNAs with different expression patterns were used, one ubiquitous and one restricted to haematopoietic cells, and a scrambled insert served as the isogenic control, which lets the same virus be silenced or not depending on the cell type it enters. Cells lacking Dicer1 confirmed that the effect depended on the microRNA pathway. NEP was lowered in two ways, by expressing it from a 2A ribosome recoding site instead of as a splice product and by small interfering RNA during wild-type infection, and raised in two ways, by supplying an extra copy from a replication-incompetent influenza-like vector grafted into segment 4 and by mutating the 5 prime splice site so that NEP became dominant and NS1 a readthrough product. Because splice site optimisation also changes an amino acid, a parental virus carrying the same substitution with the native splice site was built as the matched control. Replication was read by multicycle growth curves in culture and by titres from intranasally infected mice, host response by interferon-regulated gene expression, and the proposed timing defect by immunofluorescence of nucleoprotein localisation at early time points.", "Key findings": "1. Insertion of perfect microRNA target sites silenced NS1 at both protein and messenger RNA level without changing nucleoprotein or NEP, with silencing matching the microRNA content of the cell, complete in haematopoietic cells for both targeted viruses and restricted to the ubiquitous microRNA target in non-haematopoietic cells (Figures 1A and 1B, Figure S1C). Cells lacking Dicer1 restored NS1 in all three viruses, establishing that the effect requires microRNA processing (Figure 1C).\n2. Despite more than ninety percent loss of NS1, growth curves in lung epithelial cells showed the silenced viruses reaching titres comparable to the scrambled control and to around ten to the seventh plaque forming units per millilitre, while a virus deleted for NS1 was substantially impaired (Figure 1D). Induction of the interferon-regulated gene Mx1 was unchanged in the silenced viruses and elevated only for the deletion virus (Figure S1D).\n3. The same result held in primary lung fibroblasts and bone marrow-derived macrophages, where NS1 fell in proportion to endogenous microRNA levels without affecting nucleoprotein (Figures 2A and 2B), and in mice, where silencing was confirmed in lungs of both interferon-competent and signalling-deficient animals and titres and interferon-regulated gene induction did not differ across viruses (Figures 2C to 2E, Figures S2A and S2B). The authors read this as low levels of NS1 being sufficient to antagonise the host response, which they describe as surprising.\n4. A virus expressing NEP from a 2A recoding site produced adequate NS1 but reduced NEP and lost roughly two logs of progeny despite normal synthesis of structural protein (Figures 3A and 3B). Independently, small interfering RNAs reducing NEP during wild-type infection produced about one log of attenuation at forty-eight hours with a reported p value of 0.01 (Figures S3B and S3C).\n5. A replication-incompetent influenza-like vector carrying an extra NEP copy in place of hemagglutinin raised NEP substantially, and co-infection of this vector with wild-type virus caused approximately two logs of attenuation relative to the scrambled vector (Figures 3C and 3D). Excess NEP is therefore as damaging as insufficient NEP.\n6. Optimising the segment 8 5 prime splice site inverted the expression ratio, reducing NS1 and raising NEP, confirmed by immunoblot and by RT-PCR of the spliced products (Figure 4A, Figure S4A). The optimised virus was attenuated by about two logs relative to the matched parental virus carrying the same amino acid change (Figure 4B), and the attenuation persisted in an NS1-complementing cell line, which the authors take as evidence that the defect is caused by excess NEP rather than by the accompanying loss of NS1 (Figure 4C, Figures S4B to S4D). In mice the optimised virus never exceeded one hundred plaque forming units per millilitre while infection was confirmed by quantitative RT-PCR for nucleoprotein (Figure 4D, Figure S4E).\n7. Nucleoprotein staining in infected lung epithelial cells appeared in the cytoplasm as early as five hours after infection with the splice-optimised virus while remaining nuclear with the parental virus, and at seven hours every optimised-virus infected cell showed cytoplasmic signal against roughly half of parental-virus infected cells (Figure 4E). The virus with reduced NEP showed the converse, a delay in export relative to wild type (Figure S4F).", "Mechanistic model": "The study establishes that the quantity of NEP, and the rate at which it accumulates, determines the outcome of infection, and it establishes that the segment 8 splice site sets that rate. It does not establish the molecular step at which mistimed export becomes lethal to the infection, and the export timing data are correlative with respect to the titre phenotype.\n\nThe model the authors propose is that the virus uses a deliberately poor 5 prime splice site as a molecular timer. Because all eight segments carry comparable promoters, the virus cannot delay a protein simply by transcribing its segment later. Instead it overproduces the NS1 transcript, from which NEP is generated as a minor spliced byproduct, so that NEP concentration rises slowly and crosses the threshold for ribonucleoprotein export only after replication has proceeded. On this reading the apparent overproduction of NS1 is not waste but the mechanism by which NEP accumulation is paced, and coupling the timer to NS1 rather than to a polymerase subunit, nucleoprotein or a surface protein is advantageous because excess NS1 is tolerated while excess of the others would perturb replication or assembly. The authors also note, citing their own and others' earlier work, that the transcription to replication switch is likely triggered by NEP accumulation, and they present their NEP manipulation data as corroborating that deregulated NEP produces aberrant replication. That link is offered as consistent with the data rather than tested here.", "Conceptual or technical advance": "The work reframes an apparent inefficiency in viral gene expression as a regulatory device, and it does so by making the splice site itself the experimental variable rather than the protein it produces. It also separates the two functions carried on one segment in a way that had not previously been possible in a replication-competent virus, using host microRNA targeting to knock down NS1 while leaving NEP intact, which yields the unexpected result that the interferon antagonist is produced far in excess of what is needed for its antagonist function, at least late in infection. The set of five complementary reagents built here, the microRNA-targeted viruses, the 2A recoded virus, the NEP-bearing replication-incompetent vector, the splice-optimised virus and its matched parental control, provides a way to titrate segment 8 output in either direction and is reusable for other questions about influenza gene expression stoichiometry.", "Relationship to the broader research program": "This paper continues a line in the corpus on how influenza A virus times its own life cycle without a transcriptional clock. It cites the laboratory's earlier work identifying influenza small viral RNA and the requirement for NEP in its production, and its discussion places NEP accumulation as the likely trigger for the switch from transcription to replication that the earlier paper addressed, which ties the two studies to one question approached from opposite ends. It also draws on the laboratory's ongoing use of microRNA targeting to control viral gene expression in vivo, a technique the group had applied to influenza in earlier work and which is used here as a precision knockdown rather than as an attenuation strategy. Reading the 2010 and 2013 papers together suggests a picture in which a single segment 8 product sits at the centre of both genome synthesis regulation and export timing, which is a category 3 synthesis emerging from the pair rather than a claim either paper makes alone.", "Related publications": "- Perez and colleagues, 2010, predecessor, from the same laboratory. Identified influenza small viral RNA and showed a requirement for NEP in its production, and is cited here in the discussion of what triggers the transcription to replication switch.\n- Varble and colleagues, 2010, and Langlois and colleagues, 2012, methodological foundation, from the same laboratory. Supply the modified NS vector and the microRNA-targeting approach used to silence NS1 in a replication-competent virus.\n- Perez and colleagues, 2009, methodological foundation, from the same laboratory. Cited among the demonstrations that influenza A virus can be placed under host microRNA control.\n- Manicassamy and colleagues, 2010, methodological foundation, from another laboratory. Source of the 2A recoding strategy used to uncouple NEP expression from splicing.\n- García-Sastre and colleagues, 1998, and Egorov and colleagues, 1998, predecessor, from other laboratories. Establish the attenuation of NS1-deficient influenza virus that frames the expectation this paper tests against.\n- Robb and colleagues, 2009, predecessor, from another laboratory. Cited for the regulatory role of NEP in transcription and replication of the influenza genome.", "Limitations and boundaries": "The link between mistimed ribonucleoprotein export and loss of infectivity is inferred rather than demonstrated. Nucleoprotein localisation is scored by immunofluorescence at two early time points, and no experiment shows that restoring correct export timing rescues the titre defect. The splice-optimised virus necessarily carries an amino acid substitution, which is controlled by a matched parental virus and by an NS1-complementing cell line but not by a construct that raises NEP without altering the segment 8 sequence. Excess NEP was delivered in one experiment by a replication-incompetent vector during co-infection at high multiplicity, a setting that differs from natural infection. The conclusion that low NS1 suffices for antagonism is bounded by the readouts used, which are interferon-regulated gene induction and virus titre in the cells, primary cultures and mouse strain tested, and by the fact that silencing removes more than ninety percent of NS1 rather than all of it, so residual NS1 activity cannot be excluded as the explanation. The animal work is a sublethal intranasal mouse model, and the study does not address transmission, pathology or other host species. The proposed connection between NEP accumulation and the transcription to replication switch is drawn from prior literature and is not tested here.", "Audience summaries": "### 25 words\n\nInfluenza splices one of its genes badly on purpose. The resulting slow build-up of an export protein times the infection, and correcting the splicing cripples the virus.\n\n### 75 words\n\nInfluenza A virus segment 8 makes the interferon antagonist NS1 as its main product and the nuclear export protein NEP through a weak splice site. Silencing NS1 by over ninety percent barely affected the virus, in cells or in mice. Changing NEP in either direction crippled it. Making the splice site efficient raised NEP, sent genome complexes to the cytoplasm hours early, and cost two logs of growth. The poor splice site is a timer.\n\n### 150 words\n\nInfluenza A virus has eight segments with comparable promoters and no obvious way to schedule its life cycle. Segment 8 encodes the interferon antagonist NS1 as the unspliced transcript and the nuclear export protein NEP through a 5 prime splice site used only ten to fifteen percent of the time. Inserting host microRNA target sites allowed NS1 to be reduced by more than ninety percent without touching NEP, and this had little effect on replication or on interferon-regulated gene induction in cell lines, primary cells or mice. Reducing NEP by a 2A recoding site or by small interfering RNA lowered titres, and raising NEP by an extra gene copy or by optimising the splice site lowered them by about two logs in culture and nearly abolished replication in mice. Nucleoprotein reached the cytoplasm hours early when NEP was abundant. The authors propose that inefficient splicing serves as a molecular timer pacing NEP accumulation." }, "discoveries": [ "claim-02" ], "relationships": [ { "from": "2010-perez-influenza-a-virus-generated-small-", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "follow-up", "evidence": "stated in the Related publications section of 2010-perez-influenza-a-virus-generated-small-" }, { "from": "2013-chua-influenza-a-virus-utilizes-subopti", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-chua-influenza-a-virus-utilizes-subopti" }, { "from": "2013-langlois-microrna-based-strategy-to-mitigat", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "companion", "evidence": "stated in the Related publications section of 2013-langlois-microrna-based-strategy-to-mitigat" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-schmid-a-versatile-rna-vector-for-deliver", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-schmid-a-versatile-rna-vector-for-deliver" }, { "from": "2014-varble-influenza-a-virus-transmission-bot", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-varble-influenza-a-virus-transmission-bot" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-in-vivo-rnai-screening-identifies-", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-in-vivo-rnai-screening-identifies-" }, { "from": "2019-tenoever-synthetic-virology-building-viruse", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2019-tenoever-synthetic-virology-building-viruse" }, { "from": "2023-oishi-archaeal-kink-turn-binding-protein", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2023-oishi-archaeal-kink-turn-binding-protein" } ], "canonical_url": "https://tenoeverlab.us/publications/2013-chua-influenza-a-virus-utilizes-subopti/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "reverse-genetics", "immunofluorescence-microscopy", "sirna-knockdown", "mirna-target-site-insertion", "site-directed-mutagenesis", "in-vivo-infection-route", "growth-curve", "2a-recoding", "single-cycle-influenza-vector" ] } }, { "id": "2013-cullen-is-rna-interference-a-physiologica", "slug": "2013-cullen-is-rna-interference-a-physiologica", "url": "/publications/2013-cullen-is-rna-interference-a-physiologica/", "title": "Is RNA Interference a Physiologically Relevant Innate Antiviral Immune Response in Mammals?", "authors": [ "Bryan R. Cullen", "Sara Cherry", "Benjamin R. tenOever" ], "author_count": 3, "first_author": "Bryan R. Cullen", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Bryan R. Cullen" ], "tenoever_position": 3, "tenoever_role": "senior", "contribution_character": "collaborative", "year": 2013, "journal": "Cell Host & Microbe", "volume": "14", "issue": "4", "pages": "374-378", "doi": "10.1016/j.chom.2013.09.011", "doi_url": "https://doi.org/10.1016/j.chom.2013.09.011", "pmid": "24139396", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/24139396/", "pmcid": null, "pmc_url": null, "pmc_pdf_url": null, "publication_type": "review", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense" ], "themes": [ "does-mammalian-antiviral-rnai-exist" ], "pathogens": [ "nodamura virus", "encephalomyocarditis virus", "influenza A virus", "Ebola virus", "human immunodeficiency virus 1" ], "viral_families": [ "Nodaviridae", "Picornaviridae", "Orthomyxoviridae", "Filoviridae", "Retroviridae" ], "host_species": [ "mouse", "hamster", "human", "insect", "nematode", "plant" ], "technologies": [ "small RNA deep sequencing", "northern blot", "Argonaute knockout cells", "Dicer deficient cells", "viral suppressor mutant viruses" ], "biological_systems": [ "mouse embryonic stem cells", "embryoid bodies", "baby hamster kidney cells", "mouse embryonic fibroblasts", "suckling mice", "mouse oocytes" ], "key_concepts": [ "antiviral RNA interference", "viral suppressor of RNA silencing", "Dicer", "Argonaute and RISC", "virus-derived small interfering RNAs", "type I interferon as an alternative antiviral system", "pluripotency and RNAi competence", "criteria for demonstrating antiviral RNAi" ], "keywords": [ "RNA interference", "antiviral immunity", "embryonic stem cells", "nodamura virus B2", "Dicer", "Argonaute", "interferon", "viral small RNAs", "minireview" ], "one_sentence_contribution": "A critical appraisal of the evidence for antiviral RNA interference in mammals, accepting that mouse embryonic stem cells generate virus-derived small interfering RNAs while finding the somatic cell case unproven, because the viral proteins invoked also antagonize interferon.", "summary_25": "A 2013 Minireview weighing new claims that RNA interference fights viruses in mammals, accepting the stem cell evidence and finding the somatic cell case unproven.", "summary_75": "RNA interference protects plants and insects from viruses, but whether mammals use it the same way has been disputed. Reviewing three 2013 studies, these authors accept that mouse embryonic stem cells generate virus-derived small silencing RNAs, and argue that the parallel claim for ordinary body cells is not yet supported, because the viral proteins used to make the case also block interferon, so the two explanations have not been separated experimentally.", "summary_150": "Two 2013 Science papers reopened the question of whether RNA interference is an antiviral immune pathway in mammals, and this Minireview assesses them alongside a study in the same Cell Host and Microbe issue. The authors find the embryonic stem cell evidence convincing at the level of small RNA production, since encephalomyocarditis virus yields abundant Dicer-dependent small interfering RNAs in these cells and a nodavirus lacking its B2 protein is partly rescued when all four Argonaute proteins are removed. They are unpersuaded for somatic cells and for infected mice, because the inference there depends on B2 and on Ebola VP35 acting selectively against silencing, when both bind double-stranded RNA and also antagonize interferon, and the mutations used remove both activities together. They set out the experiments that would settle it, including challenge of interferon-deficient mice and direct demonstration that the small RNAs silence viral messenger RNA.", "citation": "Cullen BR, Cherry S, tenOever BR. Is RNA Interference a Physiologically Relevant Innate Antiviral Immune Response in Mammals? Cell Host & Microbe. 2013. 14(4), 374-378. DOI 10.1016/j.chom.2013.09.011. PMID 24139396. No PMCID recorded in the inventory. Article type is Minireview, labeled as such in the running head of the article itself. It presents no new experimental data, and every experimental result discussed in this record was produced by the laboratory named with it, not by the authors of this piece.", "sections": { "Citation": "Cullen BR, Cherry S, tenOever BR. Is RNA Interference a Physiologically Relevant Innate Antiviral Immune Response in Mammals? Cell Host & Microbe. 2013. 14(4), 374-378.\n\nDOI 10.1016/j.chom.2013.09.011. PMID 24139396. No PMCID recorded in the inventory.\n\nArticle type is Minireview, labeled as such in the running head of the article itself. It presents no new experimental data, and every experimental result discussed in this record was produced by the laboratory named with it, not by the authors of this piece.", "One-sentence contribution": "A critical appraisal of the evidence for antiviral RNA interference in mammals, accepting that mouse embryonic stem cells generate virus-derived small interfering RNAs while finding the somatic cell case unproven, because the viral proteins invoked also antagonize interferon.", "Executive summary": "RNA interference is an established antiviral mechanism in plants, nematodes, and arthropods, and whether it serves the same role in mammals had appeared settled in the negative until two 2013 Science papers reopened it. This Minireview evaluates those two papers, from the Voinnet group (Maillard and colleagues) and the Ding group (Li and colleagues), together with a study from the Sullivan group (Seo and colleagues) published in the same issue of Cell Host and Microbe. The authors accept that the evidence for small interfering RNA production in mouse embryonic stem cells is strong, since virus-derived small RNAs of the expected size and phasing were detected after encephalomyocarditis virus infection, were lost in Dicer negative cells, and a nodamura virus lacking its B2 suppressor was partly rescued in cells lacking all four Argonaute proteins. They are considerably more cautious about somatic cells and about the in vivo data, because the central inference there depends on treating the nodavirus B2 protein and the Ebola virus VP35 protein as selective inhibitors of small interfering RNA production, when both are double-stranded RNA binding proteins that also antagonize the interferon response, and the point mutation used to inactivate B2 suppressor function also abolishes double-stranded RNA binding. The authors set out the experiment they consider decisive, which is genetic rescue of a suppressor-deficient virus in RNA interference-compromised somatic cells combined with direct evidence of viral messenger RNA silencing, and note that it has not been done.", "Scientific context": "The article assembles the state of the field as of 2013. Dicer cleavage of long double-stranded RNA into small interfering RNAs, loading of one strand into an Argonaute-containing silencing complex, and the resulting cleavage of complementary RNA are established in invertebrates and plants, where viral suppressors of RNA silencing have evolved repeatedly, the nodavirus B2 protein being among the best characterized by the Ding and Schneemann groups. In mammals the picture had been different. Deep sequencing across many virus and host combinations, notably the survey by Parameswaran and colleagues, either failed to find virus-derived small interfering RNAs or found them at very low levels, and long double-stranded RNA transfected into mammalian somatic cells does not yield functional small interfering RNAs but instead triggers the interferon response, which is absent from most organisms that use RNA interference antivirally. Against this, work from the Filipowicz, Hannon and Svoboda groups had shown that germ cells, embryonic stem cells, and some embryonal carcinoma lines do process long double-stranded RNA productively, and interferon inducibility is reported to be absent in those same cell types, which the authors note as a suggestive complementarity rather than a demonstrated relationship.", "Central question": "Does RNA interference constitute a physiologically relevant antiviral immune response in mammals, and specifically, do the recently reported virus-derived small interfering RNAs in embryonic stem cells, in cultured somatic cells, and in infected mice actually direct the silencing of viral RNA and restrict virus replication?", "Experimental strategy": "The strategy of the piece is evidentiary rather than experimental. The authors take a standard for what would establish antiviral RNA interference, quoted from Maillard and colleagues, namely genetic rescue of a suppressor-deficient virus in host cells whose RNA interference machinery is compromised, and they add a second requirement of their own, direct demonstration that the detected small RNAs silence viral messenger RNAs. They then apply that standard in turn to the embryonic stem cell data, the cultured somatic cell data, and the in vivo data, and in each case identify which of the two requirements is met and which alternative explanation remains open. A recurring analytical move is to ask whether a phenotype attributed to loss of suppressor activity against RNA interference could equally be explained by loss of double-stranded RNA binding and therefore loss of interferon antagonism.", "Key findings": "The numbered entries below are the findings of the cited laboratories as the Minireview reports them, followed by the assessment the authors of the Minireview offer.\n\n1. Maillard and colleagues, from the Voinnet group, detected encephalomyocarditis virus-derived small RNAs of the expected 22 nucleotide size in mouse embryonic stem cells by both deep sequencing and northern blot, derived in near equal amounts from both strands of this positive-sense virus and showing the phased register characteristic of processive Dicer cleavage. The small RNAs were lost in Dicer negative embryonic stem cells. The Minireview accepts this as establishing Dicer-dependent production of virus-derived small interfering RNAs in these cells.\n2. Maillard and colleagues found few virus-derived small interfering RNAs after infection of embryonic stem cells with wild-type nodamura virus and higher levels with a B2-deficient mutant, and that mutant replicated to higher levels in embryonic stem cells lacking all four Argonaute proteins. The Minireview reads this as consistent with B2 acting by blocking Dicer cleavage, while noting that viral RNA cleavage and silencing were not directly assessed, so antiviral RNA interference was not itself demonstrated.\n3. Maillard and colleagues also reported that loss of Dicer in encephalomyocarditis virus-infected embryonic stem cells did not enhance virus replication. The Minireview flags this as a result that does not fit a simple protective model.\n4. Differentiation of embryonic stem cells into embryoid bodies substantially reduced encephalomyocarditis virus-derived small interfering RNA levels, though low levels persisted, again from Maillard and colleagues. The molecular basis is stated to be unknown.\n5. Li and colleagues, from the Ding group, reported that baby hamster kidney cells infected with wild-type nodamura virus produced no detectable viral small interfering RNAs while the B2-deficient mutant produced small RNAs of the characteristic size, many from the negative strand, but at hundreds of reads out of millions. Replication of the B2-deficient mutant in these cells was rescued by expression of B2 and also by expression of Ebola virus VP35.\n6. Li and colleagues found that a single B2 point mutation replacing arginine at position 59 with glutamate strongly attenuated the virus. The Minireview points out that this mutation also abolishes double-stranded RNA binding, so it cannot distinguish suppression of RNA interference from interferon antagonism, and that VP35 is itself a double-stranded RNA binding protein known to block the interferon response.\n7. Li and colleagues detected viral small interfering RNAs in suckling mice infected with the B2-deficient virus at levels visible by northern blot, and both the B2-deficient and B2 point mutant viruses were highly attenuated in vivo. The Minireview treats the small RNA detection as a real observation and treats the attribution of attenuation to loss of small interfering RNA suppression as unproven, since the decisive experiment of challenging interferon-deficient mice with the mutant viruses had not been performed.\n8. Seo and colleagues, from the Sullivan group, reported in the same issue that viral infection drives posttranslational modification and inactivation of Argonaute, with the silencing complex nonfunctional as early as 8 hours after infection in vivo, and proposed that this relieves silencing of host antiviral genes. The Minireview notes that this is hard to reconcile with RNA interference operating as a genuine antiviral defense in somatic cells over the same interval.\n9. Work from the tenOever laboratory and from the Steel laboratory, cited here rather than reported, had found that two widely proposed viral suppressors, influenza virus NS1 and human immunodeficiency virus 1 Tat, do not in fact block RNA interference. The Minireview also cites the observation by García-Sastre and colleagues that influenza lacking NS1 remains lethal in interferon-defective mice, which the authors read as arguing that any RNA interference response present is insufficient to restrict replication during a physiological infection.", "Mechanistic model": "The article does not establish a mechanism, and its purpose is the opposite, namely to specify which mechanistic claims the available data can and cannot support. The authors accept a Dicer-dependent, Argonaute-dependent pathway that generates virus-derived small interfering RNAs in mouse embryonic stem cells and is lost on differentiation, with the molecular basis of that loss unknown. For somatic cells they leave the question open and identify the specific ambiguity that prevents resolution, which is that the nodavirus B2 protein and Ebola virus VP35 bind double-stranded RNA and therefore have at least two separable potential functions, blocking Dicer access and blocking pattern recognition receptor-driven interferon induction, and that the mutants used do not separate them. The figure accompanying the article presents interferon, microRNA, and RNA interference as three systems used to different degrees across taxa, with somatic mammalian cells shown with uncertain RNA interference usage, and this is offered as a framework rather than as a result. A possible reciprocal relationship between interferon competence and RNA interference competence is raised as a suggestion and is not tested anywhere in the work discussed.", "Conceptual or technical advance": "The lasting contribution is a set of evidentiary criteria. The article states plainly what an experiment must show before a virus-derived small RNA can be called an antiviral effector, namely genetic rescue of a suppressor-deficient virus in cells whose RNA interference machinery is genetically compromised, combined with direct evidence that the small RNAs silence viral RNA, and it names the specific control that separates suppression of silencing from interferon antagonism when double-stranded RNA binding proteins are involved. It also identifies cell state rather than viral countermeasure as the more likely explanation for why embryonic stem cells and somatic cells differ, on the grounds that a strictly viral explanation cannot account for encephalomyocarditis virus producing abundant small interfering RNAs in stem cells and none in somatic cells. Those criteria make the disagreement testable rather than rhetorical.", "Relationship to the broader research program": "The question of whether mammalian cells mount antiviral RNA interference recurs throughout this corpus, and this Minireview marks the position taken in 2013, which is skeptical for somatic cells and accepting for embryonic stem cells. The tenOever laboratory contribution to the argument is the demonstration by Perez and colleagues that influenza NS1 does not suppress RNA interference, together with the laboratory's use of the small RNA machinery as an engineering tool, cited here as the ability to build viruses that express small RNAs against host messenger RNAs. Both of those threads run forward into later corpus entries on microRNA-targeted viruses and on RNase III biology. Setting this article beside later primary work from the laboratory on RNA interference and on RNase III nucleases as antiviral effectors would be category 3 synthesis, and it should be assembled centrally rather than asserted from this article, which by its nature reports the state of an argument at one moment and is not evidence about work published afterwards.", "Related publications": "- Perez et al. 2009, microRNA-mediated species-specific attenuation of influenza A virus. Predecessor from the tenOever laboratory, cited here for the finding that influenza NS1 does not block RNA interference.\n- tenOever 2013, RNA viruses and the host microRNA machinery. Review or synthesis by the tenOever laboratory, cited here for the engineering of viruses that express functional small RNAs.\n- Maillard et al. 2013, antiviral RNA interference in mammalian cells. Subject of the review, from the Voinnet group, not laboratory work.\n- Li et al. 2013, RNA interference functions as an antiviral immunity mechanism in mammals. Subject of the review, from the Ding group, not laboratory work.\n- Seo et al. 2013, intracellular antiviral signaling mediates inhibition of RNAi in mammalian cells. Companion in the same issue, from the Sullivan group, not laboratory work.", "Limitations and boundaries": "The article contains no new data and its conclusions are only as good as the three studies it evaluates, all of which rest heavily on one virus, nodamura virus, and one suppressor protein, B2, in mouse and hamster systems. The authors are explicit that the functional relevance of viral small interfering RNAs in mammalian somatic cells remains an open question, that the in vivo attenuation data do not establish that small interfering RNAs are antiviral, and that a detailed identification of which cell types in an animal produce viral small RNAs is still needed. Nothing in the article addresses human viral pathogens directly, and the authors name that as the critical unaddressed question. As a Minireview it also reflects the judgment of its three authors at the time of writing and should not be read as a consensus position of the field or as evidence about experiments published later.", "Audience summaries": "### 25 words\n\nA 2013 Minireview weighing new claims that RNA interference fights viruses in mammals, accepting the stem cell evidence and finding the somatic cell case unproven.\n\n### 75 words\n\nRNA interference protects plants and insects from viruses, but whether mammals use it the same way has been disputed. Reviewing three 2013 studies, these authors accept that mouse embryonic stem cells generate virus-derived small silencing RNAs, and argue that the parallel claim for ordinary body cells is not yet supported, because the viral proteins used to make the case also block interferon, so the two explanations have not been separated experimentally.\n\n### 150 words\n\nTwo 2013 Science papers reopened the question of whether RNA interference is an antiviral immune pathway in mammals, and this Minireview assesses them alongside a study in the same Cell Host and Microbe issue. The authors find the embryonic stem cell evidence convincing at the level of small RNA production, since encephalomyocarditis virus yields abundant Dicer-dependent small interfering RNAs in these cells and a nodavirus lacking its B2 protein is partly rescued when all four Argonaute proteins are removed. They are unpersuaded for somatic cells and for infected mice, because the inference there depends on B2 and on Ebola VP35 acting selectively against silencing, when both bind double-stranded RNA and also antagonize interferon, and the mutations used remove both activities together. They set out the experiments that would settle it, including challenge of interferon-deficient mice and direct demonstration that the small RNAs silence viral messenger RNA." }, "discoveries": [ "claim-08" ], "relationships": [ { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "review or synthesis", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "review or synthesis", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" }, { "from": "2015-aguado-microrna-function-is-limited-to-cy", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "review or synthesis", "evidence": "stated in the Related publications section of 2015-aguado-microrna-function-is-limited-to-cy" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2016-tenoever-the-evolution-of-antiviral-defense", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "review or synthesis", "evidence": "stated in the Related publications section of 2016-tenoever-the-evolution-of-antiviral-defense" }, { "from": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2017-aguado-rnase-iii-nucleases-from-diverse-k" } ], "canonical_url": "https://tenoeverlab.us/publications/2013-cullen-is-rna-interference-a-physiologica/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "emcv", "ebola-virus", "hiv-1", "nodamura-virus" ], "technologies": [ "small-rna-seq", "northern-blot", "rnai-machinery-knockout-cells", "vsr-mutant-viruses" ] } }, { "id": "2013-langlois-microrna-based-strategy-to-mitigat", "slug": "2013-langlois-microrna-based-strategy-to-mitigat", "url": "/publications/2013-langlois-microrna-based-strategy-to-mitigat/", "title": "MicroRNA-based strategy to mitigate the risk of gain-of-function influenza studies", "authors": [ "Ryan A Langlois", "Randy A Albrecht", "Brian Kimble", "Troy Sutton", "Jillian S Shapiro", "Courtney Finch", "Matthew Angel", "Mark A Chua", "Ana Silvia Gonzalez-Reiche", "Kemin Xu", "Daniel Perez", "Adolfo García-Sastre", "Benjamin R tenOever" ], "author_count": 13, "first_author": "Ryan A Langlois", "senior_authors": [ "Benjamin R tenOever" ], "corresponding_authors": [ "Daniel Perez", "Adolfo García-Sastre", "Benjamin R tenOever" ], "tenoever_position": 13, "tenoever_role": "senior", "contribution_character": "co-led", "year": 2013, "journal": "Nature Biotechnology", "volume": "31", "issue": "9", "pages": "844-847", "doi": "10.1038/nbt.2666", "doi_url": "https://doi.org/10.1038/nbt.2666", "pmid": "23934176", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/23934176/", "pmcid": "PMC3808852", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808852/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808852/pdf/", "publication_type": "methods/resource", "declared_conflicts": null, "research_areas": [ "programmable-virology" ], "themes": [ "microrna-mediated-viral-attenuation", "molecular-biocontainment" ], "pathogens": [ "influenza A virus", "influenza A virus H5N1", "influenza A virus H3N2" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "human", "mouse", "ferret", "dog" ], "technologies": [ "small RNA deep sequencing", "influenza A virus reverse genetics", "microRNA target site engineering", "northern blot", "quantitative PCR", "plaque assay", "ferret transmission study" ], "biological_systems": [ "A549 cells", "MDCK cells", "Calu3 cells", "primary human nasal epithelial cells", "primary ferret lung", "C57BL/6 mouse", "ferret" ], "key_concepts": [ "molecular biocontainment", "species-specific microRNA expression", "microRNA target site insertion", "viral tropism restriction", "gain-of-function research biosafety", "hemagglutinin segment engineering", "packaging signal duplication", "ferret transmission model", "miR-192", "viral attenuation" ], "keywords": [ "miR-192", "influenza A virus", "biocontainment", "gain-of-function", "ferret transmission", "hemagglutinin", "microRNA targeting", "biosafety", "H5N1", "reverse genetics" ], "one_sentence_contribution": "Engineering target sites for miR-192, a microRNA abundant in human and mouse respiratory tissue but absent from the ferret respiratory tract, into the influenza A virus hemagglutinin segment attenuates the virus in mice while leaving replication and transmission in ferrets intact, providing a genetic layer of biocontainment for transmission studies.", "summary_25": "A small RNA present in human and mouse airways but not in ferret airways was used to cripple influenza A virus in humans while preserving ferret transmission experiments.", "summary_75": "Influenza transmission experiments rely on ferrets, which raises concern about accidental human infection. Sequencing of small RNAs across species identified miR-192 as abundant in human and mouse respiratory tissue and absent from ferret lung. Four miR-192 target sites inserted after the hemagglutinin stop codon silenced the virus in human cells and rendered it harmless in mice at ten times a lethal dose, while leaving replication and airborne transmission in ferrets unchanged.", "summary_150": "Demonstrations that few hemagglutinin substitutions can confer ferret-to-ferret airborne transmission of H5N1 prompted debate about the biosafety of such experiments. Small RNA deep sequencing of human A549 cells, primary ferret lung and MDCK cells, followed by northern blot confirmation, identified miR-192 as expressed in human and murine respiratory tissue but not in the two carnivore-derived sources. Four perfectly complementary miR-192 sites were inserted into a duplicated packaging region downstream of the hemagglutinin stop codon, leaving the protein and packaging signal intact and tying the safety element to the segment that carries transmission determinants. The engineered virus was silenced in miR-192-expressing cells, replicated normally where the microRNA is absent, and caused no disease in mice even at tenfold the lethal dose. An H3N2 version infected, replicated and transmitted in ferrets by direct and respiratory contact like unmodified virus, and no escape variants were recovered from either animal system.", "citation": "Langlois RA, Albrecht RA, Kimble B, Sutton T, Shapiro JS, Finch C, Angel M, Chua MA, Gonzalez-Reiche AS, Xu K, Perez D, García-Sastre A, tenOever BR. MicroRNA-based strategy to mitigate the risk of gain-of-function influenza studies. Nature Biotechnology. 2013. Volume 31, issue 9, pages 844-847. DOI 10.1038/nbt.2666. PMID 23934176. PMCID PMC3808852.", "sections": { "Citation": "Langlois RA, Albrecht RA, Kimble B, Sutton T, Shapiro JS, Finch C, Angel M, Chua MA, Gonzalez-Reiche AS, Xu K, Perez D, García-Sastre A, tenOever BR. MicroRNA-based strategy to mitigate the risk of gain-of-function influenza studies. Nature Biotechnology. 2013. Volume 31, issue 9, pages 844-847. DOI 10.1038/nbt.2666. PMID 23934176. PMCID PMC3808852.", "One-sentence contribution": "Engineering target sites for miR-192, a microRNA abundant in human and mouse respiratory tissue but absent from the ferret respiratory tract, into the influenza A virus hemagglutinin segment attenuates the virus in mice while leaving replication and transmission in ferrets intact, providing a genetic layer of biocontainment for transmission studies.", "Executive summary": "Demonstrations that a small number of amino acid changes in hemagglutinin can make H5N1 influenza A virus transmissible between ferrets raised the question of how experiments of that kind can be conducted with an additional safeguard beyond physical containment. The authors asked whether the natural divergence in microRNA expression between the experimental host and humans can be used to build a virus that behaves normally in the model species but is crippled in human cells.\n\nSmall RNA deep sequencing of human A549 lung cells, primary ferret lung and MDCK cells identified candidate microRNAs abundant in human cells and scarce in the two carnivore-derived sources. Northern blotting narrowed the candidates and miR-192 was selected, since it was also detectable in mouse lung and in human bronchial, alveolar and nasal epithelium. Four fully complementary miR-192 target sites were placed in a duplicated packaging region downstream of the hemagglutinin stop codon, leaving the hemagglutinin coding sequence untouched.\n\nThe targeted virus was silenced in cells expressing miR-192 and replicated normally in cells that do not. In mice it caused no morbidity or mortality even at ten times the lethal dose of the parental virus. In ferrets an H3N2 version carrying the same modification infected, replicated and transmitted by direct and by respiratory contact indistinguishably from controls, and sequencing recovered no escape variants from either animal system.", "Scientific context": "Reports that airborne transmission of H5N1 between ferrets can be conferred by a small number of hemagglutinin substitutions prompted a broad debate about whether such gain-of-function experiments should be performed at all, including a voluntary moratorium. Physical containment at enhanced biosafety level 3 was the existing safeguard. Separately, several groups had shown that microRNA target sites inserted into a viral genome can restrict a virus to tissues lacking the relevant microRNA, work cited here from the tenOever laboratory on hematopoietic-specific targeting of influenza A virus and on species-specific attenuation, and from other laboratories on flaviviruses and on attenuated virus vaccine design. What had not been done was to exploit the difference in microRNA expression between the experimental animal and the human, so that the same virus is permissive in the model and restricted in the species of concern. The paper frames this as adding a molecular layer to existing physical precautions rather than replacing them.", "Central question": "Can a microRNA expressed in human and mouse respiratory tissue but absent from the ferret respiratory tract be used to restrict influenza A virus in human cells without compromising the replication and transmission behavior that makes the ferret a useful transmission model?", "Experimental strategy": "The strategy has a discovery step and a validation step, and the validation is deliberately two-sided. Discovery was unbiased profiling of the small RNA pool in human lung epithelium, primary ferret lung and MDCK cells, followed by northern blot confirmation, because deep sequencing read counts across species can be misleading when reference annotation differs. The two non-human sources are both from the order Carnivora, which the authors note, and MDCK cells matter because influenza A virus stocks are grown in them, so a candidate microRNA has to be absent there for the virus to be propagable.\n\nThe engineering step placed target sites in the hemagglutinin segment rather than in nucleoprotein or NS1, which earlier studies had used, specifically so that the segment carrying the transmission-determining protein cannot be separated from the safety element by reassortment. Duplicating the 5 prime packaging sequence creates an untranslated region after the stop codon in which target sites can sit without altering the hemagglutinin protein or the packaging signal.\n\nValidation used a low-pathogenicity H5 construct with the polybasic cleavage site removed for the cell culture and mouse work, which permits lethality studies under appropriate containment, and the seasonal H3N2 Wyoming strain for ferret transmission, since aerosol transmission of that strain in ferrets is established. Escape was assessed by plaque purification and sequencing from both animal systems.", "Key findings": "1. Small RNA sequencing of A549 human lung cells, primary ferret lung and MDCK cells identified miR-138, miR-193b and miR-192 as candidates abundant in human cells and low or absent in the other two (Figure 1). Northern blotting contradicted the sequencing for miR-193b, which was substantially expressed in ferret lung, leaving miR-138 and miR-192. The discrepancy is reported directly and the authors treat the blot as the arbiter.\n\n2. miR-192 but not miR-138 was detectable in murine lung at levels the authors judged sufficient for silencing, and miR-192 was robustly expressed in human bronchial and alveolar lines and in primary human nasal epithelial cells, with nasal cells carrying approximately twofold more than alveolar cells (Supplementary Figure 1).\n\n3. In a single cycle of infection, hemagglutinin and nucleoprotein expression from the wild-type, scrambled control and 192t viruses were comparable in MDCK cells, while hemagglutinin from the 192t virus was ablated in MDCK cells engineered to express miR-192 and in A549 cells (Figure 2b). The authors read this as sequence-specific post-transcriptional silencing of the hemagglutinin segment.\n\n4. Over multiple cycles, wild-type and control viruses grew equivalently regardless of miR-192, while 192t replication was blunted in MDCK cells expressing miR-192 and in A549 cells (Figure 2c).\n\n5. Mice infected intranasally with 100 plaque forming units of the 192t H5 HAlo virus showed no weight loss and no mortality, while wild-type and control infections were uniformly lethal by day eight. A tenfold higher dose of 192t still produced no morbidity or mortality (Figure 3a and 3b). Pulmonary titers and viral hemagglutinin and nucleoprotein protein were reduced at days three and five (Figure 3c and 3d).\n\n6. All 17 plaques recovered from mouse lung at day five retained perfect miR-192 target sites, and virus sequenced from ferrets at day seven after aerosol transmission showed no excision events. The observation is an absence of detected escape at these sampling depths. The authors' statement that these results demonstrate the safety of the platform goes beyond what the sampling establishes and should be read as their interpretation.\n\n7. An H3N2 Wyoming virus engineered the same way showed roughly 100-fold reduced growth in A549 cells relative to control (Supplementary Figure 4).\n\n8. In ferrets, wild-type, control and 192t H3N2 viruses all produced high nasal wash titers from day one that declined over ten days in directly inoculated animals, and all three transmitted to naive animals by direct contact and by respiratory contact (Figure 4). This is consistent with the absence of miR-192 in ferret lung and is the central requirement for the approach to be usable.", "Mechanistic model": "The silencing mechanism itself is not dissected here and the paper does not set out to establish one. The working model, supported by the cell culture data and consistent with the earlier work the authors cite, is that endogenous miR-192 loaded into the RNA-induced silencing complex recognizes the fully complementary sites in the hemagglutinin messenger RNA and directs its post-transcriptional destruction, so that hemagglutinin protein fails to accumulate and multicycle replication collapses in cells that express the microRNA. Placement downstream of the stop codon and upstream of the duplicated packaging signal is intended to leave protein sequence and segment packaging unaffected, and the equivalence of the engineered and wild-type viruses in miR-192-negative cells is the evidence offered that this intention was met.\n\nWhat the data constrain is the phenotype in each host. What they do not address is whether silencing is by cleavage or by translational repression, whether the negative-sense genomic RNA is engaged at all, and what fraction of the attenuation in mice is cell-intrinsic silencing as opposed to altered kinetics of immune engagement.", "Conceptual or technical advance": "Biocontainment becomes a property that can be written into the viral genome rather than only into the building. The specific technical contributions are the choice of a microRNA defined by a cross-species expression difference between the experimental animal and the species of concern, and the placement of target sites in the hemagglutinin segment through duplication of the packaging signal, which couples the safety element to the segment that carries the transmission phenotype under study. The authors propose that the approach generalizes to other pathogens for which gain-of-function work is contemplated, naming Ebola virus, SARS coronavirus and henipaviruses, and that targeting multiple segments or multiple microRNAs would further reduce escape. Both of these are stated as prospects and are not tested here.", "Relationship to the broader research program": "This is a direct extension of a line of work in the tenOever laboratory on inserting microRNA response elements into influenza A virus to control where the virus can replicate. Perez and colleagues 2009 established species-specific attenuation using this approach, and Langlois and colleagues 2012 used hematopoietic-specific targeting to ask what cell types must be infected for antiviral immunity to be induced. Pham, Langlois and tenOever 2012 applied the same logic to dengue virus dissemination. The technique is used in the corpus both as a safety measure and as a tool for cell-type-restricted infection, and this paper is the biosafety application of it. Category 3 synthesis, visible across those papers together, is that the laboratory treated engineered microRNA response elements as a general control layer over viral tropism rather than as a single-purpose attenuation trick.", "Related publications": "- Perez and colleagues 2009, Nature Biotechnology, MicroRNA-mediated species-specific attenuation of influenza A virus. Predecessor. Established species-specific microRNA targeting of influenza A virus, cited here as the direct precedent.\n- Langlois and colleagues 2012, PNAS, Hematopoietic-specific targeting of influenza A virus. Predecessor and methodological foundation. Source of the deep sequencing, northern blot and microRNA-expressing MDCK methods used here, and the prior demonstration of cell-type-restricted targeting.\n- Chua and colleagues 2013, Cell Reports, on influenza A virus suboptimal splicing. Companion. Cited as the source of the alternative NS1 target site placement strategy that this paper chose not to use.\n- Pham, Langlois and tenOever 2012, PLoS Pathogens, on dengue virus dissemination. Application. Extends the same targeting logic to a different virus family.", "Limitations and boundaries": "The approach depends entirely on an expression difference that holds in the tissues that matter, and the evidence for absence of miR-192 in ferret is from lung and from a small set of samples, so restriction of the virus in other ferret tissues or at other ages is not addressed. Silencing was demonstrated with four fully complementary sites, which is the most favorable configuration, and no titration of site number or of mismatch tolerance is presented. Escape was assessed from 17 plaques at a single time point in mice and from sequencing after transmission in ferrets, which bounds the escape frequency only loosely, and the ferret arm did not use a miR-192-expressing species in which selective pressure for escape would exist. The mouse and cell culture work used an H5 construct with the polybasic cleavage site removed, so behavior of a highly pathogenic H5N1 virus carrying the same modification is not tested, and the ferret transmission work used a seasonal H3N2 strain rather than an H5N1 strain of the kind that motivated the study. The human evidence is entirely from cell lines and primary cells, since no human challenge is possible, so restriction in an infected person is an inference from those systems. Mice and ferrets were not randomized and investigators were not blinded, which the methods state. Transmission experiments were performed at biosafety level 2 plus with the seasonal strain, so the containment regime under which a gain-of-function experiment would actually run was not itself evaluated.", "Audience summaries": "### 25 words\n\nA small RNA present in human and mouse airways but not in ferret airways was used to cripple influenza A virus in humans while preserving ferret transmission experiments.\n\n### 75 words\n\nInfluenza transmission experiments rely on ferrets, which raises concern about accidental human infection. Sequencing of small RNAs across species identified miR-192 as abundant in human and mouse respiratory tissue and absent from ferret lung. Four miR-192 target sites inserted after the hemagglutinin stop codon silenced the virus in human cells and rendered it harmless in mice at ten times a lethal dose, while leaving replication and airborne transmission in ferrets unchanged.\n\n### 150 words\n\nDemonstrations that few hemagglutinin substitutions can confer ferret-to-ferret airborne transmission of H5N1 prompted debate about the biosafety of such experiments. Small RNA deep sequencing of human A549 cells, primary ferret lung and MDCK cells, followed by northern blot confirmation, identified miR-192 as expressed in human and murine respiratory tissue but not in the two carnivore-derived sources. Four perfectly complementary miR-192 sites were inserted into a duplicated packaging region downstream of the hemagglutinin stop codon, leaving the protein and packaging signal intact and tying the safety element to the segment that carries transmission determinants. The engineered virus was silenced in miR-192-expressing cells, replicated normally where the microRNA is absent, and caused no disease in mice even at tenfold the lethal dose. An H3N2 version infected, replicated and transmitted in ferrets by direct and respiratory contact like unmodified virus, and no escape variants were recovered from either animal system." }, "discoveries": [ "claim-10" ], "relationships": [ { "from": "2013-langlois-microrna-based-strategy-to-mitigat", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2013-langlois-microrna-based-strategy-to-mitigat" }, { "from": "2013-langlois-microrna-based-strategy-to-mitigat", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "companion", "evidence": "stated in the Related publications section of 2013-langlois-microrna-based-strategy-to-mitigat" }, { "from": "2013-langlois-microrna-based-strategy-to-mitigat", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "application", "evidence": "stated in the Related publications section of 2013-langlois-microrna-based-strategy-to-mitigat" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2013-langlois-microrna-based-strategy-to-mitigat", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2019-tenoever-synthetic-virology-building-viruse", "to": "2013-langlois-microrna-based-strategy-to-mitigat", "relationship": "related", "evidence": "stated in the Related publications section of 2019-tenoever-synthetic-virology-building-viruse" } ], "canonical_url": "https://tenoeverlab.us/publications/2013-langlois-microrna-based-strategy-to-mitigat/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "rt-qpcr", "plaque-assay", "reverse-genetics", "small-rna-seq", "mirna-target-site-insertion", "northern-blot", "animal-transmission-model" ] } }, { "id": "2013-tenoever-rna-viruses-and-the-host-microrna-", "slug": "2013-tenoever-rna-viruses-and-the-host-microrna-", "url": "/publications/2013-tenoever-rna-viruses-and-the-host-microrna-/", "title": "RNA viruses and the host microRNA machinery", "authors": [ "Benjamin R. tenOever" ], "author_count": 1, "first_author": "Benjamin R. tenOever", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 1, "tenoever_role": "sole", "contribution_character": "lab-led", "year": 2013, "journal": "Nature Reviews Microbiology", "volume": "11", "issue": "3", "pages": "169-180", "doi": "10.1038/nrmicro2971", "doi_url": "https://doi.org/10.1038/nrmicro2971", "pmid": "23411862", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/23411862/", "pmcid": null, "pmc_url": null, "pmc_pdf_url": null, "publication_type": "review", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense" ], "themes": [ "does-mammalian-antiviral-rnai-exist", "evolution-of-antiviral-defense" ], "pathogens": [ "influenza A virus", "poliovirus", "dengue virus", "vesicular stomatitis virus", "West Nile virus", "hepatitis C virus", "bovine leukaemia virus", "herpesviruses", "poxviruses", "adenovirus" ], "viral_families": [ "Orthomyxoviridae", "Picornaviridae", "Flaviviridae", "Rhabdoviridae", "Retroviridae", "Herpesviridae", "Poxviridae", "Adenoviridae" ], "host_species": [ "human", "mouse", "chicken" ], "technologies": [ "small RNA deep sequencing", "artificial microRNAs", "short hairpin RNAs", "microRNA target site insertion", "recombinant viral vectors", "reverse genetics", "adeno-associated virus vectors", "lentiviral vectors" ], "biological_systems": [ "mammalian cell culture", "mouse models", "embryonated chicken eggs", "plants", "nematodes", "arthropods" ], "key_concepts": [ "microRNA", "virus-derived interfering RNA", "RNA interference", "antiviral innate immunity", "type I interferon", "microRNA target site engineering", "viral tropism control", "artificial microRNA delivery", "cytoplasmic microprocessor", "small RNA copy number", "RISC saturation", "live-attenuated vaccine design" ], "keywords": [ "microRNA", "RNAi", "viRNA", "chordates", "viral vectors", "tropism", "amiRNA", "interferon", "live-attenuated vaccines", "gene silencing" ], "one_sentence_contribution": "Argues that chordates, unlike plants, nematodes and arthropods, do not use small RNAs as antiviral defence, and that this absence of interplay between host microRNAs and RNA viruses is what leaves the microRNA machinery available for engineering viral tropism and small RNA delivery.", "summary_25": "Chordates abandoned small RNA antiviral defence for interferon, leaving their microRNA machinery untouched by RNA viruses and available for engineering viral tropism and delivering designed small RNAs.", "summary_75": "Plants and insects fight viruses with small RNAs, but chordates use interferon instead. This review argues that host microRNAs cannot serve antiviral roles in chordates, because they are too scarce, bind too weakly and act too slowly relative to a viral life cycle. Viruses therefore have no reason to disrupt the microRNA machinery, so it stays intact and can be exploited, by grafting target sites into viral genomes and by engineering viruses to deliver designed small RNAs.", "summary_150": "Virus-derived interfering RNAs provide antiviral immunity in plants, nematodes and arthropods, while chordates rely on pattern recognition receptors and type I interferon. This review sets out why the chordate microRNA machinery does not substitute for the lost RNA-based defence, arguing from three constraints. Genome-encoded microRNAs rarely reach the abundance needed to act on abundant viral transcripts, partial complementarity limits repression to less than twofold and precludes catalytic cleavage, and the time required to transcribe, process and load a new microRNA exceeds the life cycle of most acute RNA viruses. Published small RNA profiling from several laboratories is consistent with this position, and reported counterexamples are largely confined to DNA viruses and read as viral piracy. The consequence is practical. Because RNA viruses leave the machinery intact, microRNA target sites can be grafted into viral genomes to control tropism and attenuation, and viruses can be engineered to deliver artificial microRNAs.", "citation": "tenOever BR. RNA viruses and the host microRNA machinery. Nature Reviews Microbiology, 2013, volume 11, issue 3, pages 169-180. DOI 10.1038/nrmicro2971. PMID 23411862.", "sections": { "Citation": "tenOever BR. RNA viruses and the host microRNA machinery. Nature Reviews Microbiology, 2013, volume 11, issue 3, pages 169-180.\n\nDOI 10.1038/nrmicro2971. PMID 23411862.", "One-sentence contribution": "Argues that chordates, unlike plants, nematodes and arthropods, do not use small RNAs as antiviral defence, and that this absence of interplay between host microRNAs and RNA viruses is what leaves the microRNA machinery available for engineering viral tropism and small RNA delivery.", "Executive summary": "Small RNA silencing systems are found in all three domains of life, but their uses differ. Plants, nematodes and arthropods generate virus-derived interfering RNAs and use them as antiviral immunity, whereas chordates rely on a protein-based system built on pattern recognition receptors, type I interferons and interferon-stimulated genes. This review sets out the case that chordate microRNAs do not serve an antiviral role, organizing the argument around three constraints. Copy number is the first, since an abundance threshold of roughly 100 copies per cell is needed for a microRNA to act on a host transcript, and a higher threshold would be needed to act on the far more numerous viral transcripts. Silencing capacity is the second, since genome-encoded microRNAs bind with partial complementarity and repress by less than twofold, which prevents the catalytic cleavage that virus-derived interfering RNAs achieve. Kinetics is the third, since most viral life cycles are shorter than the time needed to transcribe, process and load a new microRNA against proteins with half-lives beyond ten hours. Work from several laboratories profiling small RNAs in infected cells found no evidence of virus-derived small RNAs in vertebrates, and reported exceptions among herpesviruses, poxviruses and adenovirus are treated as a distinct set of cases. The practical argument follows from the biological one. Because viruses leave the host small RNA landscape intact, target sites for tissue-restricted or ubiquitous microRNAs can be grafted into viral genomes to dictate tropism, and viruses can be engineered to produce artificial microRNAs.", "Scientific context": "By 2013 it was established that small RNAs silence gene expression across all three domains of life, with clustered regularly interspaced short palindromic repeat RNAs restricting phage in bacteria and archaea, PIWI-interacting RNAs restricting transposable elements and foreign nucleic acid in animal germlines, and virus-derived interfering RNAs constituting the antiviral innate immune system in nematodes, arthropods and plants. MicroRNAs are the eukaryote-conserved class, encoded in the genome, processed from polymerase II transcripts by the Drosha and DGCR8 microprocessor and then by Dicer, and loaded into RISC where they fine-tune endogenous transcripts. Because chordates possess this machinery, it had been assumed that they would also generate an RNA-based antiviral defence and that such a defence might reinforce the interferon response. The review notes that the evidence for this expectation did not materialise. It records that small RNA profiling of virus-infected cells by other laboratories found no virus-derived small RNAs in vertebrate cells, and that an independent survey across hepatitis C virus, poliovirus, dengue virus, vesicular stomatitis virus and West Nile virus recovered small RNAs only at very low abundance, Dicer independent and without silencing capacity. The unresolved question the review addresses is not whether such a defence exists, but why it does not, and what follows from its absence.", "Central question": "Do host microRNAs contribute to antiviral defence in chordates, and if the answer is no, what does the resulting absence of interplay between RNA viruses and the host small RNA machinery permit in the way of engineered virological tools?", "Experimental strategy": "Not applicable in the experimental sense, since this is a single-author review rather than a primary research report. The argumentative strategy is worth stating, because it determines what the article can and cannot support. The review does not present new data. It assembles published findings from many laboratories and organizes them around a quantitative argument. Rather than asking whether any microRNA has ever been reported to affect a virus, it asks what abundance, complementarity and timing a small RNA would need in order to matter within the life cycle of an acute RNA virus, and then evaluates the literature against those thresholds. This framing is used to set aside a body of reports that measure microRNA induction as fold change rather than copies per cell, on the grounds that a large fold induction from a negligible baseline is not physiologically meaningful. The second half of the review inverts the same logic, treating the absence of viral engagement with the microRNA machinery as an engineering resource rather than as a negative result.", "Key findings": "This is a review, so the entries below separate what the tenOever laboratory reported from what other laboratories reported, and both from the interpretive claims the review itself advances.\n\n1. Claims advanced by the review itself, presented as argument rather than as new data. Three constraints are proposed to explain why chordate microRNAs cannot function in antiviral defence. Copy number, with a conservative estimate of roughly 100 copies per cell for activity on a host transcript, probably an order of magnitude higher for a viral target, against the observation that the 20 to 30 most abundant microRNAs make up more than 90 percent of the microRNA content of a cell. Silencing capacity, since partial complementarity gives less than twofold repression and precludes the catalytic cleavage and turnover that perfect complementarity permits. Kinetics, since most viral life cycles run under 12 hours while average protein half-life under stress exceeds 10 hours, leaving only newly transcribed targets and short-lived proteins within reach.\n\n2. Reported by other laboratories. Small RNA profiling of virus-infected cells found no evidence of virus-derived interfering RNA production in vertebrates, in work the review credits to the Pfeffer and colleagues studies that identified herpesvirus-encoded microRNAs. An independent survey by Parameswaran and colleagues across six RNA viruses recovered small RNAs only at very low concentrations, Dicer independent and without silencing capacity.\n\n3. Reported by other laboratories. Exceptions to the general rule cluster among DNA viruses. Herpesviruses encode their own microRNAs and antagonize specific host microRNAs. Poxvirus infection leads to degradation of host microRNAs. Adenovirus expresses the 160 nucleotide non-coding RNA VA1, which interferes with small RNA export and RISC loading, work attributed to the Cullen laboratory and others. Hepatitis C virus uses the liver-specific miR-122 to stabilize its genome and mask the 5 prime end from host nucleases, work attributed to the Sarnow laboratory and colleagues. The review classifies these as viral piracy rather than as host defence.\n\n4. Reported by other laboratories. The engineering of microRNA target sites into viral genomes to control tropism was introduced in lentiviral vectors using the haematopoietic-restricted miR-142, attributed to Brown and colleagues in the Naldini laboratory, and extended to live-attenuated vaccine design in poliovirus by Barnes and colleagues in the Andino laboratory using let-7a and the neuron-specific miR-124. Application to flaviviruses is attributed to the Pletnev laboratory, and to oncolytic vectors to several groups.\n\n5. Reported by the tenOever laboratory. The same target-site strategy was adapted to influenza A virus to produce a vaccine candidate that is attenuated in mammals but grows to wild-type titres in fertilized eggs, by exploiting a ubiquitous microRNA absent from the allantoic membrane, addressing attenuation and a manufacturing constraint at once, reported by Perez and colleagues.\n\n6. Reported by the tenOever laboratory. MicroRNA targeting was used as a tool to assign a requirement to a cell type rather than to attenuate. Engineering four miR-142 target sites into dengue virus blocked spread in vivo, which was read as evidence that replication in haematopoietic cells is required for dissemination, reported by Pham, Langlois and tenOever. Targeting influenza A virus in antigen-presenting cells while leaving lung epithelial replication intact led to the conclusion that antigen-presenting cells acquire viral antigen by engulfing non-haematopoietic cells or debris, reported by Langlois and colleagues.\n\n7. Reported by the tenOever laboratory, with independent corroboration. RNA viruses of both nuclear and cytoplasmic origin can be engineered to produce functional microRNAs without self-cleavage or attenuation, reported by Varble and colleagues and by Shapiro and colleagues from the tenOever laboratory, and by Rouha, Thurner and Mandl independently. In vivo delivery of cytoplasmic RNA virus-derived microRNAs was reported by Langlois, Shapiro, Pham and tenOever. Mechanistic analysis of how hairpins in a cytoplasmic genome are processed, attributed to Shapiro and colleagues from the same laboratory, was interpreted as evidence for a non-nuclear microprocessor activity forming during infection.\n\n8. Reported by other laboratories. The main liabilities of vector-delivered small RNAs were identified as pathogen-associated molecular pattern generation, since polymerase I or III transcripts carry a 5 prime triphosphate and hairpins present cytoplasmic double-stranded RNA, and saturation of the export and RISC machinery, with fatal shRNA oversaturation in mice attributed to Grimm and colleagues and export bottlenecks noted as resembling the adenovirus VA1 phenotype. Artificial microRNAs delivered as microprocessor substrates are presented as the least toxic route.\n\n9. Escape from engineered targeting is described as less frequent than anticipated, with no escape mutants recoverable under ubiquitous let-7a pressure in poliovirus, single-nucleotide mismatches but retained silencing under neuron-restricted miR-124 pressure, and outright excision of the target region in dengue virus. The generalization drawn, that multiple targets placed at separate genomic locations mitigate excision, is the review's own interpretation rather than a tested rule.", "Mechanistic model": "The review does not establish a mechanism, since it presents no new data. It advances an evolutionary and quantitative account, and the status of that account should be read as author interpretation throughout.\n\nThe account runs as follows. Chordates traded an RNA-based antiviral system for a protein-based one built on interferon, and the two appear to have been mutually exclusive, a point the review states remains a mystery. The article notes as supporting circumstance that small interfering RNAs and short hairpin RNAs can themselves induce type I interferon, and that the very features marking viral RNA as foreign, double-stranded character and an exposed 5 prime triphosphate, are also the products of the RNA-dependent RNA polymerase activity that amplifies virus-derived interfering RNAs in other phyla. Because chordate microRNA sequences are fixed in the genome, they are easily evaded, and the three constraints of copy number, silencing capacity and kinetics then explain why they were not retained for defence. The corollary, which is the engineering premise of the second half of the article, is that RNA viruses of chordates experience no selective pressure to disrupt the microRNA machinery and therefore leave it intact and available.\n\nThe review is explicit that the counter-evidence is not settled. Reports that the double-stranded RNA-binding proteins of influenza A virus, vaccinia virus and Ebola virus suppress RNA silencing are described as provocative but difficult to weigh, since the same proteins mask pattern recognition and since viruses lacking them are attenuated in wild-type cells but replicate normally in interferon-deficient cells irrespective of microRNA expression. A speculative proposal is also set out in a boxed section, that ubiquitous microRNAs might function as sequence restriction elements analogous to bacterial restriction enzymes rather than as regulators with physiological targets. The review labels this as a hypothesis and proposes growing viruses in microRNA-deficient cells as a way to test it.", "Conceptual or technical advance": "The article supplies a framework rather than a result. Its contribution is to convert a negative observation, the absence of virus-derived small RNAs in chordates, into a set of quantitative criteria against which claims of antiviral microRNA activity can be judged, and to press the field toward reporting microRNA abundance as copies per cell rather than as fold change. It also gathers the engineering consequences into a single account, showing that the same fact about chordate biology underwrites two distinct classes of tool. The first is control of viral tropism by grafting target sites, which supports attenuation, vaccine manufacture and the assignment of replication requirements to specific cell lineages. The second is delivery, where viruses engineered to express artificial microRNAs address the solubility, stability and membrane-crossing problems that had limited small interfering RNA therapeutics, with the additional observation from the tenOever laboratory and others that cytoplasmic RNA viruses can serve this role without nuclear trafficking.", "Relationship to the broader research program": "The review is written from the tenOever laboratory's own line of work and reads as a statement of its organizing premise, that engineered viruses are instruments for asking biological questions and not only objects of study. Several threads visible here recur across the corpus. Microbe-restricted and tissue-restricted microRNA targeting is used as a genetic tool for cell-type attribution rather than only for attenuation. Small RNA and interferon biology are treated as alternative solutions to the same problem rather than as cooperating systems. Viral genome engineering is treated as a general-purpose capability.\n\nCategory 3 synthesis, visible only when this article is set beside other papers in the corpus. The interferon-centred account of chordate antiviral defence that this review takes as given is the same system dissected experimentally in the 2007 Science report on IKKε, where the interferon-stimulated gene set was shown to be internally structured. The review's closing position, that virus design is a route to understanding, is stated in its own right in the later Cold Spring Harbor Perspectives article on synthetic virology. Establishing these continuities requires the other papers and is not supported by this article alone.", "Related publications": "- tenOever 2019, Cold Spring Harbor Perspectives in Medicine, conceptual extension. The synthetic virology perspective develops the same premise that engineered viruses are tools for understanding, across a broader set of applications.\n- tenOever et al. 2007, Science, conceptual predecessor within the corpus. It treats the protein-based interferon system that this review presents as the chordate alternative to RNA-based defence, though the review does not cite it.\n- Perez et al. 2009, Nature Biotechnology, and Perez et al. 2010, PNAS, application and predecessor from the tenOever laboratory, on microRNA-mediated species-specific attenuation of influenza A virus and on influenza-generated small RNAs.\n- Langlois et al. 2012, PNAS, and Pham, Langlois and tenOever 2012, PLoS Pathogens, application, using microRNA targeting to assign replication requirements to haematopoietic cells.\n- Varble et al. 2010, PNAS, Shapiro et al. 2010, RNA, Shapiro et al. 2012, RNA, and Langlois et al. 2011, Molecular Therapy, methodological foundation from the tenOever laboratory for RNA virus-mediated small RNA delivery and for cytoplasmic hairpin processing.\n- Barnes et al. 2008, Cell Host Microbe, from the Andino laboratory, and Brown et al. 2006, Nature Medicine, from the Naldini laboratory, predecessors from other groups that established microRNA target-site control of viral and vector tropism.", "Limitations and boundaries": "The article is a review and presents no new experimental data, so none of its claims are demonstrated here and all of them rest on the cited primary literature. Its central negative conclusion, that chordate microRNAs do not contribute to antiviral defence, is an argument from quantitative thresholds applied to published work rather than a direct test, and the review itself acknowledges contradictory reports that it sets aside on methodological grounds. The copy number threshold of roughly 100 copies per cell is described as a conservative estimate, and the extrapolation to a tenfold higher threshold for viral targets is reasoning rather than measurement. The scope is explicitly RNA viruses of chordates. DNA viruses are treated as exceptions and the review states that the herpesvirus, poxvirus and adenovirus cases are incompletely explained, with the question of why poxviruses are threatened by host microRNAs left open. Latent and chronic infections fall outside the kinetic argument by the review's own statement. The engineering sections describe results from a period when most applications had not reached clinical use, and the article flags unresolved problems, including competing endogenous RNA effects that may preclude the approach for persistent vectors, incompletely explained toxicity from hairpins that should not be pattern recognition receptor substrates, and the need to combine artificial microRNAs with adeno-associated virus vectors in future work. Priority and emphasis language in the source, including its characterization of two profiling studies and several annotated references as first demonstrations, belongs to the review and is not adopted here.", "Audience summaries": "### 25 words\n\nChordates abandoned small RNA antiviral defence for interferon, leaving their microRNA machinery untouched by RNA viruses and available for engineering viral tropism and delivering designed small RNAs.\n\n### 75 words\n\nPlants and insects fight viruses with small RNAs, but chordates use interferon instead. This review argues that host microRNAs cannot serve antiviral roles in chordates, because they are too scarce, bind too weakly and act too slowly relative to a viral life cycle. Viruses therefore have no reason to disrupt the microRNA machinery, so it stays intact and can be exploited, by grafting target sites into viral genomes and by engineering viruses to deliver designed small RNAs.\n\n### 150 words\n\nVirus-derived interfering RNAs provide antiviral immunity in plants, nematodes and arthropods, while chordates rely on pattern recognition receptors and type I interferon. This review sets out why the chordate microRNA machinery does not substitute for the lost RNA-based defence, arguing from three constraints. Genome-encoded microRNAs rarely reach the abundance needed to act on abundant viral transcripts, partial complementarity limits repression to less than twofold and precludes catalytic cleavage, and the time required to transcribe, process and load a new microRNA exceeds the life cycle of most acute RNA viruses. Published small RNA profiling from several laboratories is consistent with this position, and reported counterexamples are largely confined to DNA viruses and read as viral piracy. The consequence is practical. Because RNA viruses leave the machinery intact, microRNA target sites can be grafted into viral genomes to control tropism and attenuation, and viruses can be engineered to deliver artificial microRNAs." }, "discoveries": [ "claim-08" ], "relationships": [ { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" }, { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "application", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" }, { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" }, { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" }, { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" }, { "from": "2013-tenoever-rna-viruses-and-the-host-microrna-", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-tenoever-rna-viruses-and-the-host-microrna-" } ], "canonical_url": "https://tenoeverlab.us/publications/2013-tenoever-rna-viruses-and-the-host-microrna-/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "vsv", "poliovirus", "adenovirus", "dengue-virus", "blv", "hcv", "herpesviruses", "poxviruses", "west-nile-virus" ], "technologies": [ "reverse-genetics", "sirna-knockdown", "small-rna-seq", "mirna-target-site-insertion", "lentiviral-transduction", "artificial-mirna", "aav-vector" ] } }, { "id": "2013-varble-an-in-vivo-rnai-screening-approach", "slug": "2013-varble-an-in-vivo-rnai-screening-approach", "url": "/publications/2013-varble-an-in-vivo-rnai-screening-approach/", "title": "An In Vivo RNAi Screening Approach to Identify Host Determinants of Virus Replication", "authors": [ "Andrew Varble", "Asiel A. Benitez", "Sonja Schmid", "David Sachs", "Jaehee V. Shim", "Ruth Rodriguez-Barrueco", "Maryline Panis", "Marshall Crumiller", "Jose M. Silva", "Ravi Sachidanandam", "Benjamin R. tenOever" ], "author_count": 11, "first_author": "Andrew Varble", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 11, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2013, "journal": "Cell Host & Microbe", "volume": "14", "issue": "3", "pages": "346-356", "doi": "10.1016/j.chom.2013.08.007", "doi_url": "https://doi.org/10.1016/j.chom.2013.08.007", "pmid": "24034620", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/24034620/", "pmcid": null, "pmc_url": null, "pmc_pdf_url": null, "publication_type": "methods/resource", "declared_conflicts": null, "research_areas": [ "programmable-virology" ], "themes": [ "in-vivo-screening-through-fitness" ], "pathogens": [ "Sindbis virus", "influenza A virus" ], "viral_families": [ "Togaviridae", "Orthomyxoviridae" ], "host_species": [ "mouse", "human", "hamster" ], "technologies": [ "artificial microRNA libraries", "alphavirus reverse genetics", "in vivo serial passage selection", "small RNA deep sequencing", "barcoded virus libraries", "messenger RNA sequencing", "small RNA Northern blotting", "multicycle growth curves" ], "biological_systems": [ "murine embryonic fibroblasts", "A549 cells", "BHK-21 cells", "Hepa 1.6 cells", "Vero cells", "Zfx conditional knockout primary fibroblasts", "Dicer conditional knockout fibroblasts", "mouse spleen" ], "key_concepts": [ "in vivo RNA interference screening", "virus-delivered artificial microRNAs", "natural selection as screen readout", "host restriction factors", "interferon-stimulated genes", "RIG-I sensing of alphavirus", "transcriptional maintenance of antiviral capacity", "barcode control for drift" ], "keywords": [ "Sindbis virus", "artificial microRNA", "RNAi screen", "Zfx", "Mga", "RIG-I", "interferon", "host factors" ], "one_sentence_contribution": "Replication-competent Sindbis viruses, each encoding an artificial microRNA against one murine open reading frame, turn viral fitness in infected mice into a selection-based screen for host restriction factors, identifying the transcription factors Zfx and Mga as maintainers of antiviral capacity.", "summary_25": "Viruses were made to carry their own gene-silencing reagents, so that surviving best in infected mice revealed which host genes had been holding infection back.", "summary_75": "Screens for host genes that limit virus infection normally use cultured cells and indirect readouts. Here roughly 10,000 Sindbis viruses, each silencing one mouse gene, were passaged through mice, letting the virus's own replication do the selecting. Barcoded control viruses showed the enrichment was real. Hits included known interferon-stimulated genes and, unexpectedly, the transcription factors Zfx and Mga, whose loss degraded interferon signaling and allowed substantially more virus growth.", "summary_150": "Conventional RNA interference screens for virus host factors require transformed cells and surrogate readouts. This work delivers the silencing reagent from inside a replication-competent alphavirus, so that a hairpin relieving host restriction increases the fitness of the virus carrying it and selection during infection of mice becomes the assay. Approximately 10,000 artificial microRNA viruses were passaged by footpad injection with recovery from spleen, alongside a barcode library that controls for drift and with recloning between passages to remove hitchhiking mutations. Hairpins were shared across independent screens 16-fold more often than barcodes. Enriched hairpins targeted interferon-stimulated genes, homeostatic genes and general transcription factors, and the two most reproducible targeted Zfx and Mga. Independent silencing, a conditional knockout and transcriptome sequencing showed that losing either factor collapses parts of the interferon induction and signaling machinery and raises viral titers, with Mga required for interferon-driven Stat1 induction and Zfx for NF-kB activity.", "citation": "Varble A, Benitez AA, Schmid S, Sachs D, Shim JV, Rodriguez-Barrueco R, Panis M, Crumiller M, Silva JM, Sachidanandam R, tenOever BR. An In Vivo RNAi Screening Approach to Identify Host Determinants of Virus Replication. Cell Host & Microbe. 2013. Volume 14, issue 3, pages 346-356. DOI 10.1016/j.chom.2013.08.007. PMID 24034620.", "sections": { "Citation": "Varble A, Benitez AA, Schmid S, Sachs D, Shim JV, Rodriguez-Barrueco R, Panis M, Crumiller M, Silva JM, Sachidanandam R, tenOever BR. An In Vivo RNAi Screening Approach to Identify Host Determinants of Virus Replication. Cell Host & Microbe. 2013. Volume 14, issue 3, pages 346-356.\n\nDOI 10.1016/j.chom.2013.08.007. PMID 24034620.", "One-sentence contribution": "Replication-competent Sindbis viruses, each encoding an artificial microRNA against one murine open reading frame, turn viral fitness in infected mice into a selection-based screen for host restriction factors, identifying the transcription factors Zfx and Mga as maintainers of antiviral capacity.", "Executive summary": "RNA interference screens for host factors affecting virus replication had used cultured, usually transformed, cells with indirect readouts such as reporter activity or surface staining, which restricts them to one cell type and to a nonphysiological infection. This study replaces exogenous delivery of small interfering RNAs with delivery by the pathogen itself. An attenuated mouse-adapted Sindbis virus was configured to express an artificial microRNA on the murine miR-124-2 backbone, and a library of approximately 10,000 such viruses, each silencing one open reading frame, was passaged through mice with recovery from spleen. Because a hairpin that relieves restriction increases the fitness of the virus carrying it, selection itself becomes the readout and viral titer rather than a surrogate is the output. A matched library of 22 nucleotide barcodes, which confer no advantage, controls for drift, and recloning surviving hairpins into a fresh genome between passages removes hitchhiking mutations. Across four parallel screens, hairpins were shared by two or more sets 16-fold more often than barcodes, and enriched hairpins targeted interferon-stimulated genes, homeostatic genes and general transcription factors. Two hairpins recovered in three or more sets targeted Zfx and Mga. Independent silencing, a Zfx conditional knockout and transcriptome profiling showed that loss of either factor reduced interferon beta induction, STAT1, RIG-I and IKK beta, and raised viral titers by about one to one and a half logs. The authors propose that viral fitness may depend on antagonizing broad cellular programs as much as specific antiviral effectors.", "Scientific context": "Vertebrate detection of RNA virus infection proceeds through pattern recognition receptors such as RIG-I and MDA5, recruitment of MAVS at the mitochondrion, activation of NF-kB and interferon regulatory factors, assembly of the interferon beta enhanceosome, and induction of hundreds of interferon-stimulated genes. Many of the host factors in this network, and many proviral factors, had been found by high-throughput RNA interference screens against influenza A virus, dengue virus, hepatitis C virus, West Nile virus and human immunodeficiency virus. Those screens share two constraints. They require cell culture, typically a transformed line, and they measure virus output indirectly. Efforts to move to physiological settings had begun to use genetic variation among outbred mice correlated with susceptibility.\n\nIn parallel, several groups including this laboratory had shown that RNA viruses of either polarity and of either replication compartment can be engineered to produce functional microRNAs, even though no RNA virus lacking a DNA intermediate has been found in nature to encode a canonical one. The paper brings these two lines together, using virus-encoded artificial microRNAs as the delivery vehicle for a genome-scale silencing library inside a living host.", "Central question": "Can a replication-competent RNA virus that delivers its own silencing reagent be used so that natural selection on virus fitness during infection of an animal identifies the host factors that restrict replication.", "Experimental strategy": "The logic is that a natural infection generates quasispecies from which the host response selects, and that a library of otherwise identical viruses differing only in an encoded hairpin mimics that process with a defined, readable genotype. Sindbis virus was chosen because it is an attenuated, mouse-adapted, cytoplasmically replicating alphavirus with a duplicated subgenomic promoter into which sequence can be grafted, and because the laboratory had already shown that it processes artificial microRNAs accurately. Hairpins were built by substituting designed 21 nucleotide guides into the murine miR-124-2 backbone, drawing on an existing miR-30 based whole-genome library for the large screens.\n\nThree screens were run in sequence. A small in vitro screen against a subset of interferon-stimulated genes, with two hairpins per open reading frame, tested whether silencing a restriction factor is enough to be selected. A roughly 4,000 hairpin library passaged through mice by footpad injection with recovery from spleen established the timing of enrichment and, through a genetic reset in which surviving hairpins were recloned into the parental genome, established that enrichment tracks the hairpin rather than unrelated mutations. The full screen used approximately 10,000 hairpins in four independent quadruplicate passage series alongside a matched barcode library, with deep sequencing of the amplified hairpin or barcode region after each 48 hour passage. The barcode arm is the control that distinguishes selection from bottleneck-driven drift, and reproducibility across independent sets is the statistic. Hits were then removed from the viral context entirely and tested with independent small interfering RNAs, with overexpression, with a conditional knockout, and with messenger RNA sequencing, so that no conclusion about a host factor depends on the screening vector.", "Key findings": "1. Sindbis virus expressing a designed anti-GFP artificial microRNA processed the hairpin accurately at every time point tested and silenced GFP protein during infection, with four of five designed hairpins effective and two producing complete knockdown (Figures 1B and 1C and Figure S1C).\n2. In vitro passage of a virus library targeting interferon-stimulated genes positively selected viruses silencing IKK beta and RIG-I (Figure S2A). RIG-I involvement was confirmed outside the virus library, since an independent small interfering RNA enhanced replication and Ddx58 deficient fibroblasts supported more than a log more virus (Figures S2B to S2D). A purpose-built virus silencing RIG-I reduced RIG-I protein and grew about an order of magnitude better than a matched control virus, and this advantage was lost in Dicer-deficient cells, tying the phenotype to microRNA production rather than to the insert (Figures 2B, 2C, S2F and S2G).\n3. In mice, deep sequencing of the roughly 4,000 hairpin library showed clear enrichment of particular hairpins by the third passage, and approximately 75 percent of library amiRNAs were processed as predicted (Figure 3A and Figure S3). A genetic reset between passages three and four preserved the population dynamic, which the authors take as evidence that fitness gains track hairpin identity rather than hitchhiking mutations. A recombinant virus carrying the single most enriched hairpin, predicted to target the interferon-stimulated gene USP32, reached titers about one log above control in mouse spleen (Figures 3B and 3C).\n4. Across four independent screens with the approximately 10,000 hairpin library, only 0.5 percent of barcoded viruses were shared by two sets and none by three or more, whereas hairpin-bearing viruses showed a 16-fold enrichment for appearing in two or more sets and some appeared in three or more (Figures 4A to 4D and Table S2). Reproducibility therefore exceeds what drift in the barcode arm produces.\n5. Enriched hairpins targeted known interferon-stimulated genes annotated as virus response genes, and the only other functional categories enriched more than fivefold by passage three were cellular homeostasis and general transcription (Figure 5A and Table S3). Candidate hairpins found in at least two sets were verified for processing and silencing, and all but one gave a significant titer increase when the target was silenced with an independent small interfering RNA (Figures S4A to S4J).\n6. The two hairpins recovered in three or more independent screens target Zfx and Mga, transcription factors previously implicated in self-renewal rather than in antiviral defense.\n7. An independent small interfering RNA against Mga raised Sindbis titers by about an order of magnitude, overexpression of Mga reduced replication, and Mga knockdown lowered RIG-I and STAT1 during infection. The effect was not virus specific, being reproduced with an attenuated influenza A virus, with double-stranded RNA, and with type I interferon treatment (Figures 5B and 5C and Figure S5).\n8. Primary fibroblasts from Zfx conditional knockout mice supported about 1.5 logs more virus after Cre delivery, with a marked reduction in interferon-stimulated genes and loss of STAT1 (Figures 5D and 5E).\n9. Loss of either factor reduced virus-induced Ifnb and Stat1 transcription, but the point of interruption differed. Interferon-driven upregulation of Stat1 was compromised only by Mga knockdown, whereas NF-kB activity after TNF alpha treatment was affected by Zfx and not by Mga (Figures 6A to 6D). Messenger RNA sequencing showed broad loss of the machinery for interferon induction and signaling, with Zfx loss reducing IKK beta and Mga knockdown reducing Irf9, Stat2, Ifnar1, Ifnar2 and Irf7 (Figures 6E and 6F and Table S4).", "Mechanistic model": "This study does not establish a mechanism by which Zfx or Mga acts, and the authors say as much. They report that the two factors are required to maintain a transcriptome capable of mounting an antiviral response, and their proposal is that these are indirect determinants, transcriptional maintenance factors whose loss degrades the network rather than antiviral effectors themselves. The epistasis experiments constrain where each one acts. Mga is needed for interferon-driven induction of Stat1, placing its requirement within or upstream of the response to type I interferon, while Zfx is needed for NF-kB activity after TNF alpha, placing its requirement in a distinct arm. Both reduce IKK beta or the interferon signaling components measured. No direct binding, occupancy or target gene assignment is performed here, and the connection to the self-renewal transcriptional module comes from cited work on Zfx and Myc occupancy rather than from data in this paper.\n\nThe authors also flag a timing problem they cannot resolve. It is unclear how silencing either factor within a single round of an acute infection provides enough time to remodel the host transcriptome, and they speculate that at least one critical factor must fall within the first six to eight hours, possibly a component of interferon signaling or a potent interferon-stimulated gene. That is explicitly speculation.\n\nThe broader reading offered, that pathogenicity may be defined by the capacity to antagonize broad cellular programs rather than specific antiviral factors, is an interpretation of the screen's output distribution. The authors note that it is consistent with how oncolytic virus selectivity is understood, since transformation compromises the same signaling networks.", "Conceptual or technical advance": "The screen inverts the usual arrangement. Instead of perturbing cells and then measuring a proxy for infection, the perturbation rides inside the pathogen and the measurement is the pathogen's own replicative success in a living animal, read out by sequencing. That removes the transformed cell line, removes the single cell type, removes the surrogate readout, and allows any cell the virus enters to contribute. It also brings a control that cell culture screens do not usually need, the matched barcode library, which quantifies how much apparent reproducibility a bottlenecked in vivo passage generates on its own.\n\nThe platform is portable in a defined way. The authors point out that the same library, with a different inoculation route or a different tissue for recovery, would select for different biology, for example factors involved in crossing the blood-brain barrier if an alphavirus were recovered from brain, and that the approach should extend to questions of tropism, transmission and persistence. They also address biosafety directly, arguing that the approach cannot enhance a natural pathogen because encoding a hairpin is itself attenuating, so selection operates only within a population of equally attenuated viruses.", "Relationship to the broader research program": "This paper converts a capability into an instrument. The engineering it depends on, that an RNA virus can be built to produce a functional small RNA without losing replication, comes from the laboratory's earlier work, and the specific proposal to deliver a library of artificial microRNAs from a virus so that evolutionary selection identifies restriction factors was stated in the discussion of the 2012 Molecular Therapy paper. The alphavirus vector and the evidence that Sindbis processes artificial microRNAs come from the laboratory's Sindbis work with Shapiro and colleagues.\n\nCategory 3 synthesis. Placed beside Varble and colleagues in 2010 and Langlois and colleagues in 2012, this paper completes a three-step arc from demonstrating that RNA viruses can make microRNAs, to delivering them in animals, to using them as a selectable genetic tool. The recurring interest in the interface between RNA viruses and the host small RNA machinery, and in why RNA viruses do not naturally exploit it, is treated conceptually in the laboratory's later perspective writing, where the attenuating cost of encoding a hairpin is one of the arguments.", "Related publications": "- Varble and colleagues, 2010, Engineered RNA viral synthesis of microRNAs, cited here. Predecessor and methodological foundation.\n- Shapiro and colleagues, 2010 and 2012, on noncanonical cytoplasmic processing of viral microRNAs, cited here and supplying both the Sindbis platform and the observation that virus-mediated amiRNA production causes low level self-targeting. Methodological foundation.\n- Langlois and colleagues, 2012, In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs, cited here. Predecessor, and the source of the explicit proposal for this kind of screen.\n- Silva and colleagues, 2005, the miR-30 based whole-genome library, cited here with Silva as a coauthor of this paper. Methodological foundation.\n- Galan-Caridad and colleagues, 2007, the Zfx conditional knockout, cited here and supplying the mice. Methodological foundation.\n- Frolova and colleagues, 2002, on Sindbis nsP2 and interferon, cited here and supplying the nsP2 mutant used for in vitro knockdown work. Methodological foundation.\n- tenOever, 2016, The Evolution of Antiviral Defense Systems. Review or synthesis, treating conceptually why RNA viruses do not encode microRNAs in nature.", "Limitations and boundaries": "The authors devote a section of the discussion to the constraints of the platform, and they are substantial. The output is the emergence of dominant strains, so the screen is biased toward hairpins that confer an advantage cell-autonomously within an infected cell, and it cannot report on factors acting in uninfected bystanders or on paracrine effects. The advantage a hairpin confers changes with time, since silencing a pattern recognition receptor is worth little once interferon has been induced, and the fitness landscape shifts as the composition of the population shifts. Successful production of an artificial microRNA does not guarantee knockdown of the target protein, off-target silencing can confound the assignment of a hairpin to a gene, and a target protein with a half-life longer than the viral life cycle would show no benefit when its transcript is silenced. These together prevent comprehensive coverage of the transcriptome, and the screen returned only a small number of reproducible hits.\n\nResults are specific to what was selected on. All in vivo selection used footpad inoculation of an attenuated mouse-adapted Sindbis virus in C57BL/6 mice with recovery from spleen at 48 hours, so the hits reflect splenic replication in an acute alphavirus infection and not neurotropic disease, other routes, other tissues, other viruses, other mouse genotypes or other species. The authors note that the screen recovered relatively few interferon-stimulated genes, and offer two competing explanations, that Sindbis nsP2 already antagonizes them and so biases the output, or that broadly acting host genes matter more, without resolving which applies. Target assignment for enriched hairpins is computational, based on free energy prediction, so a predicted target such as USP32 for the most enriched hairpin in the small screen is a prediction rather than a validated target. The mechanistic follow-up on Zfx and Mga is largely in cell culture, uses transcript and protein abundance rather than direct transcription factor assays, and does not test whether the in vivo fitness advantage of those hairpins operates through the pathways mapped in vitro. Finally, the connection drawn between the screen output and pathogenicity is an interpretation, since no pathogenesis measurement is reported.", "Audience summaries": "### 25 words\n\nViruses were made to carry their own gene-silencing reagents, so that surviving best in infected mice revealed which host genes had been holding infection back.\n\n### 75 words\n\nScreens for host genes that limit virus infection normally use cultured cells and indirect readouts. Here roughly 10,000 Sindbis viruses, each silencing one mouse gene, were passaged through mice, letting the virus's own replication do the selecting. Barcoded control viruses showed the enrichment was real. Hits included known interferon-stimulated genes and, unexpectedly, the transcription factors Zfx and Mga, whose loss degraded interferon signaling and allowed substantially more virus growth.\n\n### 150 words\n\nConventional RNA interference screens for virus host factors require transformed cells and surrogate readouts. This work delivers the silencing reagent from inside a replication-competent alphavirus, so that a hairpin relieving host restriction increases the fitness of the virus carrying it and selection during infection of mice becomes the assay. Approximately 10,000 artificial microRNA viruses were passaged by footpad injection with recovery from spleen, alongside a barcode library that controls for drift and with recloning between passages to remove hitchhiking mutations. Hairpins were shared across independent screens 16-fold more often than barcodes. Enriched hairpins targeted interferon-stimulated genes, homeostatic genes and general transcription factors, and the two most reproducible targeted Zfx and Mga. Independent silencing, a conditional knockout and transcriptome sequencing showed that losing either factor collapses parts of the interferon induction and signaling machinery and raises viral titers, with Mga required for interferon-driven Stat1 induction and Zfx for NF-kB activity." }, "discoveries": [ "claim-10" ], "relationships": [ { "from": "2013-varble-an-in-vivo-rnai-screening-approach", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-varble-an-in-vivo-rnai-screening-approach" }, { "from": "2013-varble-an-in-vivo-rnai-screening-approach", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2013-varble-an-in-vivo-rnai-screening-approach" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2013-varble-an-in-vivo-rnai-screening-approach", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2013-varble-an-in-vivo-rnai-screening-approach", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" } ], "canonical_url": "https://tenoeverlab.us/publications/2013-varble-an-in-vivo-rnai-screening-approach/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sindbis-virus" ], "technologies": [ "bulk-rna-seq", "reverse-genetics", "small-rna-seq", "small-rna-northern-blot", "growth-curve", "barcoded-virus-library", "in-vivo-rnai-screen", "serial-passage" ] } }, { "id": "2014-backes-the-mammalian-response-to-virus-in", "slug": "2014-backes-the-mammalian-response-to-virus-in", "url": "/publications/2014-backes-the-mammalian-response-to-virus-in/", "title": "The Mammalian Response to Virus Infection Is Independent of Small RNA Silencing", "authors": [ "Simone Backes", "Ryan A. Langlois", "Sonja Schmid", "Andrew Varble", "Jaehee V. Shim", "David Sachs", "Benjamin R. tenOever" ], "author_count": 7, "first_author": "Simone Backes", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 7, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2014, "journal": "Cell Reports", "volume": "8", "issue": "1", "pages": "114-125", "doi": "10.1016/j.celrep.2014.05.038", "doi_url": "https://doi.org/10.1016/j.celrep.2014.05.038", "pmid": "24953656", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/24953656/", "pmcid": "PMC4096324", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4096324/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4096324/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense" ], "themes": [ "does-mammalian-antiviral-rnai-exist" ], "pathogens": [ "vesicular stomatitis virus", "influenza A virus", "Sindbis virus", "Borna disease virus", "vaccinia virus" ], "viral_families": [ "Rhabdoviridae", "Orthomyxoviridae", "Togaviridae", "Bornaviridae", "Poxviridae" ], "host_species": [ "mouse", "hamster" ], "technologies": [ "recombinant VSV reverse genetics", "small RNA deep sequencing", "mRNA sequencing", "small RNA Northern blotting", "quantitative RT-PCR", "plaque assay", "siRNA transfection", "microRNA target site insertion" ], "biological_systems": [ "murine embryonic fibroblasts", "Dicer-deficient fibroblasts", "BHK cells", "RAW macrophage cells", "bone marrow derived macrophages", "C6 glial cells", "mouse lung", "mouse spleen", "Ifnar1 and Il28r double knockout mice" ], "key_concepts": [ "antiviral RNA interference", "interferon response", "virus-derived small RNAs", "RISC", "VP55 poly(A) polymerase", "microRNA targetome", "interferon-stimulated genes", "Dicer independence", "small RNA tailing and degradation", "evolutionary divergence of antiviral strategies" ], "keywords": [ "RNAi", "interferon", "vesicular stomatitis virus", "VP55", "NS1", "microRNA", "Dicer", "small RNA sequencing", "antiviral defense", "mammalian" ], "one_sentence_contribution": "Engineering vesicular stomatitis virus to eliminate RISC-loaded small RNAs attenuates rather than enhances replication in mice, and confers no replication advantage even when interferon signaling is removed, arguing that small RNA silencing does not contribute to mammalian antiviral defense.", "summary_25": "Giving a virus an enzyme that destroys host small RNAs made it grow worse, not better, arguing mammals do not use RNA silencing against viruses.", "summary_75": "Plants and insects fight viruses with RNA interference, while mammals use interferon. Whether mammals also retain the RNA silencing defense has been disputed. Researchers armed vesicular stomatitis virus with a poxvirus enzyme that destroys host small RNAs. The armed virus gained no advantage in cells or mice, and was attenuated because losing microRNAs raised antiviral gene expression. Removing interferon signaling in mice erased all differences, indicating no hidden silencing contribution beneath interferon.", "summary_150": "Whether mammals retain a functional antiviral RNA interference arm alongside interferon has been contested, with detectable virus-derived small RNAs on one side and the absence of viral silencing suppressors on the other. This study tests the question by arming vesicular stomatitis virus with vaccinia virus VP55, an enzyme that tails and destroys RISC-loaded small RNAs, and comparing it with a virus carrying influenza NS1 to blunt interferon induction. Small RNA profiling across four virus families found viral small RNAs that were, in the one case with an informative size distribution, Dicer independent. The VP55 virus destroyed host microRNAs efficiently yet gained no fitness in Dicer-deficient cells, fibroblasts or primary macrophages, and was attenuated about a log in mice. Transcriptome profiling attributed that attenuation to derepression of interferon-stimulated genes normally held down by microRNAs. In mice lacking type I and type III interferon receptors, all viruses replicated comparably.", "citation": "Backes S, Langlois RA, Schmid S, Varble A, Shim JV, Sachs D, tenOever BR. The Mammalian Response to Virus Infection Is Independent of Small RNA Silencing. Cell Reports. 2014. Volume 8, Issue 1, pages 114-125. DOI 10.1016/j.celrep.2014.05.038. PMID 24953656. PMCID PMC4096324.", "sections": { "Citation": "Backes S, Langlois RA, Schmid S, Varble A, Shim JV, Sachs D, tenOever BR. The Mammalian Response to Virus Infection Is Independent of Small RNA Silencing. Cell Reports. 2014. Volume 8, Issue 1, pages 114-125.\n\nDOI 10.1016/j.celrep.2014.05.038. PMID 24953656. PMCID PMC4096324.", "One-sentence contribution": "Engineering vesicular stomatitis virus to eliminate RISC-loaded small RNAs attenuates rather than enhances replication in mice, and confers no replication advantage even when interferon signaling is removed, arguing that small RNA silencing does not contribute to mammalian antiviral defense.", "Executive summary": "Plants, arthropods and nematodes defend against viruses using RNA interference, in which double-stranded viral RNA is processed into small interfering RNAs that guide cleavage of viral transcripts. Mammals detect the same double-stranded RNA but respond through interferon induction instead. Whether mammals also retain a functional antiviral RNA interference arm has been contested, and reports of virus-derived small RNAs in infected mammalian cells, together with observations that several viral double-stranded RNA binding proteins suppress silencing in heterologous systems, had kept the question open.\n\nThe study tests the question with a gain-of-function design. Rather than removing silencing components from the host, which also perturbs other biology, the authors give the virus the ability to remove small RNAs. Vaccinia virus VP55, a poly(A) polymerase previously shown by this group to tail and destroy RISC-associated small RNAs, was inserted into vesicular stomatitis virus, and a parallel virus carrying influenza A virus NS1 was built to blunt interferon induction for comparison.\n\nIf small RNAs contributed meaningfully to antiviral defense, the small RNA destroying virus should gain fitness. It did not. It replicated like the control virus in fibroblasts and in primary macrophages, and in wild-type mice it was attenuated by about a log, which transcriptome profiling attributes to derepression of interferon-stimulated transcripts normally held down by microRNAs. In mice lacking both type I and type III interferon receptors, all three viruses reached comparable titers.", "Scientific context": "In plants, arthropods and nematodes, double-stranded RNA is recognized as a pathogen-associated molecular pattern and processed by Dicer-family RNase III nucleases into virus-derived small interfering RNAs that load into RISC and direct cleavage of complementary viral RNA. Viruses of these hosts have accordingly evolved suppressors of that pathway. Mammalian cells detect the same structures but convert detection into a transcriptional program, inducing type I and type III interferons and hundreds of interferon-stimulated genes, and mammalian viruses have evolved antagonists of that program.\n\nThe paper states that whether RNA interference is also a component of the mammalian response remains controversial. Arguments in favor include the conservation of the machinery and of microRNAs, the fact that several viral double-stranded RNA binding proteins that antagonize mammalian sensing also disrupt silencing in plants and flies, and an earlier report from this group identifying a genuine poxvirus inhibitor of small RNAs. Arguments against include that an influenza A virus lacking its double-stranded RNA antagonist regains full virulence in interferon-deficient mice, and that microRNA target sites can be inserted into a wide range of viruses to attenuate them, implying those viruses never evolved countermeasures against silencing.\n\nDeep sequencing intensified rather than settled the debate. Virus-derived small RNAs are detectable in infected mammalian cells, but the paper notes they persist in the absence of Dicer and may be byproducts of interferon-stimulated genes such as RNase L. The paper identifies two recent reports, on a mutant nodavirus in stem cells and immortalized fibroblasts, as the studies that gave the idea of mammalian RNA interference renewed traction, and notes that both used multifunctional viral antagonists whose effects on the host response are not limited to small RNAs.", "Central question": "Does small RNA silencing make a physiological contribution to the mammalian cellular response to virus infection, and if any contribution exists, is it masked by the interferon system?", "Experimental strategy": "The central design choice is to perturb small RNA function from inside the virus rather than from the host genome. Removing Dicer or Argonaute from a cell changes microRNA-dependent gene regulation broadly and alters the starting state of the cell before infection begins. Encoding a small RNA destroying enzyme in the viral genome instead confines the perturbation to infected cells and to the window of infection, and it poses the question in the form evolution would pose it, namely whether a virus that can destroy host small RNAs gains fitness.\n\nThe enzyme is vaccinia virus VP55, which this group had previously shown adds non-templated adenosines specifically to RISC-associated small RNAs and triggers their degradation. A stabilized and more active variant identified by mutagenesis was used. Vesicular stomatitis virus was chosen as the vehicle because it is a vaccine strain highly sensitive to both interferon and to RNA interference in arthropod and nematode systems, so it should register any protective silencing that exists.\n\nThe comparison virus encoding influenza A virus NS1, a RIG-I antagonist, is the positive control for the logic of the experiment. It shows what a genuine gain in fitness from disabling a real antiviral system looks like in the same virus backbone and the same animals. A matched control virus carrying an RNA insert of comparable size with no open reading frame controls for the insertion.\n\nBefore that, the study characterizes the small RNA landscape across four virus families to ask whether any of these infections generates small RNAs with the size distribution and Dicer dependence expected of genuine RNA interference, and tests whether vesicular stomatitis virus encodes its own silencing suppressor, since a virus already able to block silencing would be uninformative.\n\nFinally, the interferon receptor double knockout animals address the redundancy objection directly. If small RNA silencing were a secondary layer whose contribution is hidden behind interferon, then removing interferon should expose it, and destroying small RNAs should then help the virus.", "Key findings": "1. Small RNA deep sequencing of cells infected with Borna disease virus, influenza A virus, Sindbis virus and vesicular stomatitis virus recovered reads mapping across each viral genome, at 0.04, 18.4, 24.1 and 0.05 percent of total small RNA reads respectively (Figure 1A). Only vesicular stomatitis virus showed a size preference, peaking at 22 nucleotides with enrichment at the genome ends (Figure 1A, Figure 1B). The two most abundant of these were detectable by Northern blot and were unchanged in Dicer-deficient cells (Figure 1C, Figure 1D). The observation is Dicer independence. The interpretation is that these species are not products of canonical RNA interference, and the authors use the size distribution as a reason to select this virus as the most demanding test case.\n\n2. Inserting four perfectly complementary miR-142 target sites into the 3-prime untranslated region of the L polymerase message reduced titers by two logs in miR-142-expressing RAW macrophage cells, while all viruses grew comparably in cells lacking the microRNA (Figure 2B, Figure 2C, Figure S1). A virus carrying a primary miR-124 hairpin produced mature microRNA during infection (Figure 2D). The observation is that both the silencing arm and the hairpin processing arm operate normally during infection, so the virus does not encode a suppressor and RISC function is intact at least early in infection.\n\n3. Vesicular stomatitis virus encoding VP55 expressed the protein robustly and tailed and degraded exogenously expressed miR-124 while leaving viral leader RNA and U6 intact (Figure 3B, Figure 3C). Transfected unmodified small interfering RNA against the nucleoprotein silenced the control virus but not the VP55 virus (Figure 3D), and sequencing of miR-146b from infected cells showed 82.1 percent of reads carrying non-templated adenosines (Figure 3E). The tool works as intended.\n\n4. In Dicer-deficient cells, the VP55 virus and the control virus produced equivalent viral protein and reached comparable titers near 1 times 10 to the eighth plaque-forming units per milliliter (Figure 4A, Figure 4B), establishing that the insert itself carries no cost. In Dicer-expressing wild-type fibroblasts the two viruses were again indistinguishable in protein and titer (Figure 4C, Figure 4D) and at earlier time points (Figure S3). The observation is no fitness gain from eliminating small RNAs in cell culture.\n\n5. In primary bone marrow derived macrophages the two viruses replicated comparably (Figure 5A) despite a pronounced loss of miR-142, miR-146, miR-155 and miR-93 with U6 unaffected (Figure 5B), so the absence of a phenotype is not explained by failure of the enzyme in primary cells.\n\n6. Messenger RNA sequencing of infected macrophages found that the transcripts most affected by VP55 expression were predominantly canonical interferon-stimulated genes, including guanylate-binding proteins, cytokines, and components of sensing and signaling machinery, with independent confirmation by quantitative PCR for a subset (Figure 5C, Figure 5D, Table S2). Only two genes were higher in the control infection. The observation is a directional shift. The authors interpret it as consistent with microRNAs acting as negative regulators and with a prior report that microRNAs suppress basal antiviral transcript levels, and they explicitly caution that the data do not indicate specific targeting of this gene class, only that interferon-stimulated gene changes are the most prominent in the context of infection.\n\n7. Intranasal infection of mice produced virus-derived small RNAs resembling those from fibroblasts but at levels the authors describe as vanishingly rare (Figure 6A, Table S3), and lung tissue from three animals showed significant loss of miR-146 with the VP55 virus (Figure 6B), confirming the enzyme works in vivo.\n\n8. In wild-type fibroblasts the NS1-encoding virus showed dramatic loss of interferon beta induction while the control and VP55 viruses induced it robustly, with the VP55 virus modestly higher (Figure 6C). In wild-type mice, interferon beta induction inversely tracked titer. The NS1 virus exceeded control by more than a log in lung and spleen, and the VP55 virus was reduced by about a log, with reported p values of 0.0041, 0.0015 and 0.0365 for the indicated comparisons (Figure 6D).\n\n9. In mice lacking both type I and type III interferon receptors, all three viruses reached comparable titers in lung and spleen with no significant differences (Figure 6E). The authors read this as showing that the NS1 advantage in wild-type animals was entirely attributable to muting interferon, that the VP55 attenuation was attributable to an enhanced interferon response, and that destroying small RNAs confers no benefit even with interferon removed.", "Mechanistic model": "The study does not establish a mechanism for an antiviral activity, because its conclusion is that the activity is not present under the conditions tested. What it does propose a mechanism for is the direction of the phenotype it observed.\n\nThe model is that host microRNAs act as negative regulators that hold antiviral transcripts, including many interferon-stimulated genes, below their maximum in the infected cell. Destroying those microRNAs relieves that repression, raises the antiviral transcriptional output, and therefore attenuates the virus. On this reading the VP55 virus is attenuated not because small RNAs were protecting it, but because small RNAs were dampening the system that was. The evidence for the derepression step is the transcriptome comparison and the quantitative PCR confirmation, together with the modest rise in interferon beta induction in fibroblasts and the loss of the phenotype in interferon receptor knockout animals.\n\nThe negative claim is bounded by what a negative result can support, and the authors say so, writing that it is difficult to prove the absence of a biological activity and framing their result as a strong argument rather than a proof. The design does establish that eliminating RISC-loaded small RNAs gives this virus no replication advantage in fibroblasts, in Dicer-deficient cells, in primary macrophages, in wild-type mice, or in mice with both interferon systems removed. The interpretation that no functional antiviral RNA interference system is available to act on this virus in these settings follows from that, and is the authors' conclusion.\n\nThe authors additionally offer several supporting arguments that are reasoning rather than new data. They note that microRNAs pair only partially with targets and so would repress too weakly and too slowly to matter during an acute infection given typical protein half-lives, that mammalian viruses have not evolved suppressors of silencing whereas many can be attenuated by inserted microRNA target sites, that expressing an RNA-dependent RNA polymerase in mammalian cells is itself sufficient to induce type I interferon, and that mammals do not use the 2-prime-O-methylation chemistry that extends small RNA half-life in flies and plants outside of Piwi-interacting RNAs in stem and germ cells. These are category 2 and category 4 material and are presented in the discussion as such.", "Conceptual or technical advance": "The work reframes a contested question as a fitness experiment. Instead of asking whether small RNAs derived from viral genomes can be detected, which deep sequencing had already answered affirmatively without settling anything, it asks whether a virus armed with the ability to destroy host small RNAs does better. That reframing is transferable to other proposed antiviral pathways.\n\nThe VSV-VP55 virus is itself a reusable reagent, and the authors say so, noting that it allowed the microRNA targetome to be mapped in infected cells from an in vivo infection and suggesting the same enzyme could be added to more inert vectors such as lentivirus or adenovirus for comparable studies.\n\nThe conclusion also has a practical consequence the authors draw out. If mammalian viruses do not contend with small RNA silencing, then the small RNA pathway remains available as an engineering handle, for controlling viral tropism, for building attenuated vaccines, for layered biocontainment, and for using RNA viruses as vehicles to deliver small interfering RNAs.", "Relationship to the broader research program": "This paper is the conceptual pivot between two strands of the laboratory's work. One strand established that host small RNA pathways can be recruited to control viruses, through microRNA target site insertion to restrict tropism in influenza A virus and in dengue virus and through engineered viral synthesis of microRNAs. The other strand asked what those pathways are actually doing during infection. The immediate predecessor is the identification of VP55 as a poxvirus degrader of RISC-associated microRNAs, which supplied both the reagent used here and part of the argument that motivated the question.\n\nRead together with the dengue and influenza targeting papers from the same group, the picture is coherent and is category 3 synthesis. Those studies depend on RISC functioning during infection, and this study reports directly that it does, while concluding that the virus gains nothing from shutting it down. The corpus records for those papers would be needed to support this as a formal cross-paper claim.\n\nThe finding that microRNAs restrain baseline antiviral transcript levels also connects to the laboratory's recurring interest in how the magnitude of the interferon response is set, and the microRNA targetome generated here is a resource pointed in that direction.", "Related publications": "- Backes, Shapiro, Sabin, Pham, Reyes, Moss, Cherry and tenOever 2012, Cell Host and Microbe, degradation of host microRNAs by poxvirus poly(A) polymerase. Relationship predecessor and methodological foundation. Source of VP55 and of the finding that terminal RNA methylation protects small RNAs from tailing, which dictated the unmodified small interfering RNA chemistry used here.\n- Langlois, Shapiro, Pham and tenOever 2012, Molecular Therapy, in vivo delivery of cytoplasmic RNA virus-derived microRNAs. Relationship methodological foundation. Source of the VSV construct expressing miR-124.\n- Langlois, Varble, Chua, Garcia-Sastre and tenOever 2012, Proceedings of the National Academy of Sciences, hematopoietic-specific targeting of influenza A virus. Relationship predecessor. Part of the body of engineered microRNA targeting work cited as evidence that mammalian viruses lack silencing countermeasures, and source of small RNA amplification methods used here.\n- Pham, Langlois and tenOever 2012, PLoS Pathogens, replication in cells of hematopoietic origin is necessary for dengue virus dissemination. Relationship predecessor. Cited among the engineered targeting studies that motivate the argument.\n- Cullen, Cherry and tenOever 2013, Cell Host and Microbe, on whether RNA interference is a physiologically relevant innate antiviral response in mammals. Relationship review or synthesis. The review framing of the debate that this study addresses experimentally.\n- Li and colleagues 2013 and Maillard and colleagues 2013, on virus-derived small interfering RNAs against a mutant nodavirus and against encephalomyocarditis virus. Relationship predecessors from other laboratories whose claims this study is designed to test, and whose limitations the discussion addresses directly.\n- Seo and colleagues 2013, on microRNA suppression of basal antiviral transcripts. Relationship conceptual extension from another laboratory. The transcriptome result here is presented as being in agreement with it.", "Limitations and boundaries": "The strongest conclusions rest on one virus in one backbone. Vesicular stomatitis virus was selected because it is highly sensitive to both interferon and, in invertebrate systems, to RNA interference, but a negative result in one rhabdovirus does not exclude a silencing contribution against other viruses, other tissues, or other infection kinetics. The small RNA profiling covers four families, but the functional tests do not.\n\nThe conclusion is a negative one, and the authors state plainly that it is difficult to prove the absence of a biological activity and that they regard their result as a strong argument rather than a demonstration of absence.\n\nAll in vivo work used intranasal inoculation and examined lung and spleen at 24 hours in wild-type animals and 48 hours in knockout animals, with different inoculum doses between the two, which limits direct quantitative comparison across the two mouse genotypes.\n\nThe perturbation is specific to RISC-associated small RNAs. VP55 tails and degrades small RNAs loaded into RISC, so activities of the small RNA machinery that do not proceed through that state, and any small RNA species resistant to tailing, would not be tested by this approach. Relatedly, 2-prime-O-methylated small RNAs are resistant to VP55, which is why unmodified duplexes were used in the silencing control.\n\nCell culture work relies substantially on immortalized fibroblasts, hamster kidney cells and a macrophage line in addition to primary bone marrow derived macrophages. The competing reports the study addresses proposed that RNA interference operates in undifferentiated stem cells and diminishes as cells become interferon responsive, and stem cells were not examined here.\n\nVP55 is a viral enzyme with its own biology, and expressing it from the virus adds a foreign activity to the infection. The matched no-open-reading-frame control and the equivalence in Dicer-deficient cells address the insertion and the general burden, but they do not exclude effects of the enzyme unrelated to small RNA tailing.\n\nThe transcriptome analysis is correlative with respect to the attenuation mechanism. Derepression of interferon-stimulated transcripts is observed and the attenuation disappears in interferon receptor knockout mice, which is consistent, but no experiment restores individual repressed targets to test whether they account for the titer difference.", "Audience summaries": "### 25 words\n\nGiving a virus an enzyme that destroys host small RNAs made it grow worse, not better, arguing mammals do not use RNA silencing against viruses.\n\n### 75 words\n\nPlants and insects fight viruses with RNA interference, while mammals use interferon. Whether mammals also retain the RNA silencing defense has been disputed. Researchers armed vesicular stomatitis virus with a poxvirus enzyme that destroys host small RNAs. The armed virus gained no advantage in cells or mice, and was attenuated because losing microRNAs raised antiviral gene expression. Removing interferon signaling in mice erased all differences, indicating no hidden silencing contribution beneath interferon.\n\n### 150 words\n\nWhether mammals retain a functional antiviral RNA interference arm alongside interferon has been contested, with detectable virus-derived small RNAs on one side and the absence of viral silencing suppressors on the other. This study tests the question by arming vesicular stomatitis virus with vaccinia virus VP55, an enzyme that tails and destroys RISC-loaded small RNAs, and comparing it with a virus carrying influenza NS1 to blunt interferon induction. Small RNA profiling across four virus families found viral small RNAs that were, in the one case with an informative size distribution, Dicer independent. The VP55 virus destroyed host microRNAs efficiently yet gained no fitness in Dicer-deficient cells, fibroblasts or primary macrophages, and was attenuated about a log in mice. Transcriptome profiling attributed that attenuation to derepression of interferon-stimulated genes normally held down by microRNAs. In mice lacking type I and type III interferon receptors, all viruses replicated comparably." }, "discoveries": [ "claim-08" ], "relationships": [ { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2012-backes-degradation-of-host-micrornas-by-p", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2013-varble-an-in-vivo-rnai-screening-approach", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2014-backes-the-mammalian-response-to-virus-in", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "review or synthesis", "evidence": "stated in the Related publications section of 2014-backes-the-mammalian-response-to-virus-in" }, { "from": "2015-aguado-microrna-function-is-limited-to-cy", "to": "2014-backes-the-mammalian-response-to-virus-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-aguado-microrna-function-is-limited-to-cy" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2014-backes-the-mammalian-response-to-virus-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2018-aguado-homologous-recombination-is-an-int", "to": "2014-backes-the-mammalian-response-to-virus-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2018-aguado-homologous-recombination-is-an-int" } ], "canonical_url": "https://tenoeverlab.us/publications/2014-backes-the-mammalian-response-to-virus-in/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "vsv", "sindbis-virus", "vaccinia-virus", "borna-disease-virus" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "reverse-genetics", "sirna-knockdown", "small-rna-seq", "small-rna-northern-blot", "mirna-target-site-insertion" ] } }, { "id": "2014-heaton-long-term-survival-of-influenza-vi", "slug": "2014-heaton-long-term-survival-of-influenza-vi", "url": "/publications/2014-heaton-long-term-survival-of-influenza-vi/", "title": "Long-term survival of influenza virus infected club cells drives immunopathology", "authors": [ "Nicholas S. Heaton", "Ryan A. Langlois", "David Sachs", "Jean K. Lim", "Peter Palese", "Benjamin R. tenOever" ], "author_count": 6, "first_author": "Nicholas S. Heaton", "senior_authors": [ "Peter Palese", "Benjamin R. tenOever" ], "corresponding_authors": [ "Peter Palese", "Benjamin R. tenOever" ], "tenoever_position": 6, "tenoever_role": "senior", "contribution_character": "co-led", "year": 2014, "journal": "Journal of Experimental Medicine", "volume": "211", "issue": "9", "pages": "1707-1714", "doi": "10.1084/jem.20140488", "doi_url": "https://doi.org/10.1084/jem.20140488", "pmid": "25135297", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/25135297/", "pmcid": "PMC4144728", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144728/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144728/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling", "programmable-virology" ], "themes": [ "calibration-of-interferon-in-vivo", "lineage-tracing-of-infection" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "mouse", "human" ], "technologies": [ "influenza reverse genetics", "Cre-lox lineage tracing", "tdTomato reporter mice", "diphtheria toxin receptor depletion", "fluorescence-activated cell sorting", "mRNA sequencing", "multiplex bead cytokine array", "quantitative RT-PCR", "histopathology" ], "biological_systems": [ "mouse lung", "primary mouse lung fibroblasts", "mtCC10-1 murine club cell line", "MLE-15 murine lung epithelial cell line", "H441 human club cell line", "MDCK cells", "embryonated chicken eggs" ], "key_concepts": [ "cell survival of lytic infection", "lineage tracing of infected cells", "club cells", "interferon-stimulated genes", "proinflammatory chemokines", "immunopathology", "bronchiolar epithelium", "virus clearance", "type I interferon response" ], "keywords": [ "influenza A virus", "club cells", "Clara cells", "Cre recombinase reporter virus", "tdTomato", "immunopathology", "interferon-stimulated genes", "CXCL10", "CCL20", "CCL5", "diphtheria toxin receptor", "lung pathology" ], "one_sentence_contribution": "A Cre recombinase-expressing influenza A virus combined with Cre-responsive reporter and ablation mouse strains showed that a subpopulation of directly infected lung cells, predominantly club cells, survives productive infection, sustains elevated interferon-stimulated gene and chemokine expression, and contributes to bronchiolar epithelial damage after virus is cleared.", "summary_25": "Some lung cells survive influenza infection rather than dying. These surviving airway cells keep signalling inflammation after virus is gone, and removing them reduces airway damage in mice.", "summary_75": "Influenza is usually thought to kill the cells it infects. Using a virus that permanently tags any cell it replicates in, this work showed that a small population of mouse airway cells, mostly club cells, clears the virus and lives on. Those survivors keep antiviral and inflammatory genes switched on after virus is undetectable. Genetically removing them lessened damage to the airway lining, indicating that surviving infected cells contribute to post-infection lung injury.", "summary_150": "An influenza A virus carrying Cre recombinase on the PB2 segment was used with Cre-responsive mouse strains to permanently mark cells that supported viral replication, independent of whether virus remained. Marked cells persisted at 10 and 21 days after infection, past the point when infectious virus was recoverable, and were confined to the epithelium of larger airways. RNA sequencing of sorted marked and unmarked cells from the same lungs identified Cc10 as the only retained lineage marker, implicating club cells, and showed that survivors carry an amplified interferon-stimulated gene signature and elevated Cxcl10, Ccl20 and Ccl5 without induction of Ccl2. A murine club cell line was intrinsically more interferon responsive than a comparison lung epithelial line, and murine and human club cell lines secreted the same selective chemokine set upon infection. Ablating marked survivors with a Cre-inducible diphtheria toxin receptor significantly reduced bronchiolar epithelial necrosis without altering overall infiltration scores.", "citation": "Heaton NS, Langlois RA, Sachs D, Lim JK, Palese P, tenOever BR. Long-term survival of influenza virus infected club cells drives immunopathology. Journal of Experimental Medicine. 2014. Volume 211, issue 9, pages 1707-1714. DOI 10.1084/jem.20140488. PMID 25135297. PMCID PMC4144728.", "sections": { "Citation": "Heaton NS, Langlois RA, Sachs D, Lim JK, Palese P, tenOever BR. Long-term survival of influenza virus infected club cells drives immunopathology. Journal of Experimental Medicine. 2014. Volume 211, issue 9, pages 1707-1714.\n\nDOI 10.1084/jem.20140488. PMID 25135297. PMCID PMC4144728.", "One-sentence contribution": "A Cre recombinase-expressing influenza A virus combined with Cre-responsive reporter and ablation mouse strains showed that a subpopulation of directly infected lung cells, predominantly club cells, survives productive infection, sustains elevated interferon-stimulated gene and chemokine expression, and contributes to bronchiolar epithelial damage after virus is cleared.", "Executive summary": "Influenza A virus infection of the respiratory tract produces tissue damage and proinflammatory signalling that can outlast detectable virus. Conventional tracking of infected cells relies on viral RNA or viral protein, both short-lived, so the fate of an infected cell after virus clearance has been hard to establish. Heaton and colleagues built an H1N1 virus carrying Cre recombinase fused to PB2 through a PTV-1 2A site and infected mice carrying Cre-activated reporter cassettes, which permanently marks any cell in which viral replication and protein synthesis occurred. Reporter-positive cells were present not only during active replication but also at 10 and 21 days after infection, past the point at which infectious virus could be recovered from lung. Histology placed the surviving marked cells in the epithelium of larger airways and never in alveoli. Transcriptional profiling of sorted reporter-positive and reporter-negative cells identified Cc10 as the only cell type marker retained in survivors, implicating club cells, and showed that survivors carried a higher magnitude interferon-stimulated gene signature and elevated Cxcl10, Ccl20 and Ccl5. Ablating the surviving cells with a Cre-inducible diphtheria toxin receptor reduced bronchiolar epithelial necrosis. The work reframes a subset of infected airway cells as durable local sources of inflammatory signal rather than as cells that are uniformly destroyed.", "Scientific context": "Influenza A virus infects several cell types in the respiratory tract, including ciliated epithelial cells, type I and type II alveolar cells and immune cells. Infected cells were understood to be eliminated either by replication-driven apoptosis and necrosis or by innate and adaptive immune clearance, and acute infection in humans resolves within about two weeks. The paper states the methodological gap directly. Infected cells are normally identified through virus-derived products or reporters whose half-lives are short, so those approaches cannot define infected cell types at times well after replication has ceased. Separately, severe influenza outcomes have been associated with sustained proinflammatory responses, which raised the unresolved question of what cellular source maintains that signalling once virus has been cleared.", "Central question": "Do any lung cells survive a productive influenza A virus infection, and if so, what lineage are they, what transcriptional state do they hold after virus clearance, and do they contribute to the immunopathology that persists beyond the acute phase.", "Experimental strategy": "The design rests on converting a transient viral event into a permanent host-genome mark. Cre recombinase was inserted downstream of a PTV-1 2A site at the 3 prime end of the PB2 segment of A/Puerto Rico/8/1934, so that Cre is produced only where the viral polymerase segment is expressed. Mice carrying a lox-stop-tdTomato cassette then label any cell in which that expression occurred, and the label persists whether or not the cell still contains virus. Specificity controls established that reporter activation requires active replication rather than uptake of infected cell debris, and virulence comparison with the parental strain established that the recombinant virus still produces influenza-like disease. Once the tool was validated, three readouts were layered onto it. Flow cytometry over a time course established whether marked cells persist past clearance, measured against plaque assay for infectious virus. RNA sequencing of sorted reporter-positive and reporter-negative cells from the same lungs supplied both lineage assignment through cell type marker transcripts and functional state through interferon-stimulated gene and chemokine levels, with the paired sorting controlling for shared tissue environment. Finally, substituting a Cre-inducible diphtheria toxin receptor strain for the reporter strain converted the labelling system into an ablation system, allowing the surviving population to be removed after replication had largely ended and the consequence for lung pathology to be scored by an independent pathologist.", "Key findings": "1. IAV-Cre activates the host reporter only under productive infection. Mock-infected and wild-type-infected reporter fibroblasts showed no tdTomato, IAV-Cre infection produced red fluorescence, pretreatment with type I interferon before IAV-Cre abolished the signal, and lysed debris from IAV-Cre infected cells applied under neutralizing antibody produced no signal (Figure 1 C). The authors read this as evidence that viral RNA replication and protein expression are required for labelling.\n\n2. The recombinant virus retains influenza pathogenicity. Intranasal IAV-Cre produced morbidity comparable to the parental strain, with the median lethal dose shifting from 50 to 240 PFU (Figure 1 D). The paper notes this experiment was performed once.\n\n3. Reporter-positive cells persist beyond virus clearance. Marked cells were detected at 5, 10 and 21 days after infection (Figure 2 A), while infectious virus recoverable from lung homogenate was high at days 2 and 5 and undetectable by day 10 (Figure 2 C). No marked cells appeared in uninfected mice or in reporter mice given wild-type virus (Figure 2, A and B).\n\n4. Surviving marked cells localise to the epithelium of larger airways. At day 10, tdTomato-positive cells were found in bronchial epithelium and never in alveoli (Figure 2 D).\n\n5. Surviving cells carry a transcriptional profile distinct from their reporter-negative neighbours in the same lung (Figure 3 A), and replicate sorting and sequencing of day 10 samples were reproducible (Figure 3 B).\n\n6. Viral mRNA is lost from marked cells by day 10 (Figure 3 C), and day 5 viral reads mapped across all eight segments (Figure 3 D). The authors interpret the eight-segment coverage as arguing against reporter activation by defective interfering particle entry. Sorted day 5 marked cells injected into embryonated eggs yielded virus in 11 of 12 eggs, while day 10 marked cells did not (Figure 3 E), which is read as showing the day 10 population has genuinely cleared a productive infection.\n\n7. Club cells are the predominant surviving lineage. Early after infection marked cells expressed both Sftpc and Cc10, but Sftpc was almost entirely lost by day 5 while Cc10 was maintained (Figure 3 F), and no other cell type specific marker was detected at appreciable levels in the survivor population (Table S1). This is a lineage inference from marker transcripts rather than from a lineage-restricted genetic label, and the authors support it with the concordance between club cell anatomical distribution and the observed localisation of marked cells.\n\n8. Survivors hold an amplified interferon-stimulated gene response. Interferon-stimulated genes were higher in magnitude in tdTomato-positive than in tdTomato-negative cells from the same lungs at day 5 (Figure 3 G) and the signature persisted across the time course (Table S1). In cell lines, a murine club cell line showed significantly higher Irf7 and Isg15 induction than a lung epithelial line after interferon treatment or infection (Figure 3, H and I). The authors state explicitly that the relationship between interferon-stimulated gene upregulation and cellular survival is only correlative, and describe the survival explanation as a favoured hypothesis.\n\n9. Depleting surviving cells reduces airway pathology. Diphtheria toxin given at day 5 to Cre-inducible diphtheria toxin receptor mice infected with IAV-Cre produced a significant reduction in bronchiolar epithelial pathology at day 10, with non-depleted airways showing frequent segmental necrosis and depleted airways showing only minor lesions and often unaffected terminal bronchioles (Figure 4, A and B). Overall cell infiltration scores did not differ significantly between groups. The authors flag that interpretation is complicated by possible contributions from surviving non-club cells.\n\n10. Survivors are a selective source of proinflammatory chemokines. Cxcl10, Ccl20 and Ccl5 were highly induced in marked cells relative to uninfected controls while Ccl2 was not appreciably induced (Figure 4 C). Infection of the murine club cell line raised secreted CXCL10, CCL20 and CCL5 protein without a significant change in CCL2 (Figure 4 D), and the human club cell line H441 behaved similarly (Figure 4 E).", "Mechanistic model": "The study does not establish a definitive mechanism for either survival or pathology. What the data constrain is the sequence of states. Cells that go on to survive were productively infected, as shown by the eight-segment viral read coverage and the recovery of infectious virus from day 5 sorted cells. Those cells clear virus, as shown by loss of viral mRNA and by failure of day 10 sorted cells to amplify in eggs. They are predominantly club cells by marker transcript, and they retain an elevated interferon-stimulated gene and chemokine programme after clearance. Removing them reduces bronchiolar epithelial necrosis.\n\nWhat the data do not constrain is causation in either direction. The authors state that the link between interferon-stimulated gene levels and survival is correlative, and the club cell line comparison establishes a higher intrinsic interferon responsiveness in that lineage without showing that this responsiveness is what permitted survival in vivo. Likewise the depletion experiment shows that removing surviving cells reduces damage, but it does not identify which secreted factor or which recruited cell population mediates that damage, and the authors note that non-club survivors may contribute. The model the authors propose is that club cells directly infected by influenza A virus but surviving that infection establish a local proinflammatory environment in the bronchi, useful for initial antiviral control but detrimental to bronchial remodelling once the pathogen is gone. That is presented as a proposal consistent with the data rather than as a demonstrated causal chain.", "Conceptual or technical advance": "The reporter virus converts a transient infection event into a permanent heritable mark on the host cell, which makes the post-clearance fate of infected cells an accessible experimental question rather than an inference. Pairing that label with a Cre-inducible diphtheria toxin receptor turns the same genetic logic into a functional test, so the population defined by having been infected can be removed and its contribution to disease measured. Conceptually, the finding that an acute lytic respiratory infection leaves behind a surviving, transcriptionally altered epithelial population makes it testable whether post-viral inflammatory states in the airway are maintained by cells that were themselves infected, rather than solely by immune infiltrate or by residual antigen.", "Relationship to the broader research program": "The Cre-expressing influenza virus extends a line of engineered influenza tools from the same groups that use viral genome modification to interrogate host biology, including the hematopoietic-specific targeting virus of Langlois and colleagues in 2012 and the microRNA-based biocontainment strategy of Langlois and colleagues in 2013, both cited here. The coupling of a recombinant virus to host transcriptional profiling by sequencing, with sorted infected and uninfected populations compared within the same tissue, is a recurring design in the corpus.\n\nCategory 3 synthesis. Recurring questions about how a localised infected cell population shapes tissue-level inflammatory outcome, and about post-acute sequelae of respiratory virus infection, appear elsewhere in this corpus, including in later hamster and human airway work on SARS-CoV-2. Establishing that connection properly requires those records side by side and is not something this paper supports on its own.", "Related publications": "- Langlois and colleagues, 2012, methodological foundation. Cited here for hematopoietic-specific targeting of influenza A virus, part of the same programme of engineered influenza viruses used to define host contributions to antiviral response.\n- Langlois and colleagues, 2013, methodological foundation. Cited here for a microRNA-based strategy to restrict influenza host range, reflecting the same engineering approach to the viral genome.\n- Heaton and colleagues, 2013, methodological foundation. Cited here as the source of the PB2-GLuc plasmid and of the rescue procedure used to generate IAV-Cre, and as prior genome-wide mutagenesis work establishing the plasticity of influenza segments to insertion.", "Limitations and boundaries": "The infection model is a single laboratory-adapted H1N1 strain, A/Puerto Rico/8/1934, delivered intranasally to inbred C57BL/6 mice, and the conclusions are bounded to that combination. The recombinant virus is mildly attenuated relative to the parental strain, with the median lethal dose shifting from 50 to 240 PFU, and the virulence comparison was performed once. Lineage assignment to club cells rests on cell type marker transcripts in sorted populations and on anatomical concordance rather than on an independent club cell specific genetic label, and the authors describe it as a hypothesis supported by those observations. The depletion experiment removes all Cre-marked survivors rather than club cells specifically, and the authors state that contributions from surviving non-club cells complicate interpretation. The link between the elevated interferon-stimulated gene signature and cellular survival is explicitly described by the authors as correlative. Time points extend to 21 days after infection, so nothing is established about longer-term persistence or resolution of the survivor population. Chemokine secretion in the human system was measured only in the H441 cell line, not in primary human airway tissue, and no human in vivo data are presented. Several key experiments are stated to be representative of two independent experiments, and pathology was scored on two lung sections 100 micrometres apart per animal.", "Audience summaries": "### 25 words\n\nSome lung cells survive influenza infection rather than dying. These surviving airway cells keep signalling inflammation after virus is gone, and removing them reduces airway damage in mice.\n\n### 75 words\n\nInfluenza is usually thought to kill the cells it infects. Using a virus that permanently tags any cell it replicates in, this work showed that a small population of mouse airway cells, mostly club cells, clears the virus and lives on. Those survivors keep antiviral and inflammatory genes switched on after virus is undetectable. Genetically removing them lessened damage to the airway lining, indicating that surviving infected cells contribute to post-infection lung injury.\n\n### 150 words\n\nAn influenza A virus carrying Cre recombinase on the PB2 segment was used with Cre-responsive mouse strains to permanently mark cells that supported viral replication, independent of whether virus remained. Marked cells persisted at 10 and 21 days after infection, past the point when infectious virus was recoverable, and were confined to the epithelium of larger airways. RNA sequencing of sorted marked and unmarked cells from the same lungs identified Cc10 as the only retained lineage marker, implicating club cells, and showed that survivors carry an amplified interferon-stimulated gene signature and elevated Cxcl10, Ccl20 and Ccl5 without induction of Ccl2. A murine club cell line was intrinsically more interferon responsive than a comparison lung epithelial line, and murine and human club cell lines secreted the same selective chemokine set upon infection. Ablating marked survivors with a Cre-inducible diphtheria toxin receptor significantly reduced bronchiolar epithelial necrosis without altering overall infiltration scores." }, "discoveries": [ "claim-14" ], "relationships": [ { "from": "2019-tenoever-synthetic-virology-building-viruse", "to": "2014-heaton-long-term-survival-of-influenza-vi", "relationship": "application", "evidence": "stated in the Related publications section of 2019-tenoever-synthetic-virology-building-viruse" } ], "canonical_url": "https://tenoeverlab.us/publications/2014-heaton-long-term-survival-of-influenza-vi/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "reverse-genetics", "histopathology", "multiplex-cytokine-assay", "cell-sorting", "cre-lox-lineage-tracing", "cell-ablation" ] } }, { "id": "2014-schmid-a-versatile-rna-vector-for-deliver", "slug": "2014-schmid-a-versatile-rna-vector-for-deliver", "url": "/publications/2014-schmid-a-versatile-rna-vector-for-deliver/", "title": "A Versatile RNA Vector for Delivery of Coding and Noncoding RNAs", "authors": [ "Sonja Schmid", "Lum C. Zony", "Benjamin R. tenOever" ], "author_count": 3, "first_author": "Sonja Schmid", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 3, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2014, "journal": "Journal of Virology", "volume": "88", "issue": "4", "pages": "2333-2336", "doi": "10.1128/jvi.03267-13", "doi_url": "https://doi.org/10.1128/jvi.03267-13", "pmid": "24307584", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/24307584/", "pmcid": "PMC3911536", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911536/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911536/pdf/", "publication_type": "methods/resource", "declared_conflicts": null, "research_areas": [ "programmable-virology" ], "themes": [ "rna-vectors-for-delivery" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "human", "mouse", "canine" ], "technologies": [ "influenza reverse genetics", "replication-incompetent virus-like vectors", "artificial microRNA expression", "microRNA target site attenuation", "small RNA northern blot", "quantitative PCR", "western blot", "flow cytometry", "cell viability assay", "intranasal delivery" ], "biological_systems": [ "normal human dermal fibroblasts", "MDCK cells", "HA-complementing MDCK cells", "HA and NP complementing MDCK cells", "primary mouse lung cultures from GFP transgenic mice", "bone marrow derived macrophages", "mouse lung" ], "key_concepts": [ "RNA-based gene delivery", "replication-incompetent vector", "artificial microRNA", "miR-124", "miR-302/367 cluster", "miR-93 target site attenuation", "tunable small RNA dosing", "vector cytotoxicity", "no DNA intermediate" ], "keywords": [ "virus-like vector", "influenza A virus", "RNA interference", "microRNA delivery", "artificial microRNA", "gene knockdown", "primary cells", "intranasal delivery" ], "one_sentence_contribution": "Replication-incompetent influenza-based vectors carrying no DNA intermediate deliver coding messages and functional small RNAs together to primary human and mouse cells and to mouse lung, with output and cytotoxicity tuned by microRNA target sites in the nucleoprotein segment.", "summary_25": "An influenza-derived vector that cannot replicate delivers protein-coding messages and silencing RNAs together into primary cells and mouse lung, with output tuned to avoid killing cells.", "summary_75": "Turning RNA interference into medicine depends on getting the RNA where it is needed. This work adapts a crippled influenza virus into a delivery vehicle that never becomes DNA and cannot integrate into the genome. It carried a fluorescent reporter and a silencing RNA at once, knocked down target genes in human and mouse primary cells, and reached mouse lung after nasal delivery. Building in a self-limiting switch removed the vector's toxicity without losing silencing.", "summary_150": "This short report develops replication-incompetent influenza-based virus-like vectors as a delivery system for small RNAs. Replacing the segment 4 open reading frame with a primary microRNA transcript produced roughly threefold more miR-124 in primary human fibroblasts than a replication-competent virus expressing it from segment 8, and a single vector delivered a fluorescent protein message and a microRNA at the same time, or processed a five-hairpin microRNA cluster. Delivered miR-124 lowered its endogenous target polypyrimidine tract binding protein by 75 percent. To control dose, the authors used a nucleoprotein segment bearing target sites for the ubiquitous miR-93, which cut small RNA output about fivefold, preserved target knockdown, and abolished the cytotoxicity of the untargeted vector, and they built a hemagglutinin and nucleoprotein complementing cell line to produce it. Knockdown also worked in bone marrow derived macrophages. Intranasal delivery placed miR-124 in mouse lung, though in vivo silencing was not assessed.", "citation": "Schmid S, Zony LC, tenOever BR. A Versatile RNA Vector for Delivery of Coding and Noncoding RNAs. Journal of Virology. 2014. 88(4), 2333-2336. DOI 10.1128/jvi.03267-13. PMID 24307584. PMCID PMC3911536.", "sections": { "Citation": "Schmid S, Zony LC, tenOever BR. A Versatile RNA Vector for Delivery of Coding and Noncoding RNAs. Journal of Virology. 2014. 88(4), 2333-2336.\n\nDOI 10.1128/jvi.03267-13. PMID 24307584. PMCID PMC3911536.", "One-sentence contribution": "Replication-incompetent influenza-based vectors carrying no DNA intermediate deliver coding messages and functional small RNAs together to primary human and mouse cells and to mouse lung, with output and cytotoxicity tuned by microRNA target sites in the nucleoprotein segment.", "Executive summary": "Delivering foreign RNA to a chosen tissue remains the practical bottleneck between RNA interference as a laboratory technique and RNA interference as a therapy, and the vectors in common use either integrate into the host genome or saturate the endogenous silencing machinery. This short report develops an alternative built on influenza A virus. Starting from a published hemagglutinin-deleted virus-like vector system propagated on complementing cells, the authors replaced the segment 4 open reading frame with a primary microRNA transcript and showed that the resulting vector expressed miR-124 in normal human dermal fibroblasts at levels roughly three times those from a replication-competent virus expressing the same microRNA from segment 8. The vector carried a green fluorescent protein message on one segment and a microRNA on another at the same time, and it processed all five hairpins of the miR-302/367 cluster from a single segment. Delivered miR-124 was functional, reducing its endogenous target polypyrimidine tract binding protein by 75 percent at 2 days. To control dose and toxicity the authors used a nucleoprotein segment bearing target sites for the ubiquitous miR-93, which lowered artificial microRNA output about fivefold while preserving target knockdown and removed the cytotoxicity seen with the wild-type nucleoprotein vector. Knockdown also worked in bone marrow derived macrophages, and intranasal delivery gave detectable miR-124 in whole lung. In vivo silencing was not tested.", "Scientific context": "Two facts frame the work. First, no RNA virus lacking a DNA intermediate had been found to encode a canonical microRNA, yet several groups including this laboratory had already shown that RNA viruses can be engineered to produce functional small RNAs. Second, artificial microRNAs with perfect complementarity silence far more effectively than endogenous microRNAs with partial complementarity, which makes them attractive as therapeutics, and yet in vivo delivery remains the limiting step. Existing options carry known liabilities. Vectors derived from DNA viruses and lentiviruses integrate and can perturb endogenous gene expression, and sustained high-level expression of small RNAs from integrating vectors had been reported by the Kay group to saturate the endogenous silencing pathway with lethal consequences in mice. Influenza-based vectors with limited replicative capacity had already reached regulatory approval in the vaccine setting, which the authors cite as evidence for the platform's safety profile. The specific starting material was a hemagglutinin-deleted influenza virus-like vector system and a segment 4 construct with green fluorescent protein flanked by hemagglutinin packaging sequences, both described previously by other groups and by this laboratory.", "Central question": "Can a replication-incompetent, influenza-derived RNA vector that never passes through a DNA intermediate deliver functional small RNAs, alone or together with a protein-coding message, to primary cells and to the respiratory tract, and can its output and its toxicity be tuned rather than merely accepted?", "Experimental strategy": "The design treats the influenza genome as a modular cassette system. Because the virus has eight segments, a coding message and a noncoding RNA can be assigned to different segments and tested for independent expression, and because packaging signals rather than open reading frames define a usable segment, an open reading frame can be swapped for a primary microRNA transcript. Replication incompetence comes from deleting hemagglutinin and supplying it in trans from a complementing MDCK line, which confines propagation to the producer cells. To make dose adjustable the authors reach for the reverse of a delivery function, using a previously characterized nucleoprotein segment carrying perfect target sites for miR-93, a microRNA expressed in essentially all mammalian cells, so that the host silencing machinery itself limits vector activity. Since that segment attenuates nucleoprotein, a second complementing line supplying both hemagglutinin and nucleoprotein was built to produce it. Readouts are deliberately layered. Small RNA northern blot shows that the RNA is made, western blot and quantitative PCR of an endogenous or transgenic target show that it works, a viability assay shows what the vector costs the cell, and intranasal delivery asks whether any of it survives contact with an animal.", "Key findings": "1. A virus-like vector expressing the primary miR-124-2 transcript from segment 4 produced about threefold more miR-124 in normal human dermal fibroblasts than a replication-competent influenza expressing the same microRNA from segment 8 (Fig. 1B). The authors report this comparison as unexpected.\n2. A single vector carrying green fluorescent protein on segment 4 and miR-124 on segment 8 gave both high green fluorescent protein expression and high miR-124 levels in primary human fibroblasts at 1 day (Fig. 1C and 1D), establishing simultaneous delivery of coding and noncoding RNA.\n3. The five-hairpin miR-302/367 cluster cloned into segment 4 yielded detectable miR-302 and miR-367 (Fig. 1E). Because these are the 5' and 3' ends of the transcript, the authors interpret detection of both as evidence that all five hairpins were processed, an inference the northern blot cannot confirm directly since it does not resolve miR-302a through miR-302d.\n4. Vector-delivered miR-124 was functional. Polypyrimidine tract binding protein, an established endogenous miR-124 target, fell by 75 percent at 2 days and 60 percent at 3 days after treatment (Fig. 2A), with miR-124 detectable at 1, 2, and 3 days (Fig. 2B).\n5. A nucleoprotein segment targeted by miR-93 reduced artificial microRNA against green fluorescent protein about fivefold relative to the wild-type nucleoprotein vector, bringing it to levels comparable with endogenous microRNAs (Fig. 3B), and yet both vectors knocked down green fluorescent protein transcripts comparably at 1 day in primary lung cultures from green fluorescent protein transgenic mice (Fig. 3C).\n6. Cytotoxicity separated the two vectors. The wild-type nucleoprotein vector left roughly 50 percent cell survival at 4 days, while the miR-93-targeted vector was not significantly different from mock treatment (Fig. 3D). Both gave 42 percent knockdown of green fluorescent protein protein at 4 days (Fig. 3E), and nucleoprotein expression was lower with the targeted vector as expected.\n7. In bone marrow derived macrophages, the vector expressed miR-124 at 1 day (Fig. 4A), reduced green fluorescent protein transcripts by approximately 40 percent at 1 day (Fig. 4B), and reduced green fluorescent protein protein in nucleoprotein positive cells at 3 days by flow cytometry (Fig. 4C).\n8. Intranasal treatment of C57BL/6 mice with the miR-124 vector gave detectable miR-124 in whole lung at 1 day (Fig. 4D). The authors state explicitly that demonstrating in vivo silencing will require future studies, so delivery in the animal is shown and silencing in the animal is not.\n9. The authors make a priority claim in the text, stating that this is the first demonstration that replication-incompetent virus-like vectors lacking a DNA intermediate can be used to deliver small RNAs. That claim is the paper's own and is recorded here as such.", "Mechanistic model": "This is a vector engineering report and it does not set out to establish a biological mechanism. The mechanisms it relies on are already established elsewhere and are used here as tools, namely Drosha and Dicer processing of a primary microRNA transcript delivered as part of a viral segment, loading of the mature small RNA into the silencing complex, and cleavage of a perfectly complementary target, alongside the reciprocal use of perfect host microRNA target sites in an essential viral segment to have the host silencing machinery destroy the vector's own message. What the data constrain is the input and output relationship. Small RNA abundance, target knockdown, and cytotoxicity can be moved by choice of segment and by the presence of miR-93 target sites in the nucleoprotein segment. What the data do not constrain is why a fivefold reduction in artificial microRNA levels leaves knockdown essentially unchanged, and the authors do not propose a mechanism for that dissociation. The relationship between nucleoprotein expression and the observed cytotoxicity is correlative here, since nucleoprotein level, vector activity, and toxicity all move together and were not separated.", "Conceptual or technical advance": "The report converts an attenuated influenza system into a tunable RNA delivery platform with three properties that are hard to obtain together. The vector carries no DNA intermediate and does not integrate, it can carry a protein-coding message and one or more small RNAs at once, and the amount of small RNA it produces is set by an engineered feature rather than by the dose given, which in these experiments decoupled useful silencing from measurable cytotoxicity. The introduction of a hemagglutinin and nucleoprotein complementing cell line is the enabling technical step, since it allows production of vectors whose essential nucleoprotein is itself silenced in the target cell. The authors note that a tissue-restricted microRNA could be substituted for the ubiquitous miR-93 to add tissue specificity, and that loop architecture can be used to tune processing efficiency, both of which are proposed extensions rather than results.", "Relationship to the broader research program": "This paper sits at the junction of two lines that run through the corpus. One is the engineering of RNA viruses to make small RNAs, represented in the reference list by earlier laboratory work on engineered RNA viral synthesis of microRNAs, on noncanonical cytoplasmic processing of viral microRNAs, and on in vivo delivery of cytoplasmic RNA virus-derived microRNAs. The other is the use of host microRNA target sites to restrict viral gene expression, represented by the laboratory's miR-93 attenuation of influenza and by the hematopoietic restriction of influenza through miR-142, both cited here. The novelty relative to those is the combination, since the same microRNA target site logic that was previously used to interrogate biology is used here to make a therapeutic-style vector safe. Placing this vector work beside later corpus entries that deliver or restrict RNA cargo in animals would be category 3 synthesis and belongs to central assembly.", "Related publications": "- Varble et al. 2010, engineered RNA viral synthesis of microRNAs. Methodological foundation, the source of the segment 8 microRNA-expressing influenza used here as the comparison virus.\n- Langlois et al. 2012, in vivo delivery of cytoplasmic RNA virus-derived miRNAs. Predecessor from the same laboratory, cited for the estimate of roughly fifty thousand microRNA copies per cell from influenza-based expression systems.\n- Perez et al. 2009, microRNA-mediated species-specific attenuation of influenza A virus. Methodological foundation, the source of the miR-93-targeted nucleoprotein segment used to tune vector activity.\n- Langlois et al. 2012, hematopoietic-specific targeting of influenza A virus. Companion, cited as prior use of host microRNA target sites to restrict influenza to selected cell types.\n- Chua et al. 2013, influenza A virus utilizes suboptimal splicing to coordinate the timing of infection. Methodological foundation, cited for the virus-like vector production system used here.\n- Shapiro et al. 2010, noncanonical cytoplasmic processing of viral microRNAs. Predecessor from the same laboratory on small RNA processing from RNA viral contexts.\n- Varble et al. 2013, an in vivo RNAi screening approach to identify host determinants of virus replication. Conceptual extension, cited as the source of the artificial microRNA against green fluorescent protein used here.\n- tenOever 2013, RNA viruses and the host microRNA machinery. Review or synthesis from the same laboratory, cited for the prospect of using cell or tissue-specific microRNAs.", "Limitations and boundaries": "The animal data show delivery only. Detection of miR-124 in whole lung after intranasal treatment does not demonstrate silencing of any target in vivo, and the authors say so. All silencing results come from cell culture, over a window of one to four days after treatment, and the report contains no data on durability beyond that, on repeat dosing, on immune responses to the vector, or on biodistribution. Two of the three knockdown readouts use a transgenic green fluorescent protein reporter rather than an endogenous gene, and the one endogenous target tested is polypyrimidine tract binding protein with a single microRNA, miR-124. The processing claim for the miR-302/367 cluster rests on detecting the terminal members, since the northern blot cannot distinguish the four miR-302 species. Cell survival was measured with a single luminescent viability assay at one time point and does not address sublethal effects or saturation of the endogenous silencing machinery, which is precisely the failure mode the authors invoke against integrating vectors. Finally, the system is built on one mouse-adapted influenza strain and one complementing cell background, and the safety inference drawn from approved live attenuated influenza vaccines is an argument by analogy rather than a result from this study.", "Audience summaries": "### 25 words\n\nAn influenza-derived vector that cannot replicate delivers protein-coding messages and silencing RNAs together into primary cells and mouse lung, with output tuned to avoid killing cells.\n\n### 75 words\n\nTurning RNA interference into medicine depends on getting the RNA where it is needed. This work adapts a crippled influenza virus into a delivery vehicle that never becomes DNA and cannot integrate into the genome. It carried a fluorescent reporter and a silencing RNA at once, knocked down target genes in human and mouse primary cells, and reached mouse lung after nasal delivery. Building in a self-limiting switch removed the vector's toxicity without losing silencing.\n\n### 150 words\n\nThis short report develops replication-incompetent influenza-based virus-like vectors as a delivery system for small RNAs. Replacing the segment 4 open reading frame with a primary microRNA transcript produced roughly threefold more miR-124 in primary human fibroblasts than a replication-competent virus expressing it from segment 8, and a single vector delivered a fluorescent protein message and a microRNA at the same time, or processed a five-hairpin microRNA cluster. Delivered miR-124 lowered its endogenous target polypyrimidine tract binding protein by 75 percent. To control dose, the authors used a nucleoprotein segment bearing target sites for the ubiquitous miR-93, which cut small RNA output about fivefold, preserved target knockdown, and abolished the cytotoxicity of the untargeted vector, and they built a hemagglutinin and nucleoprotein complementing cell line to produce it. Knockdown also worked in bone marrow derived macrophages. Intranasal delivery placed miR-124 in mouse lung, though in vivo silencing was not assessed." }, "discoveries": [ "claim-10" ], "relationships": [ { "from": "2014-schmid-a-versatile-rna-vector-for-deliver", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-schmid-a-versatile-rna-vector-for-deliver" }, { "from": "2014-schmid-a-versatile-rna-vector-for-deliver", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-schmid-a-versatile-rna-vector-for-deliver" }, { "from": "2014-schmid-a-versatile-rna-vector-for-deliver", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-schmid-a-versatile-rna-vector-for-deliver" } ], "canonical_url": "https://tenoeverlab.us/publications/2014-schmid-a-versatile-rna-vector-for-deliver/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "rt-qpcr", "reverse-genetics", "flow-cytometry", "small-rna-northern-blot", "mirna-target-site-insertion", "immunoblotting", "in-vivo-infection-route", "artificial-mirna", "cell-death-assays", "single-cycle-influenza-vector" ] } }, { "id": "2014-schmid-mitogen-activated-protein-kinase-m", "slug": "2014-schmid-mitogen-activated-protein-kinase-m", "url": "/publications/2014-schmid-mitogen-activated-protein-kinase-m/", "title": "Mitogen-activated Protein Kinase-mediated Licensing of Interferon Regulatory Factor 3/7 Reinforces the Cell Response to Virus", "authors": [ "Sonja Schmid", "David Sachs", "Benjamin R. tenOever" ], "author_count": 3, "first_author": "Sonja Schmid", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 3, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2014, "journal": "Journal of Biological Chemistry", "volume": "289", "issue": "1", "pages": "299-311", "doi": "10.1074/jbc.m113.519934", "doi_url": "https://doi.org/10.1074/jbc.m113.519934", "pmid": "24275658", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/24275658/", "pmcid": "PMC3879553", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879553/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879553/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling" ], "themes": [ "transcription-factor-selectivity" ], "pathogens": [ "vesicular stomatitis virus", "influenza A virus", "influenza A virus H1N1 A/Puerto Rico/8/34 NS1 R38A K41A" ], "viral_families": [ "Rhabdoviridae", "Orthomyxoviridae" ], "host_species": [ "human", "mouse" ], "technologies": [ "mRNA deep sequencing", "electrophoretic mobility shift assay", "coimmunoprecipitation", "luciferase reporter assay", "in vivo radiolabeling", "phosphatase treatment", "site-directed mutagenesis", "siRNA knockdown", "immunofluorescence microscopy", "quantitative PCR", "plaque assay" ], "biological_systems": [ "HEK293T cells", "2FTGH cells", "murine fibroblasts", "Map3k8 knockout fibroblasts", "HEK293T cells stably expressing IRF7" ], "key_concepts": [ "MAP3K8", "IRF3 and IRF7 heterodimer", "interferon regulatory factor binding element", "interferon beta enhanceosome", "feed forward amplification of innate immunity", "proline-rich hinge phosphorylation", "SP100 family", "promyelocytic leukemia nuclear bodies", "ERK activation", "threat-proportional antiviral response" ], "keywords": [ "MAP3K8", "TPL2", "IRF3", "IRF7", "interferon beta", "SP100", "PML nuclear bodies", "ERK", "vesicular stomatitis virus", "innate immunity" ], "one_sentence_contribution": "Sustained IRF7 activity induces the kinase MAP3K8, which phosphorylates the proline-rich hinge of IRF3 and redirects it from homodimers into IRF3 and IRF7 heterodimers, broadening the antiviral transcriptome and scaling the cellular response to the persistence of the viral threat.", "summary_25": "A kinase induced late in infection modifies IRF3 so that it pairs with IRF7 instead of itself, widening the set of antiviral genes a cell can reach.", "summary_75": "Cells must match the strength of an antiviral response to the size of the threat. The authors describe a loop that does this. Persistent infection keeps IRF7 active, IRF7 induces the kinase MAP3K8, and MAP3K8 causes phosphorylation of IRF3 in a flexible hinge region. The modified IRF3 pairs with IRF7 rather than with itself, and the resulting complex reaches promoters the IRF3 pair cannot. Cells lacking the kinase induce fewer antiviral genes and support more virus.", "summary_150": "IRF3 requires a stringent binding element while IRF7 tolerates degenerate ones, so which dimer forms determines how much of the antiviral genome is accessible. Building on their earlier finding that MAP3K8 is induced specifically by IRF7, the authors show that MAP3K8 is transcribed from an IRF7-selective element in its promoter, is activated during infection, and drives ERK phosphorylation. MAP3K8 suppresses interferon beta and ISG15 output that depends on IRF3 alone, an inhibition reversed by IRF7. It does not block IRF3 activation, nuclear entry or CBP binding. Instead it produces phosphorylation within the proline-rich hinge of IRF3, mapped by truncation, and hinge residues are required for the induced pairing with IRF7. Fibroblasts lacking MAP3K8 support higher vesicular stomatitis virus titers and fail to induce about seventy genes, among them Sp100, Sp110 and Sp140, and expressing SP100B or SP100C raises interferon beta and lowers titer. Direct phosphorylation of IRF3 by MAP3K8 is not established.", "citation": "Schmid S, Sachs D, tenOever BR. Mitogen-activated Protein Kinase-mediated Licensing of Interferon Regulatory Factor 3/7 Reinforces the Cell Response to Virus. Journal of Biological Chemistry. 2014. Volume 289, issue 1, pages 299-311. DOI 10.1074/jbc.m113.519934. PMID 24275658. PMCID PMC3879553.", "sections": { "Citation": "Schmid S, Sachs D, tenOever BR. Mitogen-activated Protein Kinase-mediated Licensing of Interferon Regulatory Factor 3/7 Reinforces the Cell Response to Virus. Journal of Biological Chemistry. 2014. Volume 289, issue 1, pages 299-311.\n\nDOI 10.1074/jbc.m113.519934. PMID 24275658. PMCID PMC3879553.", "One-sentence contribution": "Sustained IRF7 activity induces the kinase MAP3K8, which phosphorylates the proline-rich hinge of IRF3 and redirects it from homodimers into IRF3 and IRF7 heterodimers, broadening the antiviral transcriptome and scaling the cellular response to the persistence of the viral threat.", "Executive summary": "The mammalian antiviral response must be strong enough to clear a replicating virus and restrained enough not to shut down the cell when the trigger is minor. The authors ask how a cell scales its response to the persistence of the threat. Their entry point is MAP3K8, a kinase they had previously identified as one of the few genes induced specifically by IRF7 rather than by IRF3 or by type I interferon signaling. Working in human cells and in fibroblasts from Map3k8 knockout and wild type mice, with reporter assays, gel shift assays, coimmunoprecipitation, phosphorylation mapping and messenger RNA deep sequencing, they establish that MAP3K8 is transcribed and activated downstream of IRF7 during viral infection and that it then acts back on IRF3. MAP3K8 suppresses reporter and endogenous gene output that depends on IRF3 alone, an effect that added IRF7 reverses. It does not block IRF3 activation, nuclear entry or association with the coactivator CBP. Instead it drives phosphorylation within the proline-rich hinge of IRF3 and promotes formation of IRF3 and IRF7 heterodimers. Cells lacking MAP3K8 support higher virus titers and fail to induce roughly seventy infection-induced genes, including three SP100 family members, and expressing SP100B or SP100C raises interferon beta transcription and lowers virus titers. The authors propose a feed forward loop that upgrades the transcription factor in use once infection persists.", "Scientific context": "Interferon regulatory factors share a consensus binding element but differ in how strictly they require it. IRF3 needs an eight-nucleotide match, while IRF7 tolerates up to three deviations, so IRF7 reaches many more promoters and is often described as the master regulator of the response. IRF3 acts first, from a preexisting pool, and induces small amounts of interferon beta. Interferon beta signaling then raises IRF7, which amplifies the response. IRF3 and IRF7 were known to form both homodimers and heterodimers, and the heterodimer was known to be important for robust interferon beta induction, but what governs which dimer forms was not known. MAP3K8, also called TPL2 or COT, was known to be held inactive by the NF-kappaB1 precursor p105, to be released upon IKK activation, and to phosphorylate MEK and thereby activate ERK. Its characterized roles in innate immunity had come almost entirely from bacterial stimulation models in macrophages and dendritic cells, and the authors note that work with viral pathogens was lacking. Their own earlier comparison of the IRF3, IRF7 and ISGF3 transcriptomes had placed MAP3K8 in the small set of genes unique to IRF7, which motivated this study.", "Central question": "Does the IRF7-specific induction of MAP3K8 constitute a feedback arm of the antiviral response, and if so, how does this kinase alter the transcriptional output of the IRF3 and IRF7 system in a way that strengthens defense against a virus that has not been cleared?", "Experimental strategy": "The study moves from transcriptional regulation of the kinase, to its effect on IRF-dependent output, to the biochemical basis of that effect, and finally to consequence for virus replication and for the transcriptome. Promoter-level specificity is established with a luciferase construct driven by the human MAP3K8 promoter compared against an interferon-stimulated response element from ISG15, tested against IRF3, IRF7 and reconstituted ISGF3, and confirmed by gel shift with a 27-nucleotide element identified in the MAP3K8 promoter. Kinase activity is read out through ERK phosphorylation, with siRNA against MAP3K8 to establish that the ERK signal requires it, and with an attenuated influenza A virus carrying two substitutions in the double-stranded RNA-binding domain of NS1, chosen because it induces the innate response rather than suppressing it. Effects on output are probed with reporters for the IRF-binding region of the interferon beta promoter and for ISG15, driven by upstream activators including MDA5, constitutively active RIG-I, MAVS and synthetic double-stranded RNA, in each case with and without MAP3K8 and with IRF3 or IRF7 added back. Mechanism is dissected by asking in turn whether MAP3K8 blocks nuclear import, coactivator recruitment or dimer choice, using microscopy of an IRF3-GFP fusion, coimmunoprecipitation of CBP, and coimmunoprecipitation of tagged IRF3 and IRF7. The modification is localized by phosphatase treatment, by metabolic labeling with radiolabeled ATP, by a truncation series across the known domain structure of IRF3, and by alanine substitution of five residues in the hinge. Function is tested with an attenuated vesicular stomatitis virus that cannot block host messenger RNA export, comparing Map3k8 knockout and wild type fibroblasts by titer, by protein levels and by messenger RNA deep sequencing.", "Key findings": "1. The MAP3K8 promoter is engaged specifically by IRF7. Only activated IRF7 induced the MAP3K8 promoter reporter, while IRF3, IRF7 and ISGF3 all induced the ISG15 element reporter (Figure 1A), and in gel shift only activated IRF7 bound the newly identified IRF-binding element from the MAP3K8 promoter, while all three factors bound the comparable ISG15 element (Figure 1B,C).\n\n2. IRF7 activation drives ERK phosphorylation through MAP3K8. Activated IRF7, but not activated IRF3, produced robust ERK phosphorylation (Figure 1D), and this was abolished by a pool of three siRNAs against MAP3K8 with total ERK unchanged (Figure 1E,F). Cells stably expressing IRF7 induced MAP3K8 transcripts and phosphorylated ERK by 6 and 8 hours after infection with the NS1-mutant influenza A virus (Figure 1G,H).\n\n3. MAP3K8 suppresses output that depends on IRF3 and this is reversed by IRF7. MAP3K8 inhibited the interferon beta promoter reporter induced by double-stranded RNA, MDA5, active RIG-I or MAVS, and inhibited endogenous interferon beta transcripts (Figure 2A,C). Added IRF7 restored the response while added IRF3 did not (Figure 2B,D,E). The inhibition was dose-dependent and present at what the authors describe as physiological levels of the kinase (Figure 2F). The same pattern held for the ISG15 element (Figure 2G).\n\n4. The block is not at activation, import or coactivator recruitment. MAP3K8 did not prevent nuclear accumulation of an IRF3-GFP fusion driven by IKKε or MAVS, with roughly 80 percent of positive cells showing nuclear signal regardless of the kinase (Figure 3A,B), and did not disrupt the association between active IRF3 and CBP (Figure 3C).\n\n5. MAP3K8 promotes heterodimer formation. Low levels of IRF3 and IRF7 heterodimer were detectable without stimulus and were lost upon IRF3 activation, which the authors attribute to dominant homodimer formation. MAP3K8 strongly increased heterodimer recovery after activation, and CBP recovery fell in parallel (Figure 3D). The explanation offered for the reduced CBP, that the heterodimer presents only a single serine 396 phosphorylation event, is an interpretation drawn from prior literature rather than a measurement made here.\n\n6. MAP3K8 causes phosphorylation of IRF3 in the proline-rich hinge. IRF3 migrated more slowly in the presence of the kinase, a shift removed by calf intestinal alkaline phosphatase (Figure 4A), and metabolic labeling with radiolabeled ATP confirmed increased phosphorylation of IRF3 (Figure 4B). A truncation series placed the target region between residues 112 and 270, and comparison of constructs spanning residues 151 to 357 against 198 to 357 narrowed it to the proline-rich hinge (Figure 4C,D).\n\n7. Hinge residues are required for the induced interaction with IRF7. Substituting serine 173, serine 175, threonine 180 and serine 188 together with the associated cluster to alanine markedly reduced recovery of IRF7 with IRF3 in the presence of MAP3K8, while wild type IRF3 gave robust heterodimer (Figure 4E). The authors note that this region is distinct from the residues previously implicated in IRF7 binding at positions 306 to 357 but that published IRF dimer structures place the hinge near the dimer interface, making an intermolecular contribution plausible. They state explicitly that MAP3K8 may act directly or indirectly on IRF3, so the paper does not establish that IRF3 is a direct substrate.\n\n8. Loss of MAP3K8 increases virus replication. Fibroblasts lacking Map3k8 infected with the matrix-mutant vesicular stomatitis virus at low multiplicity produced significantly higher titers over 48 hours and higher viral glycoprotein at 12 hours (Figure 5A,B).\n\n9. The transcriptome is narrowed in the absence of the kinase. Messenger RNA sequencing at 10 hours after infection showed roughly seventy genes induced to higher levels in wild type than in knockout fibroblasts, including interferon alpha subtypes and IRF7-associated genes such as Oas2, Oas3, Herc6 and Gbp6. Three SP100 family members, Sp100, Sp110 and Sp140, were about fivefold higher in wild type cells, confirmed by quantitative PCR (Figure 6A,B).\n\n10. SP100 family promoters are IRF7 targets. Only activated IRF7 bound the previously identified IRF-binding elements from the SP100 and SP110 promoters in gel shift (Figure 6C), and activated IRF7 induced two SP100 splice variants and SP110 approximately fortyfold (Figure 6D).\n\n11. SP100B and SP100C contribute antiviral activity. Neither induced interferon beta on its own, but either raised MAVS-driven interferon beta transcripts roughly fivefold (Figure 7A). Expressing either reduced vesicular stomatitis virus titers by nearly a log and reduced viral glycoprotein while raising STAT1 (Figure 7B,C). The paper itself claims priority for the observation that SP100B and SP100C increase interferon beta expression during the antiviral response, and that claim is reported here as the paper's own.", "Mechanistic model": "The model advanced is a feed forward loop with a transcription factor upgrade at its center. IRF3, activated early by pattern recognition signaling, produces limited interferon beta because the enhanceosome is not optimized for IRF3 homodimers. Interferon beta raises IRF7. If pathogen-associated molecular patterns persist so that upstream kinases keep IRF7 active, IRF7 induces MAP3K8. MAP3K8 then phosphorylates or causes phosphorylation of the IRF3 hinge, which disfavors the IRF3 homodimer and favors the IRF3 and IRF7 heterodimer. Because IRF7 tolerates degenerate binding elements, the resulting complex reaches promoters the homodimer cannot, including those of the SP100 family, whose products in turn raise interferon beta output. MAP3K8 simultaneously activates ERK, which has been linked by others to assembly of promyelocytic leukemia nuclear bodies, the structures SP100 proteins occupy.\n\nSeveral steps are directly demonstrated. The IRF7 specificity of MAP3K8 induction, the requirement for MAP3K8 in virus-induced ERK activation, the shift in dimer composition, the localization of the phosphorylation to the hinge, the requirement for hinge residues in heterodimer formation, the transcriptome narrowing and the virus replication phenotype are all supported by experiment. Other steps are proposals. The paper does not establish that MAP3K8 phosphorylates IRF3 directly and says so. It does not show that hinge phosphorylation occurs on endogenous IRF3 during infection, nor identify which residues are modified as opposed to which are required. The connection from ERK to nuclear body assembly is drawn from other laboratories and is offered as a suggestion about how the arms of the pathway may be interconnected. The proposal that the loop scales the response to the duration of pathogen-associated molecular pattern production is a framing consistent with the data rather than a measured relationship.", "Conceptual or technical advance": "The work supplies a candidate answer to a standing question about IRF biology, namely what determines whether IRF3 acts as a homodimer or with IRF7. By locating a regulatory input in the proline-rich hinge, distinct from the C-terminal regulatory domain that receives the canonical activating phosphorylation and distinct from the mapped IRF7 contact residues, it separates dimer partner choice from activation and nuclear entry as independently controlled steps. It also reframes an apparently inhibitory effect. MAP3K8 reduces IRF3-only output, which in isolation reads as suppression of innate immunity, but in the presence of IRF7 the same activity broadens the response. That reading makes the phenotype of MAP3K8 loss interpretable and predicts that the kinase should matter only once IRF7 is available. Finally it brings SP100 family proteins into the interferon beta induction circuit as positive regulators, connecting the IRF7 and MAP3K8 arm to nuclear body biology.", "Relationship to the broader research program": "The study grows directly out of the 2010 comparison by Schmid, Mordstein, Kochs, García-Sastre and tenOever of the IRF3, IRF7 and ISGF3 transcriptomes, which supplied both the identification of MAP3K8 as an IRF7-unique target and several of the reagents and reporters used here. The recurring question across those two papers, and shared with the IKKε work from the same laboratory, is how a small set of related transcription factors with overlapping binding preferences is allocated so that the transcriptional output matches the situation rather than simply maximizing. In the IKKε papers the allocation is between STAT1-containing complexes and is set by a kinase acting on a dimer interface. Here the allocation is between IRF-containing complexes and is set by a kinase acting near a dimer interface. Reading those results as one recurring design principle is category 3 synthesis across the 2010, 2011 and 2014 papers and is offered as such.", "Related publications": "- Schmid, Mordstein, Kochs, García-Sastre and tenOever, 2010, Journal of Biological Chemistry, on transcription factor redundancy ensuring induction of the antiviral state. Predecessor. Identified MAP3K8 as an IRF7-specific target through comparison of IRF3, IRF7 and ISGF3 transcriptomes and supplied the IRF7 lentiviral vector, several expression plasmids and the reporter constructs used here.\n- Ng, Friedman, Schmid, Gertz, Myers, tenOever and Maniatis, 2011, Proceedings of the National Academy of Sciences, on IKKε and the balance between type I and type II interferon responses. Companion within the same program, sharing an author and the theme of kinase-set allocation of shared transcription factor subunits.\n- Panne, Maniatis and Harrison, 2007, on an atomic model of the interferon beta enhanceosome, and Escalante and colleagues, 2007, on the structure of IRF3 bound to the PRDIII-I element. Methodological foundation from other laboratories, the structural basis for the enhanceosome and dimer interface arguments made here.\n- Dumitru and colleagues, 2000, and related work from the Tsichlis laboratory on TPL2 signaling. Methodological foundation and predecessor from another laboratory, the source of the Map3k8 knockout fibroblasts and of the established MAP3K8 to MEK to ERK axis.", "Limitations and boundaries": "Much of the mechanistic work rests on transient overexpression in HEK293T cells, where levels of IRF3, IRF7, MAP3K8 and the upstream activators are set by transfection rather than by infection, and the paper's own statement that the inhibitory effect is evident at physiological levels of MAP3K8 is based on a dose titration in that system. Phosphorylation of the IRF3 hinge is shown for tagged IRF3 in the presence of exogenous MAP3K8 and is not demonstrated on endogenous IRF3 during infection, no phosphosite is directly identified by mass spectrometry, and whether MAP3K8 acts on IRF3 directly is left open. The alanine substitution experiment shows that those residues are required for the induced interaction but does not show that they are the modified residues. Virus work uses two attenuated viruses chosen for their inability to suppress the host response, an influenza A virus with a mutated NS1 and a vesicular stomatitis virus with a matrix substitution, so the findings speak to the behavior of the pathway rather than to the outcome of wild type infection. The Map3k8 knockout is constitutive and whole-cell, and the transcriptome comparison is a single time point at 10 hours in duplicate. The SP100 experiments rely on overexpression of two isoforms and do not test loss of function, so SP100B and SP100C are shown to be sufficient to raise interferon beta and lower titer, not to be necessary for the MAP3K8 phenotype. No animal experiments are reported, and the ERK to nuclear body link is inferred from the literature rather than tested.", "Audience summaries": "### 25 words\n\nA kinase induced late in infection modifies IRF3 so that it pairs with IRF7 instead of itself, widening the set of antiviral genes a cell can reach.\n\n### 75 words\n\nCells must match the strength of an antiviral response to the size of the threat. The authors describe a loop that does this. Persistent infection keeps IRF7 active, IRF7 induces the kinase MAP3K8, and MAP3K8 causes phosphorylation of IRF3 in a flexible hinge region. The modified IRF3 pairs with IRF7 rather than with itself, and the resulting complex reaches promoters the IRF3 pair cannot. Cells lacking the kinase induce fewer antiviral genes and support more virus.\n\n### 150 words\n\nIRF3 requires a stringent binding element while IRF7 tolerates degenerate ones, so which dimer forms determines how much of the antiviral genome is accessible. Building on their earlier finding that MAP3K8 is induced specifically by IRF7, the authors show that MAP3K8 is transcribed from an IRF7-selective element in its promoter, is activated during infection, and drives ERK phosphorylation. MAP3K8 suppresses interferon beta and ISG15 output that depends on IRF3 alone, an inhibition reversed by IRF7. It does not block IRF3 activation, nuclear entry or CBP binding. Instead it produces phosphorylation within the proline-rich hinge of IRF3, mapped by truncation, and hinge residues are required for the induced pairing with IRF7. Fibroblasts lacking MAP3K8 support higher vesicular stomatitis virus titers and fail to induce about seventy genes, among them Sp100, Sp110 and Sp140, and expressing SP100B or SP100C raises interferon beta and lowers titer. Direct phosphorylation of IRF3 by MAP3K8 is not established." }, "discoveries": [ "claim-04" ], "relationships": [ { "from": "2014-schmid-mitogen-activated-protein-kinase-m", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-schmid-mitogen-activated-protein-kinase-m" }, { "from": "2014-schmid-mitogen-activated-protein-kinase-m", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "companion", "evidence": "stated in the Related publications section of 2014-schmid-mitogen-activated-protein-kinase-m" } ], "canonical_url": "https://tenoeverlab.us/publications/2014-schmid-mitogen-activated-protein-kinase-m/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "vsv" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "immunofluorescence-microscopy", "sirna-knockdown", "luciferase-promoter-reporter", "site-directed-mutagenesis", "emsa", "co-ip", "translation-measurement", "phospho-immunoblotting" ] } }, { "id": "2014-shapiro-drosha-as-an-interferon-independen", "slug": "2014-shapiro-drosha-as-an-interferon-independen", "url": "/publications/2014-shapiro-drosha-as-an-interferon-independen/", "title": "Drosha as an interferon-independent antiviral factor", "authors": [ "Jillian S. Shapiro", "Sonja Schmid", "Lauren C. Aguado", "Leah R. Sabin", "Ari Yasunaga", "Jaehee V. Shim", "David Sachs", "Sara Cherry", "Benjamin R. tenOever" ], "author_count": 9, "first_author": "Jillian S. Shapiro", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 9, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2014, "journal": "Proceedings of the National Academy of Sciences", "volume": "111", "issue": "19", "pages": "7108-7113", "doi": "10.1073/pnas.1319635111", "doi_url": "https://doi.org/10.1073/pnas.1319635111", "pmid": "24778219", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/24778219/", "pmcid": "PMC4024876", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4024876/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4024876/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense" ], "themes": [ "rnase-iii-antiviral-effectors" ], "pathogens": [ "Sindbis virus", "vesicular stomatitis virus", "influenza A virus" ], "viral_families": [ "Togaviridae", "Rhabdoviridae", "Orthomyxoviridae" ], "host_species": [ "mouse", "human", "Drosophila melanogaster" ], "technologies": [ "conditional knockout with Cre-expressing adenoviral vectors", "small RNA northern blot", "small RNA deep sequencing", "RNA sequencing", "in vitro RNase cleavage assay", "subcellular fractionation", "immunofluorescence microscopy", "plaque assay", "RNA interference knockdown" ], "biological_systems": [ "primary mouse lung fibroblasts", "mouse embryonic fibroblasts", "HEK293T cells", "BHK cells", "Drosophila DL1 cells" ], "key_concepts": [ "Drosha", "Dicer", "antiviral RNA interference", "cytoplasmic Drosha translocation", "exportin 1 CRM1-dependent nuclear export", "interferon-independent antiviral defense", "viral genomic RNA cleavage", "host transcriptome modulation", "virus-derived small interfering RNAs", "microRNA biogenesis machinery" ], "keywords": [ "Drosha", "Dicer", "RNAi", "Sindbis virus", "CRM1", "interferon-independent", "microRNA", "innate immunity", "viral RNA cleavage", "Rnasen" ], "one_sentence_contribution": "Loss of the nuclear RNase III enzyme Drosha, but not of Dicer, increases RNA virus replication in mammalian fibroblasts, and diverse RNA viruses drive Drosha into the cytoplasm by CRM1-dependent export in a manner that does not require new protein synthesis, RIG-I, TBK1 or type I interferon signaling.", "summary_25": "Removing Drosha, an enzyme that normally makes microRNAs in the nucleus, lets RNA viruses grow better, and infection sends the enzyme into the cytoplasm.", "summary_75": "Insects and plants fight viruses with small interfering RNAs made by Dicer, a defense thought lost in mammals. In mouse fibroblasts, deleting Dicer did not change virus growth, but deleting Drosha did. Infection with several unrelated RNA viruses, or double-stranded RNA alone, pushed Drosha out of the nucleus by an export route that does not need interferon signaling. Drosha cut viral RNA and reshaped host gene expression without producing the classic small interfering RNAs.", "summary_150": "Antiviral RNA interference is well established outside chordates, and whether mammals retain a functional version remains contested. Using primary fibroblasts with conditional deletion of Drosha or Dicer, the authors found that Sindbis virus and vesicular stomatitis virus replicated to higher titers without Drosha, while Dicer loss had no significant effect. Positive sense, negative sense and segmented nuclear RNA viruses, and transfected double-stranded RNA, all triggered accumulation of Drosha in the cytoplasm. Export required CRM1 and proceeded without new protein synthesis and in cells lacking RIG-I, TBK1 or the type I interferon receptor. A Drosha mutant fixed in the cytoplasm by alanine substitution at serines 300 and 302 restricted Sindbis virus most strongly, tying localization to activity. Purified Drosha cleaved viral genomic RNA in vitro and Drosha shaped the infected host transcriptome, yet small RNA sequencing in fibroblasts and in Drosophila cells revealed no virus-derived small interfering RNA signature.", "citation": "Shapiro JS, Schmid S, Aguado LC, Sabin LR, Yasunaga A, Shim JV, Sachs D, Cherry S, tenOever BR. Drosha as an interferon-independent antiviral factor. Proceedings of the National Academy of Sciences. 2014. Volume 111, issue 19, pages 7108-7113. DOI 10.1073/pnas.1319635111. PMID 24778219. PMCID PMC4024876.", "sections": { "Citation": "Shapiro JS, Schmid S, Aguado LC, Sabin LR, Yasunaga A, Shim JV, Sachs D, Cherry S, tenOever BR. Drosha as an interferon-independent antiviral factor. Proceedings of the National Academy of Sciences. 2014. Volume 111, issue 19, pages 7108-7113. DOI 10.1073/pnas.1319635111. PMID 24778219. PMCID PMC4024876.", "One-sentence contribution": "Loss of the nuclear RNase III enzyme Drosha, but not of Dicer, increases RNA virus replication in mammalian fibroblasts, and diverse RNA viruses drive Drosha into the cytoplasm by CRM1-dependent export in a manner that does not require new protein synthesis, RIG-I, TBK1 or type I interferon signaling.", "Executive summary": "Antiviral RNA interference in plants, nematodes and arthropods depends on Dicer-generated virus-derived small interfering RNAs, and chordates were thought to have replaced this with the protein-based type I interferon system. Reports of residual mammalian antiviral RNA interference reopened the question of whether the small RNA machinery still contributes to defense in mammalian somatic cells, and if so through which enzyme.\n\nThe authors compared virus replication in primary fibroblasts conditionally deleted for Drosha or for Dicer using Cre-expressing adenoviral vectors, and followed the subcellular behavior of Drosha during infection. Sindbis virus and vesicular stomatitis virus both replicated to higher levels in the absence of Drosha, while deletion of Dicer had no significant effect. Infection with positive sense, negative sense and segmented nuclear RNA viruses, and transfection of double-stranded RNA, all drove Drosha from the nucleus into the cytoplasm within hours. Export depended on CRM1 and did not require de novo translation, RIG-I, TBK1 or the type I interferon receptor. Cytoplasmic localization was required for full antiviral activity, and mutation of serines 300 and 302 to alanine produced a constitutively cytoplasmic Drosha with the strongest restriction of Sindbis virus.\n\nDrosha cleaved Sindbis virus genomic RNA in vitro and altered the host transcriptome during infection, but small RNA sequencing found no virus-derived small interfering RNA signature in either fibroblasts or Drosophila cells. The antiviral activity is therefore attributed to something other than canonical RNA interference.", "Scientific context": "In plants, nematodes and arthropods, Dicer cleaves viral double-stranded RNA into small interfering RNAs that direct RNA-induced silencing complex cleavage of homologous viral RNA. Chordates retain much of this machinery but use it predominantly for microRNA biogenesis, with Drosha acting first in the nucleus and Dicer second in the cytoplasm. Virus-derived small interfering RNAs have been hard to detect in interferon-competent cells, yet two 2013 reports from other laboratories described antiviral RNA interference in mammalian cells, and a commentary co-authored by tenOever with Cullen and Cherry framed the open question of whether this is physiologically relevant. Separately, the tenOever laboratory had shown that a cytoplasmic RNA virus can be engineered to produce a functional microRNA and that this depends on Drosha appearing in the cytoplasm. The gap the paper addresses is whether either RNase III enzyme contributes to antiviral defense in ordinary somatic cells and what the responsible activity is.", "Central question": "Do the mammalian RNase III enzymes Drosha and Dicer contribute to the cellular response to RNA virus infection in somatic cells, and if the contribution is Drosha-dependent, does it operate through the production of virus-derived small interfering RNAs or through some other activity?", "Experimental strategy": "The design separates the two enzymes genetically and then asks what the responsible enzyme is doing. Conditional floxed alleles for Drosha and for Dicer in primary fibroblasts, disrupted by replication-incompetent adenoviral vectors delivering Cre, allow a clean loss-of-function comparison in the same cell background, with loss of the Drosha- and Dicer-dependent miR-93 as the functional readout of deletion and the Drosha- and Dicer-independent U6 as the control. Multicycle growth curves at low multiplicity then report on replication.\n\nSindbis virus was chosen because it had been shown to tolerate engineered microRNA production, which argues it does not encode a suppressor of RNA silencing, and the authors confirmed this by showing that inserted miR-124 target sites silence the virus when the microRNA is supplied. Vesicular stomatitis virus provides a negative sense comparator and a translation-independent test, since it packages its own polymerase and can therefore make transcripts under cycloheximide.\n\nLocalization was addressed with immunofluorescence across a virus panel, with subcellular fractionation after CRM1 knockdown, and with a genetic epistasis panel using Ddx58, Tbk1 and Ifnar1 deficient cells, reading out cytoplasmic Drosha activity through processing of a virus-encoded cytoplasmic primary microRNA rather than through imaging alone. Phosphosite mutants at serines 300 and 302 test whether localization and antiviral activity are coupled. Mechanism was probed by in vitro cleavage of viral genomic RNA with purified Drosha and by small RNA and messenger RNA sequencing with and without Drosha, in mammalian cells and in Drosophila cells.", "Key findings": "1. Drosha was nuclear in mock-treated murine fibroblasts and accumulated in the cytoplasm within six hours of infection with Sindbis virus, vesicular stomatitis virus or an NS1-deficient influenza A virus, and after transfection of double-stranded RNA (Figure 1A). The generality across genome types and the response to a pathogen-associated molecular pattern alone are direct observations.\n\n2. Drosophila DL1 cells infected with a Sindbis virus encoding primary miR-124 produced mature miR-124, and depleting Drosha abolished mature microRNA production while increasing unprocessed virus-derived primary microRNA and viral RNA (Figure 1B and 1C). The authors read this as cytoplasmic Drosha activity being conserved in arthropods.\n\n3. Sindbis virus carrying two or four miR-124 target sites was silenced only when miR-124 was supplied exogenously, indicating the virus does not encode a suppressor of RNA silencing (Figure 2A).\n\n4. Deletion of Drosha in conditional knockout fibroblasts raised Sindbis virus titers significantly across a multicycle growth curve and raised capsid protein levels, while deletion of Dicer did not significantly change titers or protein (Figure 2C to 2F). Vesicular stomatitis virus behaved the same way (Supplementary Figure 2). This is the central loss-of-function result.\n\n5. Knockdown of CRM1 prevented cytoplasmic accumulation of Drosha after infection and abolished processing of the cytoplasmic virus-derived primary microRNA while leaving endogenous miR-93 intact (Figure 3A to 3C). Cycloheximide did not prevent production of vesicular stomatitis virus-derived miR-124 despite loss of viral G protein (Figure 3D). Together these place the source of cytoplasmic Drosha in active nuclear export rather than in retention of newly made protein.\n\n6. Cytoplasmic Drosha activity was retained in cells lacking RIG-I, TBK1 or IFNAR1 (Supplementary Figure 3). The authors interpret this as evidence that the response is a separate, interferon-independent arm. The experiment shows that these three components are not required, and does not exclude other sensing routes.\n\n7. GFP-tagged Drosha that is wild type or phosphomimetic at serines 300 and 302 was nuclear, while the alanine mutant was constitutively cytoplasmic. Infection moved the wild-type protein to the cytoplasm but not the phosphomimetic form (Figure 4A). In a transfected genomic RNA assay, wild-type and phosphomimetic Drosha attenuated Sindbis virus by about one log while the constitutively cytoplasmic alanine mutant attenuated it by more than two logs (Figure 4B and 4C). The authors infer a virus-inducible phosphatase acting on these residues. No phosphatase is identified and the inference is not tested.\n\n8. Purified Drosha cleaved Sindbis virus genomic RNA in vitro, producing the expected pre-miR-124 product of about 55 nucleotides from the embedded hairpin and also generating species of about 20 to 25 nucleotides from the nonstructural polyprotein region, while purified GFP did not (Figure 5A).\n\n9. Small RNA deep sequencing of infected fibroblasts recovered over 675,000 reads across the Sindbis virus genome with no enrichment of the 21 nucleotide species characteristic of virus-derived small interfering RNAs, and loss of Drosha increased total viral small RNA reads without changing their profile or genomic distribution. The same was true in Drosophila cells (Figure 5B and 5C). This is the principal negative result and it is what separates the observed activity from canonical antiviral RNA interference.\n\n10. RNA sequencing of infected cells with and without Drosha showed more than 25 messenger RNAs induced greater than fivefold in the absence of Drosha, a comparable number downregulated, and more than 25 noncoding RNAs of unknown function upregulated, with Itga2 and Hspa1a confirmed by quantitative PCR (Figure 5D and Supplementary Figure 4).", "Mechanistic model": "The study does not establish a definitive mechanism for the antiviral effect, and the authors say as much, describing their account as speculation. What the data constrain is the following. Drosha limits RNA virus replication in primary fibroblasts, this restriction does not require Dicer and does not correlate with production of canonical virus-derived small interfering RNAs, and full restriction requires that Drosha be in the cytoplasm, where infection places it through CRM1-dependent export that is triggered independently of RIG-I, TBK1 and type I interferon signaling. Two non-exclusive activities are consistent with the data. Drosha can cleave structured regions of the viral genome directly, which would be expected to expose unprotected ends and promote decay, and Drosha shapes the host transcriptome during infection in ways that include induction and repression of dozens of transcripts.\n\nSeveral things are explicitly unresolved. The authors do not know whether the transcriptome effect originates in the nucleus or the cytoplasm. They do not identify the phosphatase implied by the serine 300 and 302 data, nor the sensing pathway that triggers export. They raise as an open question how cytoplasmic Drosha distinguishes viral from host RNA, suggesting adenine and uracil content or the primary sequence determinants recently described for primary microRNA hairpins, and they note it would be interesting to know whether viral genomes avoid such motifs. None of that is tested here. The claim that the response is conserved from arthropods to chordates rests on the Drosophila microRNA processing and small RNA data and is an extrapolation from those to a conserved defense.", "Conceptual or technical advance": "A microRNA biogenesis enzyme is shown to have a function in virus restriction that is separable from its canonical pathway, since Dicer loss has no equivalent effect and no small interfering RNA signature appears. This makes the subcellular relocalization of a nuclear RNase III enzyme a measurable, virus-inducible event with a defined transport requirement and a defined phosphorylation switch, and it supplies a tractable assay for that event in the form of processing of a virus-encoded cytoplasmic primary microRNA. It also sharpens the ongoing debate about mammalian antiviral RNA interference by showing that a genuine small RNA machinery contribution to antiviral defense can exist without the small interfering RNA products that the debate has centered on.", "Relationship to the broader research program": "The paper grows directly from earlier tenOever laboratory work showing that cytoplasmic RNA viruses can be engineered to make microRNAs, which required the unexpected observation of Drosha outside the nucleus, reported by Shapiro and colleagues in 2010 and 2012. The engineered microRNA target site approach used here as a control for suppressor of RNA silencing activity is the same tool the laboratory developed for tropism restriction and biocontainment. Alongside Schmid and colleagues 2010, which asked what antiviral transcription persists without interferon signaling, this paper asks what antiviral activity persists without interferon signaling at the level of RNA, and shares an author between the two. Category 3 synthesis, visible only when these papers are placed side by side, is a sustained interest in arms of the antiviral response that operate outside the interferon axis and in repurposing the RNA silencing machinery both as a tool and as a subject.", "Related publications": "- Shapiro and colleagues 2010, RNA, Noncanonical cytoplasmic processing of viral microRNAs. Predecessor. Established that a cytoplasmic RNA virus can yield a functional microRNA, which is the observation that led here.\n- Shapiro and colleagues 2012, RNA, Evidence for a cytoplasmic microprocessor of pri-miRNAs. Predecessor. Identified the Drosha translocation event whose generality and function are examined here.\n- Schmid and colleagues 2010, Journal of Biological Chemistry. Conceptual extension. Shares the question of interferon-independent antiviral responses and shares an author.\n- Langlois and colleagues 2012, Molecular Therapy, In vivo delivery of cytoplasmic RNA virus-derived miRNAs. Methodological foundation. Source of the VSV124 virus used in the translation-independence experiment.\n- Perez and colleagues 2009 and Langlois and colleagues 2013. Methodological foundation. Established the engineered microRNA target site approach used here to test for a suppressor of RNA silencing.\n- Cullen, Cherry and tenOever 2013, Cell Host and Microbe. Review or synthesis. Frames the question of whether RNA interference is a physiologically relevant mammalian antiviral response, which this paper addresses.\n- Sabin and colleagues 2013, PLoS ONE. Companion. Fly work from a co-author's laboratory on Dicer-2 processing of viral RNA species, cited as agreeing with the Drosophila results here.", "Limitations and boundaries": "The mammalian work is confined to fibroblasts, predominantly primary mouse lung fibroblasts and transformed human and hamster lines, and the authors qualify their Dicer conclusion as holding at least in the context of primary fibroblasts. No animal infection is performed, so the physiological weight of the effect in a whole organism is not addressed. The replication phenotype rests on two cytoplasmic RNA viruses, Sindbis virus and vesicular stomatitis virus, and the influenza A virus experiment addresses only Drosha localization, not replication. The magnitude of the effect on titer is modest relative to the effect of ectopic expression of the cytoplasmic Drosha mutant, and the ectopic experiments involve overexpression in transfected cells with viral genomic RNA rather than natural infection. Deletion of Drosha removes all microRNAs as well as the nuclease activity, so secondary consequences of microRNA loss cannot be excluded as contributors, and the authors' own RNA sequencing confirms that Drosha shapes the transcriptome even without infection. The in vitro cleavage assay uses purified enzyme and naked RNA, which does not establish that the same cleavage occurs on replicating genomes within cells. The absence of a virus-derived small interfering RNA signature is a negative result at the sequencing depth and in the cell types used, and other laboratories reported such signatures in different systems. Finally, the interferon independence claim rests on three specific genetic lesions and does not exclude other sensors or signaling routes.", "Audience summaries": "### 25 words\n\nRemoving Drosha, an enzyme that normally makes microRNAs in the nucleus, lets RNA viruses grow better, and infection sends the enzyme into the cytoplasm.\n\n### 75 words\n\nInsects and plants fight viruses with small interfering RNAs made by Dicer, a defense thought lost in mammals. In mouse fibroblasts, deleting Dicer did not change virus growth, but deleting Drosha did. Infection with several unrelated RNA viruses, or double-stranded RNA alone, pushed Drosha out of the nucleus by an export route that does not need interferon signaling. Drosha cut viral RNA and reshaped host gene expression without producing the classic small interfering RNAs.\n\n### 150 words\n\nAntiviral RNA interference is well established outside chordates, and whether mammals retain a functional version remains contested. Using primary fibroblasts with conditional deletion of Drosha or Dicer, the authors found that Sindbis virus and vesicular stomatitis virus replicated to higher titers without Drosha, while Dicer loss had no significant effect. Positive sense, negative sense and segmented nuclear RNA viruses, and transfected double-stranded RNA, all triggered accumulation of Drosha in the cytoplasm. Export required CRM1 and proceeded without new protein synthesis and in cells lacking RIG-I, TBK1 or the type I interferon receptor. A Drosha mutant fixed in the cytoplasm by alanine substitution at serines 300 and 302 restricted Sindbis virus most strongly, tying localization to activity. Purified Drosha cleaved viral genomic RNA in vitro and Drosha shaped the infected host transcriptome, yet small RNA sequencing in fibroblasts and in Drosophila cells revealed no virus-derived small interfering RNA signature." }, "discoveries": [ "claim-07" ], "relationships": [ { "from": "2010-schmid-transcription-factor-redundancy-en", "to": "2014-shapiro-drosha-as-an-interferon-independen", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2010-schmid-transcription-factor-redundancy-en" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2012-langlois-in-vivo-delivery-of-cytoplasmic-rn", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2012-perez-a-small-rna-enhancer-of-viral-poly", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" }, { "from": "2014-shapiro-drosha-as-an-interferon-independen", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "review or synthesis", "evidence": "stated in the Related publications section of 2014-shapiro-drosha-as-an-interferon-independen" }, { "from": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "to": "2014-shapiro-drosha-as-an-interferon-independen", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2017-aguado-rnase-iii-nucleases-from-diverse-k" } ], "canonical_url": "https://tenoeverlab.us/publications/2014-shapiro-drosha-as-an-interferon-independen/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "vsv", "sindbis-virus" ], "technologies": [ "bulk-rna-seq", "plaque-assay", "immunofluorescence-microscopy", "sirna-knockdown", "small-rna-seq", "small-rna-northern-blot", "in-vitro-reconstitution", "conditional-knockout", "subcellular-fractionation" ] } }, { "id": "2014-varble-influenza-a-virus-transmission-bot", "slug": "2014-varble-influenza-a-virus-transmission-bot", "url": "/publications/2014-varble-influenza-a-virus-transmission-bot/", "title": "Influenza A Virus Transmission Bottlenecks Are Defined by Infection Route and Recipient Host", "authors": [ "Andrew Varble", "Randy A. Albrecht", "Simone Backes", "Marshall Crumiller", "Nicole M. Bouvier", "David Sachs", "Adolfo García-Sastre", "Benjamin R. tenOever" ], "author_count": 8, "first_author": "Andrew Varble", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 8, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2014, "journal": "Cell Host & Microbe", "volume": "16", "issue": "5", "pages": "691-700", "doi": "10.1016/j.chom.2014.09.020", "doi_url": "https://doi.org/10.1016/j.chom.2014.09.020", "pmid": "25456074", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/25456074/", "pmcid": "PMC4272616", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272616/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272616/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "viral-populations-evolution" ], "themes": [ "transmission-bottlenecks", "fitness-landscapes" ], "pathogens": [ "influenza A virus", "influenza A/California/04/2009 (H1N1pdm09)" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "guinea pig", "ferret", "mouse", "chicken embryo", "canine cells", "human cells" ], "technologies": [ "reverse genetics", "genetic barcoding", "deep sequencing", "Illumina MiSeq", "plaque assay", "Monte Carlo simulation", "nebulized aerosol exposure" ], "biological_systems": [ "MDCK cells", "A549 cells", "embryonated chicken eggs", "guinea pig", "ferret", "mouse", "nasal wash", "bronchus tissue" ], "key_concepts": [ "transmission bottleneck", "viral quasispecies", "founder population", "stochastic transmission", "host adaptation", "hemagglutinin receptor specificity", "aerosol transmission", "contact transmission", "upper respiratory tract replication", "pandemic emergence" ], "keywords": [ "influenza A virus", "transmission bottleneck", "barcoded virus library", "ferret transmission model", "guinea pig transmission model", "aerosol transmission", "quasispecies", "deep sequencing", "egg adaptation", "H5N1 pandemic risk" ], "one_sentence_contribution": "Genetically barcoded influenza A virus libraries tracked through cell culture, embryonated eggs, guinea pigs, ferrets and mice show that transmission bottlenecks differ by route and recipient, with airborne transmission reducing a diverse inoculum to as few as two founder clones.", "summary_25": "Barcoded influenza viruses tracked through animals show that transmission passes only a handful of virus particles, with airborne spread the most restrictive and the recipient imposing the limit.", "summary_75": "Influenza A virus circulates as a mixed population, but how much of that mixture survives a transmission event was unclear. Inserting neutral genetic barcodes into more than a hundred otherwise identical viruses allowed the population to be read by sequencing at each step. Cell culture passed the population intact, egg passage selected avian adapted variants, and animal to animal transmission collapsed it to a few founders, with airborne transmission the narrowest route.", "summary_150": "Over one hundred influenza A viruses in the 2009 pandemic H1N1 background were rescued individually with unique neutral barcodes in an engineered intergenic region of the NS segment, pooled, and tracked by deep sequencing. Propagation in canine and human cells preserved the population. Propagation in embryonated chicken eggs collapsed it to five to thirteen clones, and all survivors carried hemagglutinin changes at residues associated with avian receptor binding, indicating genetically driven selection during host adaptation. Transmission between guinea pigs and between ferrets also collapsed the population, but without convergent hemagglutinin change, and three recipients exposed to one donor received different clone sets, placing the restriction at the recipient. Airborne transmission was more stringent than direct contact, with as few as two clones establishing infection. Transmission probability tracked with a clone's abundance in donor nasal wash rather than bronchus, implicating the upper respiratory tract as the source of transmitted virus.", "citation": "Varble A, Albrecht RA, Backes S, Crumiller M, Bouvier NM, Sachs D, García-Sastre A, tenOever BR. Influenza A Virus Transmission Bottlenecks Are Defined by Infection Route and Recipient Host. Cell Host & Microbe. 2014. Volume 16, issue 5, pages 691-700. DOI 10.1016/j.chom.2014.09.020. PMID 25456074. PMCID PMC4272616.", "sections": { "Citation": "Varble A, Albrecht RA, Backes S, Crumiller M, Bouvier NM, Sachs D, García-Sastre A, tenOever BR. Influenza A Virus Transmission Bottlenecks Are Defined by Infection Route and Recipient Host. Cell Host & Microbe. 2014. Volume 16, issue 5, pages 691-700.\n\nDOI 10.1016/j.chom.2014.09.020. PMID 25456074. PMCID PMC4272616.", "One-sentence contribution": "Genetically barcoded influenza A virus libraries tracked through cell culture, embryonated eggs, guinea pigs, ferrets and mice show that transmission bottlenecks differ by route and recipient, with airborne transmission reducing a diverse inoculum to as few as two founder clones.", "Executive summary": "Influenza A virus circulates as a genetically diverse population, and the size of the viral population that actually founds a new infection sets limits on how quickly the virus can move through genetic space. Before this study the physiological constraints on that founder population had been inferred largely from sequence surveys of natural infections rather than measured directly under controlled conditions. The authors built a library of more than one hundred influenza A viruses of the 2009 pandemic H1N1 background, each carrying a unique 22 nucleotide barcode in an engineered intergenic region of the NS segment, and confirmed that barcoding did not alter replication. Deep sequencing of the barcode pool then allowed the composition of the viral population to be read out at any point in propagation or transmission. Amplification in canine and human cell lines imposed no detectable bottleneck. Amplification in embryonated chicken eggs collapsed the library to between five and thirteen clones, and hemagglutinin sequencing of the survivors showed convergent changes at residues associated with avian receptor binding, indicating a genetically driven bottleneck during host adaptation. Transmission between susceptible mammals also collapsed the population, but without convergent hemagglutinin selection, and separate recipients exposed to the same donor received different clone sets. Airborne transmission was more restrictive than direct contact. The work separates adaptation bottlenecks from transmission bottlenecks and gives an experimental floor for how few virions initiate a natural influenza infection.", "Scientific context": "RNA virus populations carry standing genetic variation generated by an error prone polymerase, and that variation is a substrate for adaptation. Bottlenecks that repeatedly sample only a few genomes constrain that substrate, and severe repeated bottlenecks had been shown in other systems to reduce viral fitness. Natural bottlenecks had been described for human immunodeficiency virus, Venezuelan equine encephalitis virus, poliovirus, hepatitis C virus and several plant viruses, and genetic tagging approaches had been used to track poliovirus and Venezuelan equine encephalitis virus populations. For influenza A virus the practical parameters of transmission had been approached from other directions, including estimates of viral copies in exhaled breath and estimates of minimal infectious dose in human volunteers and in ferrets. What remained unresolved, as the paper states, was the physiological dynamics of the viral population during transmission as it occurs in an animal host, including whether observed restrictions reflect genetic selection on particular variants or a sampling process, and whether the restriction acts in the donor or in the recipient.", "Central question": "How severe are the population bottlenecks that influenza A virus encounters during amplification and transmission, and are those bottlenecks driven by genetic selection on particular viral variants or by stochastic sampling, and do they act at the level of the donor or the recipient?", "Experimental strategy": "The design rests on making viral genotype trackable without making it consequential. The NS segment was split so that NS1 and NS2 occupy separate reading frames with a noncoding intergenic region between them, following an approach the laboratory had established previously, and a 22 nucleotide GC content matched barcode was inserted into that region. Over one hundred such viruses were rescued individually by reverse genetics in the A/California/04/2009 background, then pooled at equivalent titers. Because the barcodes are intended to be neutral, any change in the barcode distribution between an input population and an output population reports on population sampling rather than on the fitness of the tag. Multicycle growth curves against wild type virus, pairwise comparison of amplified against nonamplified clones, and duplicate sequencing of the same population were used to test that assumption. The library was then passed through a graded series of settings, namely canine and human cell monolayers, embryonated chicken eggs, guinea pig transmission under conditions permitting droplet and aerosol spread, guinea pig cocaging that allowed one donor to seed three recipients, ferret transmission partitioned into direct contact and airborne arms in the same experiment, and mouse infection comparing intranasal instillation against nebulized delivery. Hemagglutinin sequencing was used to distinguish selection on receptor binding from sequence independent sampling. A Monte Carlo simulation was used to ask whether the observed distribution of successful transmissions is compatible with a model in which the only determinant of transmission is a clone's starting proportion in the donor.", "Key findings": "1. Barcode insertion into the split NS segment did not detectably alter replication. Multicycle growth of barcoded A/California/04/2009 matched wild type virus in human lung epithelial cells (Figure 1B), and the pooled library was stoichiometrically balanced with no clone above five percent of the population. Duplicate deep sequencing of the same population returned comparable barcode profiles, which the authors take as evidence that the readout is an accurate surrogate measure of the quasispecies.\n\n2. Propagation in cell culture imposed no detectable bottleneck. Infection of MDCK cells at low multiplicity gave uniform and reproducible barcode distributions across three independent experiments, and the same was seen in a human lung epithelial line (Figures 2A and 2B).\n\n3. Propagation in embryonated chicken eggs imposed a severe bottleneck, with only five to thirteen clones recovered per egg and different clone sets emerging in different replicates (Figure 2C). Clones that were and were not amplified showed no intrinsic replication difference when tested individually, so the barcode itself does not explain the outcome.\n\n4. The egg bottleneck was accompanied by convergent hemagglutinin change. Every virus sequenced from egg passage carried amino acid changes at residues previously implicated in the switch from mammalian to avian receptor specificity, whereas hemagglutinin sequences from MDCK passage showed no divergence from wild type (Figure S2B). A library pre-passaged once in eggs retained about fifty percent of its members on subsequent egg amplification against about ten percent for the original library. The authors read this as selective pressure for entry into the avian host rather than a sampling effect, with the caveat they state that beneficial mutations in other segments cannot be excluded.\n\n5. Transmission between guinea pigs in neighboring cages imposed a stringent bottleneck. Three of four exposed animals became infected, and those animals carried only two to five clones at day six (Figure 3B), against roughly three quarters of the library recoverable from the nasal wash of inoculated donors.\n\n6. The transmission bottleneck acts at the recipient and is not explained by viral genetics. When three naive guinea pigs were cocaged with a single donor, all three became infected and all three carried markedly different barcode profiles despite identical exposure (Figures 4C and 4D). The authors note that some selection at the point of secretion cannot be ruled out. Barcode diversity in recipients rose over time to an average of about twenty five barcodes per animal, which is interpreted as ongoing transmission or amplification after initial seeding.\n\n7. Route of transmission sets bottleneck stringency. In ferrets, contact recipients carried seven to twenty four clones where donors carried seventy one to one hundred (Figure 5B). Airborne transmission occurred in two of three exposed ferrets and reduced the recovered population to as few as two barcodes (Figure 5D). Mice infected with equivalent doses by nebulizer rather than by intranasal instillation likewise showed stronger bottlenecks (Figures S3C to S3E).\n\n8. Unlike the egg bottleneck, the ferret transmission bottleneck did not show convergent hemagglutinin selection. Viruses from directly inoculated, contact infected and airborne infected animals carried disparate mutations rather than the complete penetrance of particular residues seen in eggs (Figure S3A).\n\n9. The observed distribution of successful transmissions is statistically consistent with a stochastic bottleneck. Transmission probability correlated positively with a clone's starting proportion in the donor, and when that relationship was fitted and used in a Monte Carlo simulation, the observed distribution fell at the thirtieth percentile of simulated outcomes (Figures 6A to 6C), which the authors take as consistent with initial proportion being the only contributing factor.\n\n10. Transmitted virus tracks with the upper respiratory tract. Comparing barcode populations in donor nasal wash against donor bronchus tissue, only nasal wash proportions correlated significantly with transmission for both contact and airborne routes (Figure 7). There were eight nasal wash only transmission events and no examples of a bronchus only population transmitting. Simulation using bronchus proportions as the predictor made the observed transmissions unlikely, while nasal wash proportions gave consistent rates.", "Mechanistic model": "The study does not establish a single molecular mechanism for the transmission bottleneck, and the authors state their account of it as a hypothesis. What the data do constrain is a separation of two distinct kinds of restriction. During adaptation to a new host, here modeled by a mammalian virus amplifying in the avian egg, the restriction is genetically driven, with viruses that first acquire avian receptor binding changes in hemagglutinin outcompeting the rest of the inoculum. During transmission between already susceptible mammalian hosts, the restriction is sequence independent by the evidence available, since no convergent selection appears in hemagglutinin and since identically exposed recipients receive different clones. The authors propose that donors excrete virus with roughly equal opportunity per virion, that the founder population is therefore drawn in proportion to each clone's abundance at the site of shedding, and that the restriction is imposed at the recipient. The correlation of transmission with nasal wash rather than bronchus proportions is interpreted as indicating the upper respiratory tract as the source of transmitted virus for both contact and airborne routes. The data do not resolve whether an additional selection step occurs at secretion, do not identify a physical or immune barrier in the recipient that performs the sampling, and do not explain how viral fitness is maintained across repeated bottlenecks. The authors raise two alternatives for that last point, namely purifying selection favoring the fittest transmitted viruses, or heterogeneity among hosts such that some individuals impose weaker restriction and act as local reservoirs.", "Conceptual or technical advance": "A neutral barcode library in a replication competent influenza A virus, read out by deep sequencing, converts the size and composition of a founder population into a directly measurable quantity in live animals. That makes it possible to compare bottleneck stringency across routes and hosts within a single experimental frame rather than inferring it from consensus sequence divergence. The separation of genetically driven adaptation bottlenecks from sequence independent transmission bottlenecks is the conceptual result, and it reframes the pandemic emergence problem as two sequential and differently governed constraints. The observation that airborne transmission can be founded by very few genomes bears directly on estimates of how likely it is that a multi mutation avian phenotype present at low frequency in a donor would be carried into a new host, and the authors connect this to why an airborne transmissible H5N1 has not emerged despite numerous human infections.", "Relationship to the broader research program": "The barcoding approach builds directly on the laboratory's engineered split NS segment, which was developed to allow foreign sequence to be carried in the influenza A virus genome without disrupting NS1 and NS2 expression. That platform recurs across the corpus as a general tool for making the influenza genome carry a readable payload. The study also connects to the laboratory's interest in how the NS segment coordinates the timing of infection through suboptimal splicing, since the one poorly growing barcoded clone is attributed to a possible splicing effect. Category 3 synthesis, across the corpus rather than from this paper alone, is that the recurring strategy is to add a genetically tractable readout to a replicating virus and then let animal physiology rather than cell culture define the result. Confirming that synthesis requires setting this paper beside the other engineered influenza A virus work in the corpus, and it is not asserted from this paper in isolation.", "Related publications": "- Varble et al. 2010, engineered RNA viral synthesis of microRNAs, methodological foundation. The split NS segment with a noncoding intergenic region used to carry the barcodes is taken from this earlier work.\n- Chua et al. 2013, influenza A virus utilizes suboptimal splicing to coordinate the timing of infection, predecessor. Cited by the authors as the likely explanation for the single barcoded clone that replicated poorly.\n- Wilker et al. 2013, selection on hemagglutinin imposes a bottleneck during mammalian transmission of reassortant H5N1 viruses, companion. Cited in the discussion as an independent report of selective pressure on an adapting avian hemagglutinin within a mammalian host.", "Limitations and boundaries": "The findings rest on one viral background, the 2009 pandemic H1N1 strain A/California/04/2009, carrying an engineered split NS segment rather than the wild type segment, so generalization to other subtypes and to unmodified viral genomes is an extrapolation. Animal numbers are small, with three or four cages per transmission arm, and the airborne arm produced only two infected ferrets. The barcodes report clone identity and not full genome sequence, so within clone variation and reassortment are invisible to the readout, and only the hemagglutinin segment was sequenced to test for selection. The claim that the transmission bottleneck is sequence independent therefore rests on the absence of convergent hemagglutinin change rather than on genome wide evidence, and the authors explicitly allow that selection at the point of secretion may still occur and that beneficial mutations in segments other than hemagglutinin cannot be excluded in the egg experiments. The limit of detection was set at one hundred reads, which sets a floor on the minority clones that can be counted. The egg experiments model adaptation across a host species barrier in a specific and artificial setting rather than natural avian infection. Guinea pig, ferret and mouse models each recapitulate only parts of human influenza transmission, and no human data are presented. Finally, the stochastic model is a statistical consistency argument, since showing that observed outcomes fall within the range simulated under a proportion only model does not exclude other contributing factors.", "Audience summaries": "### 25 words\n\nBarcoded influenza viruses tracked through animals show that transmission passes only a handful of virus particles, with airborne spread the most restrictive and the recipient imposing the limit.\n\n### 75 words\n\nInfluenza A virus circulates as a mixed population, but how much of that mixture survives a transmission event was unclear. Inserting neutral genetic barcodes into more than a hundred otherwise identical viruses allowed the population to be read by sequencing at each step. Cell culture passed the population intact, egg passage selected avian adapted variants, and animal to animal transmission collapsed it to a few founders, with airborne transmission the narrowest route.\n\n### 150 words\n\nOver one hundred influenza A viruses in the 2009 pandemic H1N1 background were rescued individually with unique neutral barcodes in an engineered intergenic region of the NS segment, pooled, and tracked by deep sequencing. Propagation in canine and human cells preserved the population. Propagation in embryonated chicken eggs collapsed it to five to thirteen clones, and all survivors carried hemagglutinin changes at residues associated with avian receptor binding, indicating genetically driven selection during host adaptation. Transmission between guinea pigs and between ferrets also collapsed the population, but without convergent hemagglutinin change, and three recipients exposed to one donor received different clone sets, placing the restriction at the recipient. Airborne transmission was more stringent than direct contact, with as few as two clones establishing infection. Transmission probability tracked with a clone's abundance in donor nasal wash rather than bronchus, implicating the upper respiratory tract as the source of transmitted virus." }, "discoveries": [ "claim-11" ], "relationships": [ { "from": "2014-varble-influenza-a-virus-transmission-bot", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2014-varble-influenza-a-virus-transmission-bot" }, { "from": "2014-varble-influenza-a-virus-transmission-bot", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2014-varble-influenza-a-virus-transmission-bot" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2014-varble-influenza-a-virus-transmission-bot", "relationship": "companion", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" } ], "canonical_url": "https://tenoeverlab.us/publications/2014-varble-influenza-a-virus-transmission-bot/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "plaque-assay", "reverse-genetics", "viral-population-deep-sequencing", "barcoded-virus-library", "animal-transmission-model", "short-read-sequencing-platform", "population-diversity-statistics" ] } }, { "id": "2015-aguado-microrna-function-is-limited-to-cy", "slug": "2015-aguado-microrna-function-is-limited-to-cy", "url": "/publications/2015-aguado-microrna-function-is-limited-to-cy/", "title": "microRNA Function Is Limited to Cytokine Control in the Acute Response to Virus Infection", "authors": [ "Lauren C. Aguado", "Sonja Schmid", "David Sachs", "Jaehee V. Shim", "Jean K. Lim", "Benjamin R. tenOever" ], "author_count": 6, "first_author": "Lauren C. Aguado", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 6, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2015, "journal": "Cell Host & Microbe", "volume": "18", "issue": "6", "pages": "714-722", "doi": "10.1016/j.chom.2015.11.003", "doi_url": "https://doi.org/10.1016/j.chom.2015.11.003", "pmid": "26651947", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/26651947/", "pmcid": "PMC4683400", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683400/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683400/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense" ], "themes": [ "limits-of-microrna-function" ], "pathogens": [ "vaccinia virus", "adenovirus type 5" ], "viral_families": [ "Poxviridae", "Adenoviridae" ], "host_species": [ "human", "mouse" ], "technologies": [ "adenoviral vector delivery", "VP55 poly(A) polymerase microRNA degradation", "messenger RNA sequencing", "small RNA sequencing", "small RNA Northern blot", "luciferase reporter assay", "Luminex cytokine panel", "quantitative PCR", "gene ontology enrichment" ], "biological_systems": [ "BJ human foreskin fibroblasts", "293T cells", "NoDice 293T cells", "mouse lung" ], "key_concepts": [ "microRNA depletion", "post-transcriptional silencing", "intrinsic antiviral response", "type I interferon signalling", "cytokine derepression", "RNA-induced silencing complex inactivation", "kinetics of microRNA action", "let-7 regulation of IL6", "miR-23 regulation of IRF1" ], "keywords": [ "microRNA", "VP55", "interferon", "cytokine", "IL6", "let-7", "IRF1", "adenoviral vector", "transcriptome", "innate immunity" ], "one_sentence_contribution": "Rapid vector-delivered destruction of the entire cellular microRNA population shows that post-transcriptional silencing makes no measurable contribution to the acute transcriptional response to double-stranded RNA or to type I interferon, while sustained loss of microRNAs derepresses a broad set of chemokines and proinflammatory cytokines in fibroblasts and in mouse lung.", "summary_25": "Stripping cells of all microRNAs leaves the immediate antiviral response unchanged but releases a broad set of cytokines, placing microRNA control on inflammation rather than on defence itself.", "summary_75": "MicroRNAs tune protein levels, and their role in antiviral immunity has been argued both ways. Using a harmless adenovirus vector carrying a poxvirus enzyme that destroys microRNAs within a day, the authors removed them from primary human fibroblasts and from mouse lung. The response to double-stranded RNA and to interferon was essentially unaffected. Only after days of depletion did large changes appear, concentrated in chemokines and cytokines that recruit and activate immune cells.", "summary_150": "A replication-incompetent adenovirus delivering the vaccinia poly(A) polymerase subunit VP55 removes roughly 90 percent of abundant microRNAs from primary cells within a day without inducing interferon-stimulated genes and with minimal direct action on messenger RNAs, as judged in Dicer-deficient cells. In primary fibroblasts, microRNA loss altered 12 of 1,548 genes induced by transfected double-stranded RNA and 12 of 179 induced by interferon beta, with IRF1 among them and a miR-23 site in its untranslated region confirmed by reporter. Nine days of depletion, by contrast, changed over 1,700 transcripts while leaving most core antiviral components untouched, and the responsive set was dominated by chemokines and cytokines including IL6, whose repression was restored by a VP55-resistant let-7 mimic. Intranasal vector delivery raised six lung cytokines at the protein level in mice, in some cases without a matching transcript change. The authors frame microRNA control as acting on cytokine output over extended timescales.", "citation": "Aguado LC, Schmid S, Sachs D, Shim JV, Lim JK, tenOever BR. microRNA Function Is Limited to Cytokine Control in the Acute Response to Virus Infection. *Cell Host & Microbe* 2015, volume 18, issue 6, pages 714-722. DOI 10.1016/j.chom.2015.11.003. PMID 26651947. PMCID PMC4683400.", "sections": { "Citation": "Aguado LC, Schmid S, Sachs D, Shim JV, Lim JK, tenOever BR. microRNA Function Is Limited to Cytokine Control in the Acute Response to Virus Infection. *Cell Host & Microbe* 2015, volume 18, issue 6, pages 714-722.\n\nDOI 10.1016/j.chom.2015.11.003. PMID 26651947. PMCID PMC4683400.", "One-sentence contribution": "Rapid vector-delivered destruction of the entire cellular microRNA population shows that post-transcriptional silencing makes no measurable contribution to the acute transcriptional response to double-stranded RNA or to type I interferon, while sustained loss of microRNAs derepresses a broad set of chemokines and proinflammatory cytokines in fibroblasts and in mouse lung.", "Executive summary": "Whether microRNAs participate in mammalian antiviral defence has been contested. Some reports assign microRNAs a substantial share of interferon's antiviral activity, others find no effect on virus replication, and separate work shows that the silencing complex is inactivated by cellular stress and by infection. Testing the question requires removing microRNAs quickly, in cells that are not transformed and not already responding to a replicating virus. The authors built that tool by placing the vaccinia virus poly(A) polymerase subunit VP55, which tails and degrades Argonaute-associated small RNAs, into a replication-incompetent adenovirus vector. The vector removes the abundant microRNAs within about a day, does not itself induce interferon-stimulated genes, and shows essentially no off-target transcriptional activity in cells already lacking Dicer. Applied to primary human foreskin fibroblasts, microRNA removal changed only twelve of the roughly 1,500 genes induced by transfected double-stranded RNA, and only twelve of the genes induced by six hours of interferon beta. Extending microRNA loss to nine days, by contrast, altered more than 1,700 transcripts, with central components of the antiviral machinery mostly unchanged but with a broad set of chemokines and cytokines strongly derepressed. Delivered intranasally to mice, the vector raised six of eighteen measured cytokines in whole lung within 48 hours and changed more than 2,400 transcripts, with the strongly induced set enriched for immune activation and adhesion categories. The authors conclude that microRNA control in the antiviral setting operates on cytokine output rather than on the intrinsic response itself.", "Scientific context": "Mammals defend against viruses chiefly with a protein-based stratified system of intrinsic effectors, innate cellular responses and adaptive immunity, in contrast to organisms that rely on pathogen-derived small RNAs. Whether the microRNA arm of the small RNA machinery nonetheless contributes has been disputed. Prior reports had claimed microRNAs account for as much as half of type I interferon's antiviral activity, while other work found many human viruses refractory to inhibition by endogenous microRNAs. Meanwhile, two lines of evidence suggested that the silencing complex is not available during infection at all, since it is inactivated by cellular stress and is ribosylated beyond roughly eight hours of infection. Earlier work from this laboratory had shown that poxviruses tail and degrade microRNAs through VP55 and that delivering VP55 from a replicating vesicular stomatitis virus attenuated that virus, but the inflammatory environment created by the replicating vector confounded any attempt to separate microRNA function on cytokines from secondary interferon-stimulated gene induction. The gap the paper addresses is therefore a clean, fast, inert removal of microRNAs in primary cells.", "Central question": "Do endogenous microRNAs shape the mammalian cellular response to virus infection, and if their influence is limited, what part of that response do they actually control?", "Experimental strategy": "The strategy is subtractive and depends entirely on the quality of the subtraction. VP55 removes microRNAs post-transcriptionally rather than blocking their synthesis, so depletion is fast and does not require waiting out the turnover of existing pools. Putting VP55 in a replication-incompetent adenovirus with a modified fibre for use in primary cells gives delivery into terminally differentiated cells without provoking an antiviral response, which the authors verify by transcriptome comparison of vector-treated and untreated fibroblasts. Specificity for small RNAs rather than messenger RNAs is tested in Dicer-deficient cells, where any transcriptome change caused by VP55 must be microRNA-independent. Stimuli are chosen to isolate different arms. Transfected double-stranded RNA drives the intrinsic response at a point that mimics peak viral replication but is cytotoxic, so interferon beta treatment is used in parallel because it produces a comparable transcriptional response without cell death and therefore allows a longer window. Because microRNA effects are individually small, the design then varies time rather than stimulus, comparing depletion at one day with depletion at nine days to ask whether the acute-phase null result reflects an absence of targets or simply insufficient time. Finally, the vector is given intranasally to mice so that cytokine protein, not just transcript, can be measured in tissue.", "Key findings": "1. VP55 expressed as a fluorescent fusion degraded both abundant endogenous and overexpressed microRNAs and abolished let-7-mediated repression of a reporter, and in Dicer-deficient cells it altered under 0.35 percent of the transcriptome, with the notable exceptions being histone cluster transcripts that lack a poly(A) tail but contain a VP55 consensus site (Figure 1, A and B). The tool therefore acts on small RNAs with limited direct action on messenger RNAs.\n2. Delivered from the adenovirus vector, VP55 reduced the most abundant microRNAs, which make up more than half of the total, by about 90 percent, with degradation visible by 16 hours and uniform across the microRNA population by sequencing. The control vector did not perturb microRNA biogenesis, in contrast to replication-competent adenovirus, and neither vector was toxic (Figure 1, C and D).\n3. Control vector treatment changed only 48 genes relative to mock in fibroblasts, none of them interferon-stimulated genes or intrinsic response components, which is the basis for treating the platform as inert in this context.\n4. Removing microRNAs in immortalised fibroblasts changed about 10 percent of expressed genes, 1,346 of 13,185, over 24 hours (Figure 1E). The scale of this baseline effect makes the subsequent null results in stimulated cells informative rather than a failure of the tool.\n5. In primary BJ fibroblasts, transfected double-stranded RNA induced 1,548 differentially expressed genes, and removing microRNAs altered only 12 of them (Figure 2C). The authors state this makes microRNA-mediated repression unlikely to be a physiological contributor to the intrinsic response to a replicating virus.\n6. Six hours of interferon beta induced 179 genes by more than twofold, and microRNA loss significantly changed only 12, most notably IRF1, confirmed at transcript and protein level (Figure 2, F through H). A reporter carrying the IRF1 three prime untranslated region was repressed by a miR-23 mimic and the effect was lost when the predicted site was mutated (Figure 2I). Differentially regulated transcripts changed only two to fourfold.\n7. Extending interferon treatment to 24 hours raised the number of microRNA-sensitive genes to 78, still without central mediators of the interferon response among them. The authors read the combined datasets as showing that microRNA function requires time to produce significant transcriptome change.\n8. Nine days of microRNA loss changed 1,058 genes up and 699 down by at least twofold, compared with 49 induced at 24 hours (Figure 3, A and B). Even at nine days, IFIH1, IRF3, IRF7, RELA, RELB, IFNB, IFNAR1, STAT2 and IRF9 were unchanged, with modest changes in RIG-I, IRF1 and STAT1.\n9. The transcripts that did respond were dominated by chemokines and cytokines, including IL8, CXCL1, CXCL2, CXCL6, CCL2 and CCL7, which recruit antigen-presenting cells and neutrophils, regulators of haematopoiesis including IL1B, IL11, IL33, CSF1 and CSF2, and IL6 (Figure 3C). These were absent from the Dicer-deficient control dataset, which argues they reflect genuine microRNA targeting rather than direct VP55 action.\n10. IL6 was induced roughly sixfold by microRNA depletion during interferon treatment, and supplying a chemically protected let-7 mimic that resists VP55 restored repression of IL6. A reporter carrying the IL6 three prime untranslated region was repressed by let-7 and the effect was lost on mutating the predicted site (Figure 3, D through G).\n11. Intranasal delivery of the VP55 vector to mice significantly raised six of eighteen measured lung cytokines at 48 hours, including CCL20, CCL7, CCL4, CXCL1, CXCL9 and CCL2 (Figure 4A). For CCL2 and CCL4 the protein increase was matched by transcript increase, whereas CXCL1 and CCL7 protein rose while transcript was unchanged or reduced. The authors interpret this split as microRNA relief acting through both messenger RNA stability and translation, which is a reasonable reading of the discordance rather than a direct measurement of either process.\n12. Lung transcriptome profiling showed 50 genes changed by the control vector against 2,427 by the VP55 vector. The 139 genes induced most strongly were enriched for immune response regulation, leukocyte and lymphocyte activation and cell adhesion categories, while the 138 most strongly decreased were enriched for morphogenesis and development (Figure 4, C and D).", "Mechanistic model": "The paper does not establish a mechanism for how microRNA loss is coupled to cytokine output during infection, and the central proposal in the discussion is explicitly framed as an idea the data support rather than demonstrate. What the data constrain is the target set and the timescale. Post-transcriptional silencing exerts a constitutive, individually modest repression on a diverse group of cytokines and chemokines, demonstrated here by derepression on microRNA removal and, for IL6 and IRF1, by rescue with a protected mimic and by reporter assays with mutated target sites. That repression requires days rather than hours to change transcript levels appreciably, so it cannot shape the acute transcriptional response to double-stranded RNA or interferon, which unfolds over hours. From this the authors propose that the known inactivation of the silencing complex during infection, reported by others to occur beyond about eight hours, is itself a mechanism by which the host derepresses inflammatory transcripts without requiring new transcription, and they extend this to suggest that the influenza A virus NS1 protein, reported by others to block that inactivation, would thereby dampen cytokine output. Neither the causal link from silencing complex inactivation to cytokine burst during a real infection nor the NS1 consequence is tested in this paper. The split between transcript and protein responses in lung is described but its basis is not resolved.", "Conceptual or technical advance": "The vector is a general tool. Rapid, near-complete and uniform removal of the cellular microRNA population in terminally differentiated primary cells, achieved post-transcriptionally and without provoking an antiviral response, makes it possible to define stimulus-specific microRNA target sets by subtraction, and it can be applied in vivo. Conceptually, the work separates two questions that had been conflated, whether microRNAs act during the antiviral response and whether they act on the antiviral response, and answers them differently. It also supplies a timescale argument that reconciles the conflicting literature, since assays run over hours would find nothing even where genuine targets exist. The reframing of microRNA function as a link between the intrinsic response and the recruitment and activation of immune cells makes the cytokine target set, rather than interferon-stimulated genes, the place to look for microRNA effects on infection outcome.", "Relationship to the broader research program": "The work continues a line in the laboratory asking whether small RNA silencing is a physiologically relevant antiviral mechanism in mammals. Its own citations include earlier reports from the group on poxvirus degradation of host microRNAs through VP55 and terminal RNA methylation as a protective feature, on the mammalian response to virus infection being independent of small RNA silencing when VP55 is delivered from a replicating vesicular stomatitis virus, on noncanonical cytoplasmic processing of viral microRNAs, and on RNA viruses and the host microRNA machinery. The present paper is presented by the authors as an improvement on the earlier vesicular stomatitis virus delivery, because the inert adenovirus vector removes the confound of a replicating virus. Placing this against the laboratory's later work on antiviral RNA interference would be category 3 synthesis and is not attempted here.", "Related publications": "- Backes and colleagues, 2012, methodological foundation. The discovery that poxvirus VP55 tails and degrades host microRNAs, which supplies the enzymatic basis for the tool and for the protected-mimic rescue strategy.\n- Backes and colleagues, 2014, predecessor. Delivery of VP55 from a replicating vesicular stomatitis virus, whose inflammatory confound this paper is explicitly designed to remove.\n- Shapiro and colleagues, 2010, predecessor. Cited in support of the conclusion that loss of Dicer does not affect RNA virus titres in culture.\n- tenOever, 2013, review or synthesis. The laboratory's review of RNA viruses and the host microRNA machinery, cited for the framing of the stratified mammalian defence system.\n- Cullen, Cherry and tenOever, 2013, review or synthesis. A co-authored perspective on whether RNA interference is a physiologically relevant innate antiviral response in mammals, cited for the state of the question.", "Limitations and boundaries": "The conclusions rest on removing microRNAs and observing what changes, which detects only repression that is active at baseline and would miss a microRNA whose function requires induction that the vector timeline does not allow. VP55 acts on poly(A) substrates and does affect some non-microRNA transcripts, notably histone cluster messages, so the Dicer-deficient control bounds but does not eliminate direct effects. The acute experiments use transfected double-stranded RNA and recombinant interferon beta as surrogates for infection rather than a replicating virus, which was a deliberate choice to avoid confounds but means no virus replication phenotype is measured here. Cell systems are human foreskin fibroblasts, immortalised fibroblasts and 293T lines, so the findings do not on their own extend to immune cell types, epithelium or other tissues. The nine-day depletion regime, while informative about kinetics, is not a physiological state for an infected cell and the authors accordingly frame the relevance as chronic infection or persistence rather than acute disease. The in vivo work is a single 48-hour timepoint in mouse lung after vector delivery with no pathogen present, with five animals per group and eighteen cytokines measured, and it therefore shows derepression rather than a consequence for infection outcome. The mechanistic claims about silencing complex inactivation during infection and about NS1 are taken from other laboratories' work and are not tested here.", "Audience summaries": "### 25 words\n\nStripping cells of all microRNAs leaves the immediate antiviral response unchanged but releases a broad set of cytokines, placing microRNA control on inflammation rather than on defence itself.\n\n### 75 words\n\nMicroRNAs tune protein levels, and their role in antiviral immunity has been argued both ways. Using a harmless adenovirus vector carrying a poxvirus enzyme that destroys microRNAs within a day, the authors removed them from primary human fibroblasts and from mouse lung. The response to double-stranded RNA and to interferon was essentially unaffected. Only after days of depletion did large changes appear, concentrated in chemokines and cytokines that recruit and activate immune cells.\n\n### 150 words\n\nA replication-incompetent adenovirus delivering the vaccinia poly(A) polymerase subunit VP55 removes roughly 90 percent of abundant microRNAs from primary cells within a day without inducing interferon-stimulated genes and with minimal direct action on messenger RNAs, as judged in Dicer-deficient cells. In primary fibroblasts, microRNA loss altered 12 of 1,548 genes induced by transfected double-stranded RNA and 12 of 179 induced by interferon beta, with IRF1 among them and a miR-23 site in its untranslated region confirmed by reporter. Nine days of depletion, by contrast, changed over 1,700 transcripts while leaving most core antiviral components untouched, and the responsive set was dominated by chemokines and cytokines including IL6, whose repression was restored by a VP55-resistant let-7 mimic. Intranasal vector delivery raised six lung cytokines at the protein level in mice, in some cases without a matching transcript change. The authors frame microRNA control as acting on cytokine output over extended timescales." }, "discoveries": [ "claim-08" ], "relationships": [ { "from": "2015-aguado-microrna-function-is-limited-to-cy", "to": "2012-backes-degradation-of-host-micrornas-by-p", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-aguado-microrna-function-is-limited-to-cy" }, { "from": "2015-aguado-microrna-function-is-limited-to-cy", "to": "2014-backes-the-mammalian-response-to-virus-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-aguado-microrna-function-is-limited-to-cy" }, { "from": "2015-aguado-microrna-function-is-limited-to-cy", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-aguado-microrna-function-is-limited-to-cy" }, { "from": "2015-aguado-microrna-function-is-limited-to-cy", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "review or synthesis", "evidence": "stated in the Related publications section of 2015-aguado-microrna-function-is-limited-to-cy" }, { "from": "2016-tenoever-the-evolution-of-antiviral-defense", "to": "2015-aguado-microrna-function-is-limited-to-cy", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2016-tenoever-the-evolution-of-antiviral-defense" } ], "canonical_url": "https://tenoeverlab.us/publications/2015-aguado-microrna-function-is-limited-to-cy/", "controlled_vocabulary": { "pathogens": [ "vaccinia-virus", "adenovirus" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "small-rna-seq", "small-rna-northern-blot", "pathway-enrichment-analysis", "luciferase-promoter-reporter", "multiplex-cytokine-assay", "adenoviral-vector", "vp55-mirna-ablation" ] } }, { "id": "2015-benitez-engineered-mammalian-rnai-can-elic", "slug": "2015-benitez-engineered-mammalian-rnai-can-elic", "url": "/publications/2015-benitez-engineered-mammalian-rnai-can-elic/", "title": "Engineered Mammalian RNAi Can Elicit Antiviral Protection that Negates the Requirement for the Interferon Response", "authors": [ "Asiel Arturo Benitez", "Laura Adrienne Spanko", "Mehdi Bouhaddou", "David Sachs", "Benjamin Robert tenOever" ], "author_count": 5, "first_author": "Asiel Arturo Benitez", "senior_authors": [ "Benjamin Robert tenOever" ], "corresponding_authors": [ "Benjamin Robert tenOever" ], "tenoever_position": 5, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2015, "journal": "Cell Reports", "volume": "13", "issue": "7", "pages": "1456-1466", "doi": "10.1016/j.celrep.2015.10.020", "doi_url": "https://doi.org/10.1016/j.celrep.2015.10.020", "pmid": "26549455", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/26549455/", "pmcid": "PMC4654977", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654977/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654977/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense", "viral-populations-evolution" ], "themes": [ "reconstructing-antiviral-rnai", "recombination-and-escape" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "human", "mouse", "dog", "chicken" ], "technologies": [ "influenza reverse genetics", "microRNA target site insertion", "virus-encoded artificial microRNA", "luciferase reporter assay", "small RNA Northern blotting", "multicycle growth curves", "plaque assay", "mRNA sequencing", "flow cytometry", "lung histology" ], "biological_systems": [ "MDCK cells", "MDCK cells expressing miR-124", "A549 cells", "HEK293T cells", "NoDice Dicer-deficient cells", "murine embryonic fibroblasts", "Irf3 and Irf7 double knockout fibroblasts", "embryonated chicken eggs", "C57BL/6 mice", "Ifnar1 knockout mice" ], "key_concepts": [ "antiviral RNA interference", "type I interferon system", "engineered RNAi", "self-targeting virus", "escape mutants", "target complementarity threshold", "species-specific microRNA attenuation", "NS1 and small RNA silencing", "evolution of antiviral strategies", "live attenuated vaccine design" ], "keywords": [ "RNAi", "influenza A virus", "interferon", "miR-124", "microRNA targeting", "attenuation", "escape mutants", "Dicer", "Ifnar1", "evolution" ], "one_sentence_contribution": "Recreating a small RNA antiviral response in mice, using either host microRNAs repurposed as virus-specific guides or a virus-encoded artificial small interfering RNA, attenuates influenza A virus by more than five logs and prevents disease without any requirement for type I interferon signaling.", "summary_25": "Influenza was engineered so host small RNAs silence it. The resulting virus caused no disease in mice, including animals unable to respond to type I interferon.", "summary_75": "Plants and insects fight viruses with RNA interference while mammals use interferon, and why the switch happened is unknown. Researchers rebuilt a small RNA defense by inserting microRNA target sites into influenza and by having the virus produce a guide against itself. Attenuation reached more than five logs, escape occurred only by deleting the guide rather than altering the target, and protection in mice was complete even without type I interferon signaling.", "summary_150": "Chordates retain the small RNA machinery but defend against viruses through type I interferon rather than RNA interference, and it is unclear whether that reflects an incompatibility with mammalian cells. This study reconstructs a small RNA defense against influenza A virus in two ways, by inserting complementary target sites for cell-restricted or species-restricted host microRNAs and by engineering the virus to encode a small interfering RNA against its own nucleoprotein segment. Silencing required about 16 nucleotides of contiguous complementarity. Self-targeting attenuation was lost in Dicer-deficient cells, and escape variants always disabled guide production rather than altering the target. A virus carrying five mammalian microRNA target sites grew in eggs but was undetectable in mammalian cells and caused no morbidity in mice at 25,000 plaque-forming units, including in animals lacking the type I interferon receptor. The authors conclude that chordates could have used RNA interference in place of interferon.", "citation": "Benitez AA, Spanko LA, Bouhaddou M, Sachs D, tenOever BR. Engineered Mammalian RNAi Can Elicit Antiviral Protection that Negates the Requirement for the Interferon Response. Cell Reports. 2015. Volume 13, Issue 7, pages 1456-1466. DOI 10.1016/j.celrep.2015.10.020. PMID 26549455. PMCID PMC4654977. RNA sequencing data under GEO accession GSE73698. Asiel Arturo Benitez and Laura Adrienne Spanko are designated co-first authors in the paper's author block.", "sections": { "Citation": "Benitez AA, Spanko LA, Bouhaddou M, Sachs D, tenOever BR. Engineered Mammalian RNAi Can Elicit Antiviral Protection that Negates the Requirement for the Interferon Response. Cell Reports. 2015. Volume 13, Issue 7, pages 1456-1466.\n\nDOI 10.1016/j.celrep.2015.10.020. PMID 26549455. PMCID PMC4654977. RNA sequencing data under GEO accession GSE73698.\n\nAsiel Arturo Benitez and Laura Adrienne Spanko are designated co-first authors in the paper's author block.", "One-sentence contribution": "Recreating a small RNA antiviral response in mice, using either host microRNAs repurposed as virus-specific guides or a virus-encoded artificial small interfering RNA, attenuates influenza A virus by more than five logs and prevents disease without any requirement for type I interferon signaling.", "Executive summary": "Prokaryotes use CRISPR and plants, arthropods and nematodes use RNA interference to mount pathogen-specific nucleic acid defenses. Chordates retain most of the small RNA machinery but respond to viruses instead through the type I interferon system, a protein-based defense that is not tailored to the incoming pathogen. Why that substitution occurred is unknown, and one possible explanation is that a small RNA defense simply would not work well in a mammalian cell.\n\nThis study tests that explanation by building the missing response rather than by looking for it. Complementary target sites for cell-restricted or species-restricted host microRNAs were inserted into influenza A virus, converting endogenous microRNAs into virus-specific guides, and separately a virus was engineered to encode its own small interfering RNA directed against one of its own segments.\n\nSilencing required roughly 16 nucleotides of contiguous complementarity, and attenuation in cells reached several logs. Serial passage and mixed-culture passage failed to yield escape mutants that had altered the target site. When escape did occur in self-targeting viruses, it always came from deleting the hairpin that produced the guide rather than from mutating the target. A virus carrying five distinct mammalian microRNA target sites could still be grown in eggs but produced no detectable plaques or nucleoprotein in mammalian cells, caused no morbidity in mice up to 25,000 plaque-forming units, and was equally harmless in mice lacking the type I interferon receptor.", "Scientific context": "The paper sets out the comparative picture. Prokaryotes use CRISPR to guide a Cas nuclease with a pathogen-specific RNA template. Many eukaryotes use RNA interference, in which a Dicer-family RNase III enzyme processes viral RNA into 21 to 24 nucleotide small interfering RNAs that load into an Argonaute-containing RISC. Chordates retain much of that machinery, yet antiviral use of it appears limited to plants, arthropods and nematodes, and chordates rely instead on type I interferon.\n\nChordates do run a small RNA defense against transposable elements through PIWI-interacting RNAs, but that is confined to germ cells. The paper notes that some results support a small RNA antiviral response in pluripotent cells while evidence from differentiated cells is lacking, and that removing Dicer from mammalian fibroblasts does not change replication of most viruses. It further notes evidence that the two systems may be incompatible, citing observations that stem cells process double-stranded RNA without making interferon while differentiated cells do the reverse, that the interferon response shuts down RISC, and that expressing an antiviral Dicer induces interferon.\n\nOn timing, the paper cites data from chickens placing the emergence of the interferon system before the divergence of mammals and birds around 350 million years ago, supported by interferon induction in fish, which would place it before the recombination-based adaptive immune system of jawed vertebrates. The stated gap is that the basis for why chordates apparently abandoned RNA interference in favor of interferon remains unknown.", "Central question": "Is there something about mammalian cells that prevents a small RNA antiviral defense from working, or could chordates have used RNA interference in place of the type I interferon system with comparable protective effect?", "Experimental strategy": "The strategy is reconstruction rather than ablation. Instead of asking whether an endogenous mammalian RNA interference response exists, the study builds a functionally equivalent one and measures how well it protects, including in animals where the interferon arm has been removed.\n\nTwo independent routes into that state are used, which matters because each has a different failure mode. The first repurposes host microRNAs by inserting perfectly complementary target sites into the virus, so the guide is present in the cell before infection begins. The second has the virus encode an artificial hairpin that produces a small interfering RNA against one of its own segments, which places guide production downstream of infection and therefore under kinetics closer to a genuine RNA interference response.\n\nBefore building viruses, the silencing requirement is calibrated with a luciferase reporter carrying miR-124 target sites of graded complementarity, which fixes the threshold and lets the viral constructs be built with either full complementarity or the minimum that still silences. Building both, and building two-site and four-site versions, sets up a selection experiment in which escape should be easiest for the weakest and least redundant design.\n\nCell systems are chosen so that single variables differ. A canine kidney line and the same line expressing miR-124 differ only in the presence of one microRNA. Dicer-deficient cells test whether attenuation depends on the small RNA machinery. Fibroblasts lacking interferon regulatory factors 3 and 7 remove the transcriptional arm of innate sensing so that silencing can be measured without it, and a virus encoding an RNA-binding mutant of NS1 tests the claim that NS1 antagonizes silencing.\n\nFor the animal work, target sites were selected for microRNAs abundant in human and mouse lung but low in embryonated eggs, so the virus can still be propagated for stock production while being silenced in the host. Mice lacking the type I interferon receptor are the decisive comparison, since protection there cannot be attributed to interferon.", "Key findings": "1. In a luciferase reporter, a single 20 nucleotide fully complementary miR-124 site silenced more than 80 percent of activity, sites of 14 nucleotides or fewer gave no repression, and sites of 15, 16 and 17 nucleotides gave a graded relationship between pairing length and silencing (Figure 1A). Two sites were no better than one in this readout (Figure 1B). The threshold for silencing is therefore near 16 nucleotides of contiguous complementarity.\n\n2. Influenza A virus carrying a scrambled insert in the nucleoprotein 3-prime untranslated region replicated indistinguishably from wild-type virus, reaching 10 to the seventh through 10 to the eighth plaque-forming units per milliliter with robust nucleoprotein (Figure S1A, Figure S1B), so the insertion itself is not the source of any phenotype.\n\n3. All targeted viruses replicated normally in canine kidney cells but dropped to between 10 squared and 10 to the fourth plaque-forming units per milliliter with no detectable nucleoprotein in the miR-124-expressing derivative (Figure 1C, Figure 1D). Attenuation increased with target site number but did not differ significantly between full complementarity and 16 contiguous bases.\n\n4. Roughly ten passages in miR-124-expressing cells yielded no recoverable virus, reported as data not shown. Passage in a mixed culture at about three untargeted cells to one targeted cell also produced no escape mutants and instead led to selective death of the permissive cells and to a population dominated by microRNA-expressing cells by 72 hours (Figure 1E).\n\n5. A self-targeting virus expressing a small interfering RNA against the nucleoprotein open reading frame from the NS segment produced the guide during infection (Figure 2B) and was strongly attenuated in wild-type cells but not in Dicer-deficient cells (Figure 2C), giving more than a tenfold loss within 48 hours in cells and in animals (Figure 2D, Figure 2E, Figure S2A). The Dicer dependence establishes that attenuation runs through the small RNA machinery.\n\n6. Sequencing plaque-purified escape variants from self-targeting viruses recovered only guide-side mutations. The nucleoprotein-directed virus escaped through a large deletion in the artificial hairpin that abolished guide production and restored wild-type replication (Figure 2E, Figure 2F, Figure 2G). The 16-nucleotide four-site design escaped through six dispersed deletions in the hairpin (Figure 3B, Figure 3C, Figure 3D) and the 20-nucleotide four-site design by excising the hairpin entirely (Figure 3E, Figure 3F). The authors report they were unable to identify any virus that had mutated the target site itself, even with only a single site present.\n\n7. A timing experiment transfecting guide-expressing plasmids at 3, 6, 12 or 24 hours before infection found attenuation only when transfection preceded infection by 12 hours or more (Figure S2C), with guides visible by Northern blot by 12 hours post-transfection (Figure S2B). From comparison with a loading control the authors estimate 100 to 1,000 copies per cell and infer that a cell would need to accumulate that many guides within the first 6 hours of infection for targeting to succeed. The copy number figure is an estimate calibrated against published quantification of other microRNAs, not a direct measurement.\n\n8. In wild-type and Irf3 and Irf7 double knockout fibroblasts, comparing self-targeting viruses encoding wild-type NS1 with those encoding an RNA-binding mutant of NS1 showed that NS1 functionality had minimal impact on the extent of attenuation once the interferon regulatory factor arm was removed, with a 20-fold to 50-fold titer loss in a single cycle in knockout cells and a 50-fold to 100-fold loss when both silencing and an intact interferon system were present (Figure 3G, Figure 3H). The authors interpret the larger loss with both systems as the two pathways complementing one another, and interpret the small NS1-dependent difference as more likely reflecting defective interfering particles arising without functional NS1 than direct antagonism of silencing.\n\n9. A virus carrying five distinct target sites for miR-93, miR-192, miR-21, miR-31 and miR-29b in the nucleoprotein transcript grew in eggs to roughly 2 times 10 to the fifth egg infectious dose 50 per milliliter against 6 times 10 to the fifth for control (Figure 4A), yet produced no plaques, no hemagglutination activity and no detectable nucleoprotein in mammalian cells (Figure 4B, Figure 4C, Figure S4C). Titer in eggs had to be estimated by egg infectious dose because the targeted virus could not be plaqued at all.\n\n10. In C57BL/6 mice, 250 plaque-forming units of control virus, five times the reported lethal dose 50 of 50 plaque-forming units, caused severe weight loss and complete mortality, while the targeted virus caused no morbidity or mortality at 250, 2,500 or 25,000 plaque-forming units (Figure 5A). Lung histology at 2 and 9 days showed no inflammation or damage with the targeted virus against severe bronchiolar epithelial lesions and perivascular and alveolar edema with untargeted virus (Figure 5B).\n\n11. In Ifnar1 knockout mice the targeted virus caused no morbidity or mortality even at 25,000 plaque-forming units, a dose the authors note is more than 2,500 times the lethal dose 50 in that background, with no detectable viral messenger RNA at day 2 (Figure 5C, Figure S5A) and attenuation confirmed by plaque assay at days 2 and 9 (Figure S5B, Figure S5C). This is the central result, since protection here cannot be attributed to interferon.\n\n12. Transcriptome profiling showed robust interferon-stimulated gene induction with untargeted virus, for example Mx1 induced more than 40-fold against 2-fold for the targeted strain, and Ifit1, Oas1b, Oas2 and Isg15 induced around 10-fold against baseline for the targeted strain (Figure 6A, Table S1). By day 9 elevated antiviral and chemokine transcripts persisted with untargeted virus and were absent with the targeted strain. In Ifnar1 knockout mice the overall response was muted and differences between cohorts were less pronounced for genes such as Ifit1 (Figure 6B, Table S2), which the authors attribute to direct induction by interferon regulatory factors 3 and 7 independent of interferon feedback. Quantitative PCR for the matrix gene found untargeted virus not fully cleared by day 9 in either background while the targeted virus was at background (Figure S6A, Figure S6B).", "Mechanistic model": "The mechanism at the level of the engineered response is direct. Guide small RNAs, whether supplied as host microRNAs repurposed by inserting complementary sites or produced by the virus itself, load into RISC and cleave or silence the targeted viral transcript, and this requires Dicer, as shown by the loss of self-targeting attenuation in Dicer-deficient cells. Silencing requires approximately 16 nucleotides of contiguous pairing.\n\nThe study does not establish a mechanism for the evolutionary question it raises, and the authors treat that question as open. What the data establish is a possibility claim, that a small RNA response can protect a mammal from a lethal virus challenge with no contribution from type I interferon signaling, and therefore that the historical substitution cannot be explained by the mammalian cell being unable to support such a defense. The authors state this as the conclusion, that chordates could have used RNA interference in place of interferon.\n\nAn additional mechanistic reading concerns where selection acts. Escape arose only by destroying guide production, never by altering the target, across several independent designs including one with a single target site. The authors interpret this as reflecting the strength of the selective pressure and the difficulty of escaping a guide that is present before infection begins. The explanation for why the target was never mutated is not established by these experiments, and alternative explanations, such as functional constraint on the targeted coding sequence or on the inserted untranslated region, are not separately excluded here.\n\nThe discussion offers several hypotheses for why mammals use interferon, and these are explicitly speculative. They include that error-prone RNA viruses escape small RNA targeting readily and may have driven the emergence of a more general system, that DNA viruses including poxviruses, adenoviruses and herpesviruses do encode antagonists of small RNA pathways and one such ancient pathogen might have disabled an RNA interference response, that chordates lack the RNA-dependent RNA polymerase needed to amplify guides and lack the Dicer gene family expansion seen in plants, and that a non-viral pathogen such as an ancient protozoan or bacterium might have demanded a more general response. The authors write that such evolutionary questions are near impossible to address.\n\nOn NS1, the paper reports that it does not find NS1 to be an effective inhibitor of small RNA silencing, notes the small residual difference between wild-type and mutant NS1 genotypes, and attributes that difference more plausibly to defective interfering particles than to antagonism. That is an interpretation.", "Conceptual or technical advance": "The work converts an evolutionary question into a testable protection experiment, and the result narrows the space of explanations. Because a reconstructed small RNA defense fully protected animals with no functional type I interferon receptor, the hypothesis that mammals abandoned RNA interference because it could not work in their cells is no longer available in that simple form.\n\nPractically, the study produces an influenza strain that can be propagated in eggs at usable titers while being undetectable by plaque assay, protein expression or transcript level in mammalian cells, and that causes no disease at 25,000 plaque-forming units even without interferon signaling. The consistent failure of target-site escape across designs, contrasted with the ready guide-side escape of self-targeting viruses, bears directly on how attenuated strains and biocontainment layers built on microRNA targeting should be designed.\n\nThe calibration work is also reusable. The complementarity threshold near 16 nucleotides, and the estimate that a guide must reach roughly 100 to 1,000 copies per cell within the first six hours of infection to be effective, give quantitative design parameters for anyone building such systems.", "Relationship to the broader research program": "This paper is the counterpart to the laboratory's own negative result. The earlier study by Backes and colleagues concluded that the mammalian response to virus infection is independent of small RNA silencing, and it is cited here in exactly that role. Taken together, the pair makes a two-part statement, that mammals do not use small RNA silencing against viruses and that they could have. Placing the two side by side is category 3 synthesis, and the corpus record for the earlier paper supports it.\n\nThe study also draws on and extends the laboratory's long-running engineering line, which includes microRNA-mediated species-specific attenuation of influenza A virus, hematopoietic-specific targeting of influenza, microRNA-based mitigation of gain-of-function influenza risk, engineered RNA viral synthesis of microRNAs, and the dengue tropism work. The self-targeting design here, in which the virus encodes a guide against its own genome, is a further step in that line.\n\nBenitez and colleagues 2015 in the same journal, on in vivo RNA interference screening identifying MDA5 as a contributor to influenza defense, is cited here by the same first author and supports the statement that microRNA insertion does not inherently alter influenza biology.", "Related publications": "- Backes, Langlois, Schmid, Varble, Shim, Sachs and tenOever 2014, Cell Reports, the mammalian response to virus infection is independent of small RNA silencing. Relationship predecessor. Cited for the conclusion that the dominant intrinsic mammalian response is interferon based, and the direct conceptual complement to this study.\n- Varble and colleagues 2010, engineered RNA viral synthesis of microRNAs. Relationship methodological foundation. Source of the modified NS segment used for the self-targeting and guide-expressing viruses, and of the estimate of guide production kinetics during infection.\n- Perez and colleagues 2009, Nature Biotechnology, microRNA-mediated species-specific attenuation of influenza A virus. Relationship predecessor. Source of target site designs and of the small RNA sequencing that identified microRNAs differentially expressed between eggs and mammalian lung.\n- Langlois, Varble, Chua, Garcia-Sastre and tenOever 2012, Proceedings of the National Academy of Sciences, hematopoietic-specific targeting of influenza A virus. Relationship methodological foundation. Source of the approach to inserting target sites as an artificial 3-prime untranslated region of nucleoprotein.\n- Langlois and colleagues 2013, Nature Biotechnology, microRNA-based strategy to mitigate the risk of gain-of-function influenza studies. Relationship predecessor and application. Source of miR-93 and miR-192 target designs.\n- Pham, Langlois and tenOever 2012, PLoS Pathogens, replication in cells of hematopoietic origin is necessary for dengue virus dissemination. Relationship predecessor. Cited in the discussion as a case where virus escape occurred by excision.\n- Benitez, Panis, Xue, Varble, Shim, Frick, Lopez, Sachs and tenOever 2015, Cell Reports, in vivo RNA interference screening identifies MDA5 as a contributor to cellular defense against influenza A virus. Relationship companion. Cited to support that microRNA insertion does not inherently perturb influenza biology.\n- Bogerd, Whisnant, Kennedy, Flores and Cullen 2014, RNA, derivation of Dicer- and microRNA-deficient human cells. Relationship methodological foundation from another laboratory. Source of the NoDice cells.\n- Varble and colleagues 2013. Relationship methodological foundation. Source of the control target sequence used for the five-site control virus.", "Limitations and boundaries": "The response studied here is engineered, not endogenous. The guides are either host microRNAs already present before infection or a virus-encoded hairpin, and neither is generated by host recognition of viral double-stranded RNA. The study therefore tests whether a small RNA defense can protect a mammal, not whether mammals possess one, and the authors keep that distinction.\n\nThe kinetics differ from a genuine RNA interference response in a way the authors themselves raise. Host microRNAs are present before the virus arrives, which imposes selective pressure earlier and more strongly than a response that must first detect the pathogen. The timing experiment quantifies this and shows that guides supplied only 6 hours before infection fail to attenuate.\n\nAll of the virology is one virus, influenza A/Puerto Rico/8/34, a segmented negative-sense RNA virus with a nuclear replication cycle. Conclusions about other virus families, and in particular about the DNA viruses the discussion invokes as possible drivers of evolutionary change, are not tested.\n\nThe evolutionary conclusion is a possibility claim and cannot be more than that. The discussion offers several competing hypotheses for the emergence of the interferon system and the authors state that answers to such questions are near impossible to address.\n\nQuantitative estimates carry caveats. The guide copy number of 100 to 1,000 per cell is inferred from Northern blot signal compared with a loading control and calibrated against published values for other microRNAs. The titer of the five-site targeted virus in eggs had to be converted from egg infectious dose because the virus would not plaque, so egg and cell titers are not directly comparable.\n\nThe absence of target-site escape is bounded by the passage regimes and sequencing depth used. Approximately ten passages in microRNA-expressing cells and one mixed-culture experiment are reported, with escape mutants characterized by plaque purification and sequencing rather than by deep sequencing of the population, so rare variants could be missed. The failure to recover virus after ten passages is reported as data not shown.\n\nThe animal work is confined to intranasal infection of C57BL/6 and Ifnar1 knockout mice at 6 to 8 weeks of age, with lungs assessed at days 2 and 9. Mice lacking the type I interferon receptor still have type III interferon and adaptive immunity, so this is a removal of one arm rather than of all antiviral defense, and the study does not test whether the engineered response would suffice in an animal more broadly immunocompromised.\n\nFinally, the claim that the two pathways complement one another rests on comparing fold reductions across cell genotypes in single-cycle assays, which is consistent with additivity but is not a formal test of interaction.", "Audience summaries": "### 25 words\n\nInfluenza was engineered so host small RNAs silence it. The resulting virus caused no disease in mice, including animals unable to respond to type I interferon.\n\n### 75 words\n\nPlants and insects fight viruses with RNA interference while mammals use interferon, and why the switch happened is unknown. Researchers rebuilt a small RNA defense by inserting microRNA target sites into influenza and by having the virus produce a guide against itself. Attenuation reached more than five logs, escape occurred only by deleting the guide rather than altering the target, and protection in mice was complete even without type I interferon signaling.\n\n### 150 words\n\nChordates retain the small RNA machinery but defend against viruses through type I interferon rather than RNA interference, and it is unclear whether that reflects an incompatibility with mammalian cells. This study reconstructs a small RNA defense against influenza A virus in two ways, by inserting complementary target sites for cell-restricted or species-restricted host microRNAs and by engineering the virus to encode a small interfering RNA against its own nucleoprotein segment. Silencing required about 16 nucleotides of contiguous complementarity. Self-targeting attenuation was lost in Dicer-deficient cells, and escape variants always disabled guide production rather than altering the target. A virus carrying five mammalian microRNA target sites grew in eggs but was undetectable in mammalian cells and caused no morbidity in mice at 25,000 plaque-forming units, including in animals lacking the type I interferon receptor. The authors conclude that chordates could have used RNA interference in place of interferon." }, "discoveries": [ "claim-08", "claim-09" ], "relationships": [ { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2014-backes-the-mammalian-response-to-virus-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2013-langlois-microrna-based-strategy-to-mitigat", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2013-varble-an-in-vivo-rnai-screening-approach", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2015-benitez-in-vivo-rnai-screening-identifies-", "relationship": "companion", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2014-varble-influenza-a-virus-transmission-bot", "relationship": "companion", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" } ], "canonical_url": "https://tenoeverlab.us/publications/2015-benitez-engineered-mammalian-rnai-can-elic/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "bulk-rna-seq", "plaque-assay", "reverse-genetics", "flow-cytometry", "small-rna-northern-blot", "mirna-target-site-insertion", "luciferase-promoter-reporter", "histopathology", "growth-curve", "artificial-mirna" ] } }, { "id": "2015-benitez-in-vivo-rnai-screening-identifies-", "slug": "2015-benitez-in-vivo-rnai-screening-identifies-", "url": "/publications/2015-benitez-in-vivo-rnai-screening-identifies-/", "title": "In Vivo RNAi Screening Identifies MDA5 as a Significant Contributor to the Cellular Defense against Influenza A Virus", "authors": [ "Asiel A. Benitez", "Maryline Panis", "Jia Xue", "Andrew Varble", "Jaehee V. Shim", "Amy L. Frick", "Carolina B. López", "David Sachs", "Benjamin R. tenOever" ], "author_count": 9, "first_author": "Asiel A. Benitez", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 9, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2015, "journal": "Cell Reports", "volume": "11", "issue": "11", "pages": "1714-1726", "doi": "10.1016/j.celrep.2015.05.032", "doi_url": "https://doi.org/10.1016/j.celrep.2015.05.032", "pmid": "26074083", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/26074083/", "pmcid": "PMC4586153", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586153/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586153/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling", "programmable-virology" ], "themes": [ "sensing-aberrant-rna", "in-vivo-screening-through-fitness" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "mouse", "human", "dog" ], "technologies": [ "influenza A virus reverse genetics", "artificial microRNA library", "in vivo RNAi screening", "small RNA sequencing", "messenger RNA sequencing", "quantitative PCR", "plaque assay", "gene knockout mice", "small interfering RNA transfection" ], "biological_systems": [ "A549 cells", "mouse embryonic fibroblasts", "MDCK cells", "HEK293 and 293T cells", "NoDice 293T cells", "mouse lung" ], "key_concepts": [ "pattern recognition receptor", "RIG-I-like receptor", "MDA5", "RIG-I", "NS1 antagonist", "interferon-stimulated gene amplification", "OAS and RNase L system", "fitness-based genetic selection", "virus-encoded small interfering RNA", "self-targeting virus" ], "keywords": [ "influenza A virus", "MDA5", "Ifih1", "RIG-I", "RNAi screen", "artificial microRNA", "interferon", "NS1", "in vivo screening", "OAS" ], "one_sentence_contribution": "An attenuated influenza A virus engineered to deliver individual artificial small interfering RNAs enables a fitness-based loss-of-function screen inside an infected mouse, and that screen identifies MDA5 as a contributor to the antiviral response to influenza A virus despite the established role of RIG-I as the sensor that induces interferon beta.", "summary_25": "Influenza viruses carrying individual silencing RNAs were allowed to compete inside mice, and those disabling MDA5 won, revealing a role for this sensor beyond interferon induction.", "summary_75": "Screens for host genes that block a virus are usually done in cell lines. Here an attenuated influenza A virus was engineered to carry one silencing RNA each against a hundred host antiviral genes, then the library was given to mice and the winners sequenced. Viruses silencing MDA5 dominated. MDA5 is not needed to switch on interferon beta during influenza infection, but it is needed for the full induction of downstream antiviral genes.", "summary_150": "An influenza A virus attenuated by mutation of its NS1 antagonist was engineered so that segment eight carries an artificial microRNA, yielding a library in which each virus silences one of a hundred mouse antiviral genes. Because host restriction is what attenuates the virus, silencing a genuine restriction factor restores fitness, so enrichment after four days in mouse lung is the readout. Viruses targeting Ifih1, encoding MDA5, were enriched over fiftyfold in four independent screens and one came to dominate. The advantage disappeared in MDA5 knockout mice, was not attributable to an off-target match to Setd7, and was reproduced by conventional silencing in human A549 cells. MDA5 loss left interferon beta induction largely intact, consistent with RIG-I being the sensor, but reduced induction of Irf7, OAS isoforms, Ifit1, Stat1 and Isg15. The effect on Oas2 required RNase L. The ligand MDA5 recognises during infection was not determined.", "citation": "Benitez AA, Panis M, Xue J, Varble A, Shim JV, Frick AL, López CB, Sachs D, tenOever BR. In Vivo RNAi Screening Identifies MDA5 as a Significant Contributor to the Cellular Defense against Influenza A Virus. *Cell Reports* 2015, volume 11, issue 11, pages 1714-1726. DOI 10.1016/j.celrep.2015.05.032. PMID 26074083. PMCID PMC4586153.", "sections": { "Citation": "Benitez AA, Panis M, Xue J, Varble A, Shim JV, Frick AL, López CB, Sachs D, tenOever BR. In Vivo RNAi Screening Identifies MDA5 as a Significant Contributor to the Cellular Defense against Influenza A Virus. *Cell Reports* 2015, volume 11, issue 11, pages 1714-1726.\n\nDOI 10.1016/j.celrep.2015.05.032. PMID 26074083. PMCID PMC4586153.", "One-sentence contribution": "An attenuated influenza A virus engineered to deliver individual artificial small interfering RNAs enables a fitness-based loss-of-function screen inside an infected mouse, and that screen identifies MDA5 as a contributor to the antiviral response to influenza A virus despite the established role of RIG-I as the sensor that induces interferon beta.", "Executive summary": "Loss-of-function screens for host restriction factors have almost all been performed in transformed cell lines and read out indirectly. This work builds a screening platform in which the readout is virus fitness inside an animal. Influenza A virus was attenuated by mutating NS1 so that it can no longer block pathogen pattern detection, and segment eight was modified to carry an artificial microRNA cassette. Each virus in the resulting library is identical at the protein level but encodes a different mouse-specific small interfering RNA against one of one hundred host genes induced by infection. Because the virus is attenuated by the host antiviral response, a virus that silences a gene contributing to that response should gain replicative advantage and rise in the population. The pooled library was given to mice, and after four days the virus populations in lung were sequenced. Viruses targeting the gene Ifih1, which encodes MDA5, were enriched more than fiftyfold in every one of four screens, with one strain rising from about a quarter of a percent of input to roughly a third of output. Follow-up work confirmed the advantage is lost in mice lacking MDA5, that the advantage is not due to an off-target hit on Setd7, and that MDA5 loss does not impair interferon beta induction but does mute induction of a subset of interferon-stimulated genes including OAS isoforms, in a manner linked to RNase L. Silencing MDA5 in human A549 cells reproduced the increased replication and the reduced antiviral gene induction.", "Scientific context": "Detection of influenza A virus in most cells had been assigned to RIG-I, which recognises the exposed triphosphate on the ends of the viral genome segments. Two prior studies had asked directly whether an influenza A virus lacking functional NS1 is detected by MDA5 and had concluded that detection is exclusively through RIG-I, a conclusion that rested on transcriptional induction of Ifnb as the readout. Approaches to defining the antiviral repertoire had relied either on large-scale overexpression screens, which cannot implicate multi-subunit effectors whose single components have no activity alone, or on high-throughput small interfering RNA screens, which the authors note are limited to indirect measures of virus output and are not performed in vivo. The laboratory had previously established that influenza A virus can be engineered to make a functional microRNA and that the miR-124 scaffold tolerates sequence substitution, which supplied the technical basis for turning the virus itself into the delivery vehicle for a small interfering RNA library.", "Central question": "Which host factors impose significant restriction on influenza A virus replication during a genuine infection in an animal, and can virus fitness under that restriction be used directly as the selection signal in a loss-of-function screen?", "Experimental strategy": "The design turns the screen into a competition. Attenuation is imposed by a three-amino-acid substitution in NS1 that impairs double-stranded RNA binding, which removes the virus's principal means of blocking pattern detection and costs it roughly three logs of replication. The NS1 and NEP open reading frames were split to create an insertion point for a hairpin while preserving small viral RNA and NEP levels, both of which matter to the viral life cycle. Because the attenuation is imposed by the host response, silencing a gene that contributes to that response should restore fitness, making enrichment in the population the direct readout. Hairpins were modelled on mmu-miR-124-2 rather than the more commonly used hsa-miR-30a after a side-by-side comparison favoured the former, and were designed with thermodynamic asymmetry so that the intended strand loads into the silencing complex. All small interfering RNAs were made mouse-specific, which both restricts the screen to the animal and addresses biosafety, since the viruses cannot silence the corresponding human transcripts. Validation of the platform proceeded through a self-targeting virus, in which a virus carrying GFP on segment three and an anti-GFP hairpin on segment eight should attenuate itself, with Dicer-deficient cells serving as the control that the effect is small RNA dependent. Follow-up on the screen hit combined mouse genetics, transcriptome profiling of infected fibroblasts, comparison across single and double sensor knockouts, and conventional small interfering RNA silencing in human cells.", "Key findings": "1. The split NS1 mutant virus induced strongly elevated Ifnb, Ifit1 and Ifit2 relative to wild-type virus, and this elevation was unchanged when the virus also encoded an anti-GFP hairpin, showing that carrying a small RNA cassette does not itself further attenuate the virus (Figure 1A). Replication was inversely proportional to the induced response, and the difference between mutant and wild-type virus was absent in mice lacking a functional type I interferon receptor (Figure 1, B and C).\n2. A virus encoding GFP on segment three together with an anti-GFP hairpin on segment eight attenuated itself and lost nucleoprotein and NS1 expression, whereas the matched virus carrying miR-124 did not. The attenuation was abolished in Dicer-deficient 293T cells (Figure 2). This is direct evidence that silencing occurs within the time course of a productive infection, which the authors note was not obvious given a prior report that NS1-deficient virus triggers ribosylation of the silencing complex.\n3. Small RNA sequencing of cells infected with the assembled library showed that 83 percent of hairpins produced only the intended strand, 13 percent both strands, 2 percent only the unintended strand and 2 percent no small RNA, and that virus-derived small RNA abundance was comparable to the most abundant endogenous microRNAs (Figure 3A). Direct silencing was verified for more than ten percent of hairpins at the transcript level and for ISG15 at the protein level (Figure 3, B and C).\n4. In four independent in vivo screens, both viruses targeting Ifih1 were enriched more than fiftyfold, and one of them rose from 0.26 percent of the input population to between 26 and 31 percent of output (Figure 4A). More than thirty further host factors were enriched more than twenty-fivefold across all four screens, six of them represented by both of their gene-specific hairpins.\n5. Three additional screens using sub-libraries of about eighty hairpins each, with both Ifih1 viruses excluded, selected for viruses targeting Ddx58, the nuclear factor kappa B subunits p50 and p65, and the interferon regulatory factors IRF5 and IRF7. The authors present this as evidence that the platform selects genuine antiviral factors rather than one idiosyncratic winner.\n6. The selected Ifih1-targeting virus carried no mutations relative to the parent beyond the engineered hairpin stems, showed no replication advantage in canine MDCK cells where the mouse-specific hairpin cannot act, and gave a modest but significant titre increase over the control virus in wild-type mice at 24 and 48 hours (Figure 4B). No significant difference between the two viruses remained in Ifih1 knockout mice (Figure 4C), and viruses carrying no hairpin at all showed elevated titre in Ifih1 knockout mice when NS1 was non-functional (Figure 4D).\n7. The unintended strand of the selected hairpin was a near-perfect match to Setd7, but the virus silenced Ifih1 potently and failed to reduce Setd7 at transcript or protein level, and messenger RNA sequencing confirmed no change in Setd7 between conditions. This argues the fitness gain comes from loss of MDA5.\n8. In fibroblasts lacking MDA5, Ifnb induction by the NS1 mutant virus was only moderately reduced and was abolished only in cells lacking both MDA5 and RIG-I, while loss of RIG-I alone abolished it (Figure 5, A and B). The paper therefore agrees with the earlier conclusion that RIG-I is the sensor responsible for interferon beta induction by influenza A virus.\n9. Despite intact Ifnb induction, loss of MDA5 reduced Irf7 induction by more than twentyfold and reduced induction of Oas2, Ifit1, Stat1 and Isg15 (Figure 5, C through F). The authors interpret this as MDA5 contributing to amplification of the antiviral state downstream of RIG-I-mediated recognition rather than to sensing itself.\n10. Depletion of MDA5 reduced Oas2 transcript by about half in wild-type cells but had no effect in RNase L deficient fibroblasts, and silencing RNase L reduced Oas2 only when MDA5 was present (Figure 5, G and H). The authors read this as crosstalk in which MDA5 enhances the antiviral response through the OAS and RNase L system, and they propose in the discussion that MDA5 may detect the RNA by-products of RNase L cleavage. That proposal is not tested here.\n11. Messenger RNA sequencing of fibroblasts infected with the MDA5-targeting virus, and independently of MDA5 knockout cells infected with a hairpin-free virus, showed a common set of virus-induced genes reduced without a defect in interferon beta induction (Figure 6).\n12. Transfecting a human-directed small interfering RNA against IFIH1 into A549 cells increased replication of the NS1 mutant virus by about one log at every timepoint, an effect absent with wild-type virus, and reduced IFIT1 induction after treatment with viral pattern RNA and interferon beta (Figure 7). The authors take the loss of phenotype with wild-type virus as further support for NS1 interfering with MDA5 function, which is an interpretation and not a direct demonstration.\n13. A virus targeting human RIG-I reduced RIG-I levels and gave about a one log replication increase, and immunoblotting of whole lung showed abundant RIG-I with no detectable MDA5 at baseline. The authors offer high basal RIG-I in vivo as the explanation for why RIG-I-targeting viruses did not dominate the screen.", "Mechanistic model": "The study does not establish a mechanism by which MDA5 acts, and the authors are explicit that MDA5 is not directly responsible for type I interferon induction during influenza A virus infection. What the data constrain is the placement of the contribution. RIG-I is required for Ifnb induction and MDA5 is not, yet MDA5 is required for the full induction of a defined subset of interferon-stimulated genes including Irf7, Oas2, Oas3, Ifit1, Stat1 and Isg15, and that requirement translates into measurable restriction of virus replication in mouse lung and in human cells. The genetic interaction between MDA5 and RNase L, in which the effect of each on Oas2 depends on the presence of the other, is a correlation between two perturbations rather than a demonstrated biochemical link. The authors propose that MDA5, being both virus-inducible and interferon-inducible, may detect aberrant RNA species generated during the interferon response itself, including RNase L cleavage products, and they cite work from other groups suggesting MDA5 can displace viral proteins that mask double-stranded RNA and can detect caps lacking 2'O-methylation. None of these candidate ligands is tested here. Similarly, the claim that NS1 antagonises MDA5 rests on the observation that phenotypes present with the NS1 mutant virus disappear with wild-type virus.", "Conceptual or technical advance": "The platform is the principal advance. By making the pathogen itself the delivery vehicle for the silencing reagent and by using an attenuation that the host response imposes, the screen converts host gene function into viral fitness and can therefore be run inside an animal with selection over twelve viral generations, without a transformed cell line and without an indirect surrogate readout. The demonstration of a self-inactivating virus establishes that silencing is fast enough to matter within one infection cycle. Biologically, the result reopens a question that had been treated as settled, showing that an interferon-induction readout can miss a sensor's contribution, and it makes MDA5's role in the influenza A virus response testable as an amplification function rather than as a sensing function. The authors also note that the approach cannot be used to enhance wild-type virus pathogenesis, since the selection operates only within a population already crippled by loss of NS1.", "Relationship to the broader research program": "The work rests on a line of engineering from the same laboratory in which RNA viruses are built to express small RNAs, and it extends that line from delivery and species-specific attenuation into forward genetics. Its own reference list cites earlier reports from the group on engineered RNA viral synthesis of microRNAs, on influenza A virus small viral RNAs and the transcription to replication switch, on suboptimal splicing timing of infection, on hematopoietic-specific targeting of influenza A virus, and on transcription factor redundancy in induction of the antiviral state. Setting this screen beside the laboratory's later transcriptional profiling of infected tissue and its work on RNA interference as an antiviral pathway would be category 3 synthesis and is not attempted from this paper alone.", "Related publications": "- Varble and colleagues, 2010, methodological foundation. Cited as the origin of engineered RNA viral synthesis of microRNAs and of the modified segment eight cloning site used to build the library.\n- Shapiro and colleagues, 2012, and Langlois and colleagues, 2012, predecessor. Cited for the finding that influenza A virus infection does not substantially perturb host microRNA levels, which the discussion uses to argue the approach does not itself impair the antiviral response.\n- Chua and colleagues, 2013, predecessor. Cited for the constraints on segment eight engineering and for the potency of NS1 antagonism.\n- Schmid and colleagues, 2010, conceptual extension. Cited for interferon-independent induction of antiviral genes downstream of IRF activation, the framework used to interpret the Irf7 result.\n- Perez and colleagues, 2010, predecessor. Cited for small viral RNA function on segment eight, a constraint the virus design had to respect.", "Limitations and boundaries": "The screen surveys one hundred selected host genes chosen for induction by infection and low baseline expression, so it is not genome-wide and cannot speak to constitutively abundant restriction factors. The authors demonstrate this limitation directly with RIG-I, whose high basal level in lung they offer as the reason a RIG-I-targeting virus did not dominate, which means absence from the enrichment list is not evidence of no antiviral role. Selection depends on silencing efficiency, target protein abundance and protein stability, all of which vary between genes and none of which the enrichment magnitude separates. The whole platform requires a virus lacking functional NS1, so the results describe restriction that operates when the principal viral antagonist is disabled, and the phenotypes in human cells were indeed lost with wild-type virus. Some off-target effects were present in the screen output, and the paper only excludes the specific off-target concern for the winning hairpin. The in vivo work uses one mouse-adapted H1N1 strain in one mouse background over four days, with cell culture work in A549, MDCK and fibroblast lines, so the findings do not extend on their own to other influenza subtypes, to other species or to natural infection doses. The MDA5 effect on replication is modest, roughly one log or less, and the link between MDA5 and the OAS and RNase L system is genetic rather than biochemical. The proposed ligand for MDA5 during influenza A virus infection remains undetermined.", "Audience summaries": "### 25 words\n\nInfluenza viruses carrying individual silencing RNAs were allowed to compete inside mice, and those disabling MDA5 won, revealing a role for this sensor beyond interferon induction.\n\n### 75 words\n\nScreens for host genes that block a virus are usually done in cell lines. Here an attenuated influenza A virus was engineered to carry one silencing RNA each against a hundred host antiviral genes, then the library was given to mice and the winners sequenced. Viruses silencing MDA5 dominated. MDA5 is not needed to switch on interferon beta during influenza infection, but it is needed for the full induction of downstream antiviral genes.\n\n### 150 words\n\nAn influenza A virus attenuated by mutation of its NS1 antagonist was engineered so that segment eight carries an artificial microRNA, yielding a library in which each virus silences one of a hundred mouse antiviral genes. Because host restriction is what attenuates the virus, silencing a genuine restriction factor restores fitness, so enrichment after four days in mouse lung is the readout. Viruses targeting Ifih1, encoding MDA5, were enriched over fiftyfold in four independent screens and one came to dominate. The advantage disappeared in MDA5 knockout mice, was not attributable to an off-target match to Setd7, and was reproduced by conventional silencing in human A549 cells. MDA5 loss left interferon beta induction largely intact, consistent with RIG-I being the sensor, but reduced induction of Irf7, OAS isoforms, Ifit1, Stat1 and Isg15. The effect on Oas2 required RNase L. The ligand MDA5 recognises during infection was not determined." }, "discoveries": [ "claim-10" ], "relationships": [ { "from": "2015-benitez-engineered-mammalian-rnai-can-elic", "to": "2015-benitez-in-vivo-rnai-screening-identifies-", "relationship": "companion", "evidence": "stated in the Related publications section of 2015-benitez-engineered-mammalian-rnai-can-elic" }, { "from": "2015-benitez-in-vivo-rnai-screening-identifies-", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2015-benitez-in-vivo-rnai-screening-identifies-" }, { "from": "2015-benitez-in-vivo-rnai-screening-identifies-", "to": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-in-vivo-rnai-screening-identifies-" }, { "from": "2015-benitez-in-vivo-rnai-screening-identifies-", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2015-benitez-in-vivo-rnai-screening-identifies-" }, { "from": "2015-benitez-in-vivo-rnai-screening-identifies-", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2015-benitez-in-vivo-rnai-screening-identifies-" } ], "canonical_url": "https://tenoeverlab.us/publications/2015-benitez-in-vivo-rnai-screening-identifies-/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "reverse-genetics", "sirna-knockdown", "small-rna-seq", "in-vivo-rnai-screen", "knockout-mice" ] } }, { "id": "2016-tenoever-the-evolution-of-antiviral-defense", "slug": "2016-tenoever-the-evolution-of-antiviral-defense", "url": "/publications/2016-tenoever-the-evolution-of-antiviral-defense/", "title": "The Evolution of Antiviral Defense Systems", "authors": [ "Benjamin R. tenOever" ], "author_count": 1, "first_author": "Benjamin R. tenOever", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 1, "tenoever_role": "sole", "contribution_character": "lab-led", "year": 2016, "journal": "Cell Host & Microbe", "volume": "19", "issue": "2", "pages": "142-149", "doi": "10.1016/j.chom.2016.01.006", "doi_url": "https://doi.org/10.1016/j.chom.2016.01.006", "pmid": "26867173", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/26867173/", "pmcid": null, "pmc_url": null, "pmc_pdf_url": null, "publication_type": "perspective", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling", "small-rna-antiviral-defense" ], "themes": [ "interferon-and-cell-identity", "evolution-of-antiviral-defense" ], "pathogens": [ "bacteriophage", "RNA viruses", "DNA viruses" ], "viral_families": [], "host_species": [ "bacteria", "archaea", "plants", "Drosophila", "chordates", "human", "mouse" ], "technologies": [ "comparative genomics", "phylogenetic inference", "evolutionary parsimony analysis" ], "biological_systems": [ "bacteria", "archaea", "eukaryotes", "plants", "arthropods", "chordates", "mammalian somatic cells", "stem cells" ], "key_concepts": [ "antisense RNA defense", "restriction modification systems", "prokaryotic Argonaute", "CRISPR-Cas", "piRNA pathway", "RNA interference", "pattern recognition receptors", "type I interferon", "V(D)J recombination", "host-pathogen arms race", "RNA-dependent RNA polymerase", "incompatibility of RNAi and interferon" ], "keywords": [ "evolution of immunity", "antiviral defense", "RNA interference", "interferon", "CRISPR-Cas", "transposable elements", "adaptive immunity", "Red Queen hypothesis" ], "one_sentence_contribution": "A synthesis arguing that antiviral defenses across the three domains of life reuse a small set of designs, antisense recognition joined to nuclease activity and later to transcriptional and secreted responses, and proposing that chordates lost RNA interference through incompatibility with interferon.", "summary_25": "Defenses against viruses, from bacterial antisense RNA to antibodies, reuse a few designs. This Perspective asks why vertebrates traded RNA silencing for interferon, and proposes incompatibility.", "summary_75": "Across bacteria, archaea and eukaryotes, defenses against genetic parasites repeatedly pair a sequence-specific guide with a nuclease, from antisense RNA and restriction enzymes to Argonaute, CRISPR-Cas, piRNAs and RNA interference. Vertebrates instead use pattern recognition receptors, interferons and antibodies. This single-author Perspective, synthesizing work from many laboratories, argues that vertebrates could not keep RNA interference because systemic small RNA defense needs a viral-type polymerase, and expressing one triggers innate immunity.", "summary_150": "This Perspective reads the succession of antiviral defense systems against the changing composition of the virome, arguing that early cells faced mobile DNA and could answer only with antisense transcription, that DNA phages drove sequence-specific endonucleases and then restriction and modification, and that Argonaute, CRISPR-Cas, piRNAs and RNA interference are successive fusions of a nucleic acid guide with a nuclease. Chordates instead built pattern recognition receptors from Toll-like proteins and coupled them to secreted cytokines, with tumor necrosis factor among the earliest and interferons and recombinatorial adaptive immunity following. The article proposes that RNA interference was not merely made redundant but was incompatible, since protecting a large organism would require an RNA-dependent RNA polymerase to amplify and circulate small RNAs, and expressing such a polymerase in mammalian somatic cells triggers innate immunity. Supporting observations come from many groups, including two studies from the author's own laboratory. The evolutionary argument is presented as hypothesis.", "citation": "tenOever BR. The Evolution of Antiviral Defense Systems. Cell Host & Microbe. 2016. Volume 19, issue 2, pages 142-149. DOI 10.1016/j.chom.2016.01.006. PMID 26867173. Note on provenance. The source file is named tenOever_CHM2015 but the article was published in February 2016, and the inventory metadata is the authority. This is a single-author Perspective, so the great majority of the science discussed was produced by other laboratories and is attributed to them throughout this record.", "sections": { "Citation": "tenOever BR. The Evolution of Antiviral Defense Systems. Cell Host & Microbe. 2016. Volume 19, issue 2, pages 142-149.\n\nDOI 10.1016/j.chom.2016.01.006. PMID 26867173.\n\nNote on provenance. The source file is named tenOever_CHM2015 but the article was published in February 2016, and the inventory metadata is the authority. This is a single-author Perspective, so the great majority of the science discussed was produced by other laboratories and is attributed to them throughout this record.", "One-sentence contribution": "A synthesis arguing that antiviral defenses across the three domains of life reuse a small set of designs, antisense recognition joined to nuclease activity and later to transcriptional and secreted responses, and proposing that chordates lost RNA interference through incompatibility with interferon.", "Executive summary": "This Perspective traces antiviral defense across bacteria, archaea and eukaryotes and asks how the changing nature of genetic parasites shaped the systems that oppose them. The argument is organized by evolutionary parsimony, the principle that a component present in all three domains probably existed in their last universal ancestor, which the author notes is probabilistic and not universally accepted. Drawing chiefly on comparative genomic work by Koonin and colleagues, the article sets out a trajectory in which self-replicating RNA gave rise to polymerases and then to DNA-based parasites, so that the earliest defenses faced mobile DNA and could do little better than transcribe complementary antisense RNA. Sequence-specific endonucleases, Argonaute proteins guided by small nucleic acids, CRISPR-Cas, and the eukaryotic piRNA and RNA interference systems are then presented as successive combinations of the same two elements, a guide and a nuclease. In chordates, pattern recognition receptors derived from Toll-like proteins, antiviral cytokines including tumor necrosis factor and the interferons, and recombinatorial adaptive immunity replaced small RNA defense. The article's own proposal, labeled an attractive hypothesis, is that this replacement was forced by incompatibility. Chordate size and receptor-mediated virus spread would require an RNA-dependent RNA polymerase for systemic small RNA amplification, and expressing such a polymerase in mammalian somatic cells triggers innate immunity. Work from the tenOever laboratory supplies two supporting observations, that engineered mammalian RNA interference can protect against virus without interferon, and that microRNA function in acute infection appears limited to cytokine control.", "Scientific context": "Whether chordates retain an antiviral function for RNA interference was, at the time of writing, actively contested, and the article says so. Reports from the laboratories of Ding and of Voinnet argued for antiviral RNA interference in mammalian cells, while a joint opinion piece by Cullen, Cherry and tenOever questioned its physiological relevance, and work from Bogerd, Cullen and colleagues reported that replication of many human viruses is refractory to inhibition by endogenous cellular microRNAs. Separately, comparative genomics had begun to resolve the deep history of both viruses and defense systems. Koonin and colleagues had argued that viruses have no single common ancestor, that their diversity arose through genetic exchange, that Polintons may be the ancestors of most eukaryotic DNA viruses, and that most eukaryotic RNA viruses may descend from DNA phages that acquired an RNA-dependent RNA polymerase. Koonin and Krupovic had proposed that both known adaptive immune systems, CRISPR-Cas and the immunoglobulin system, were built when transposable elements conferred recombination capacity on pre-existing innate responses. The Perspective assembles these separate literatures into one narrative and adds a specific evolutionary argument about the loss of RNA interference in chordates.", "Central question": "Given that the composition of the virome changed over evolutionary time, from mobile DNA elements in early cells to the RNA viruses that dominate eukaryotes, how did that change shape the succession of antiviral defense systems observed across the tree of life, and why did chordates abandon RNA interference in favor of the interferon system.", "Experimental strategy": "No experiments are reported. The method is inference from conservation and phylogeny under evolutionary parsimony, applied to defense systems and, in parallel, to the evolution of the parasites themselves, since the argument is that the two histories constrain each other. The article states explicitly that the trajectory it proposes for each defense system is necessarily speculative and rests on observed conservation rather than on demonstration. Two figures organize the synthesis. One presents cartoon models of antisense RNA, restriction nucleases, Argonaute-based targeting, CRISPR-Cas, piRNAs, RNA interference, interferons and immunoglobulins side by side. The other places the appearance of each system on a timeline against the three domains of life.", "Key findings": "This is a Perspective, so the entries below are the article's principal arguments and the evidence it marshals, not results generated by the author.\n\n1. Applying parsimony, a component found in bacteria, archaea and eukaryotes likely existed in the last universal ancestor, and the best route to reconstructing the first antiparasite defense is comparative analysis of genes that process nucleic acid. The article credits this framework to Woese and colleagues, to Koonin, and to Anantharaman, Koonin and Aravind, and notes that the approach is probabilistic and contested.\n2. Virus diversity does not trace to a single ancestor and arose through genetic exchange, with DNA viruses dominating prokaryotes and RNA viruses abounding in eukaryotes. From this the article infers that the first genuine genetic parasite of unicellular life was DNA based, and that the scarcity of RNA phages suggests early cellular environments were poorly suited to RNA spread. The underlying phylogenetics is attributed to Koonin and colleagues, Edwards and Rohwer, and Krupovic and colleagues.\n3. The earliest defense available to a protocell would have been transcription of antisense RNA producing steric interference with a mobile element, a strategy still widely evident in bacteria and archaea, where small RNAs under about 150 nucleotides control plasmid, transposon and phage amplification. Attributed to Wagner and colleagues, Gottesman and Storz, and Thomason and Storz.\n4. The emergence of DNA phages is proposed as the driver for sequence-specific endonucleases, with self protection arising by modification of host DNA, creating the two-component restriction and modification design. Attributed to Kobayashi, Ishikawa and colleagues, and Mochizuki and colleagues, with the causal reading offered by the author as speculative.\n5. Three later systems are presented as fusions of antisense specificity with nuclease activity. Prokaryotic Argonaute proteins use small DNA or RNA guides against mobile elements, work attributed to Makarova and colleagues, Olovnikov and colleagues, and Swarts and colleagues. CRISPR-Cas, present in about 90 percent of sequenced archaeal and 40 percent of bacterial genomes, incorporates phage or plasmid fragments into genomic loci and transcribes them as guides, attributed to Marraffini, Grissa and colleagues, and van der Oost and colleagues. The eukaryotic piRNA pathway uses clustered transposon-derived small RNAs of 25 to 30 nucleotides with a Piwi nuclease, largely in germline cells, attributed to Ishizu and colleagues, Iwasaki and colleagues, and Gunawardane and colleagues. The article states that piRNA and CRISPR share no common ancestry and are analogous rather than homologous.\n6. RNA interference is presented as an elaboration on the prokaryotic Argonaute theme, with Dicer generating 19 to 21 nucleotide fragments loaded into RISC. A key distinction drawn is amplification. Plants and worms encode RNA-dependent RNA polymerases acquired from phage, while arthropods do not, so their defense depends on transport of small interfering RNAs and weakens with distance from infection, a point attributed to Saleh and colleagues and to Tomoyasu and colleagues.\n7. Whether chordates retain antiviral RNA interference is described as unresolved and controversial, with conflicting positions cited from Cullen, Cherry and tenOever, from Li and Ding and colleagues, and from Maillard, Voinnet and colleagues. The article's own position is that eukaryotic microRNAs descend from the ancestral antiviral system but no longer act in an antiviral capacity, supported by work from the tenOever laboratory reported by Aguado and colleagues and by Bogerd, Cullen and colleagues, and consistent with the finding of Obbard and colleagues that in Drosophila only Dicer-2, the antiviral one, shows the selective signature of immune function.\n8. In place of RNA interference, chordates use pattern recognition receptors evolved from Toll-like proteins that originated roughly 700 million years ago in development and were independently co-opted as sentinels, attributed to Leulier and Lemaitre and to Putnam and colleagues, coupled to secreted cytokines. Tumor necrosis factor is cited as an early antiviral cytokine with about 550 million years of functional conservation, attributed to Quistad and colleagues, with interferons and interleukins following.\n9. The article's own hypothesis is that loss of RNA interference in chordates reflects incompatibility with the interferon system rather than simple redundancy. The reasoning is that large chordates would need polymerase-driven amplification and circulation of small interfering RNAs, that receptor-mediated cell entry prevents the kind of passive spread plants exploit, and that expression of an RNA-dependent RNA polymerase in mammalian somatic cells itself triggers innate immunity, shown independently by Painter and colleagues and by Yu and colleagues. Supporting evidence for mutual exclusivity is drawn from Billy and colleagues, Paddison and colleagues and Maillard and colleagues on the divergent responses of stem and somatic cells to long double-stranded RNA, and from Girardi and colleagues and Seo and colleagues on reciprocal inhibition between RISC and interferon signaling. The author notes that work from his own laboratory, reported by Benitez and colleagues, showed that engineered mammalian RNA interference can protect against virus when the requirement for an RNA-dependent RNA polymerase is removed.\n10. Both known adaptive immune systems are argued to derive from the very elements they defend against, with CRISPR-Cas built largely from casposon-derived genes and the RAG recombinase derived from a Transib family transposon. Attributed to Koonin and Krupovic, Krupovic and colleagues, Kapitonov and Jurka, and Kapitonov and Koonin. The article also notes that comparative analysis of gene duplication shows defense systems diverging faster than any other process, citing Daugherty and Malik and Siddle and Quintana-Murci.", "Mechanistic model": "The Perspective does not establish a mechanism and says of its central evolutionary claims that they are necessarily speculative. What it offers is a structural account and one testable hypothesis.\n\nThe structural account is that antiviral defense has repeatedly been assembled from a small parts list. A guide that recognizes foreign sequence, first as an antisense transcript and later as a small RNA or DNA, combined with an effector, first steric hindrance and later a nuclease, then extended in multicellular organisms by a transcriptional response and finally by a secreted signal that protects cells that are not yet infected. Adaptive systems in this account are innate systems that acquired recombination from a transposable element.\n\nThe specific hypothesis, about why chordates replaced RNA interference with interferon, is presented explicitly as an attractive hypothesis whose evolutionary cause cannot be determined. It links three premises, that systemic small RNA defense in a large organism requires amplification by an RNA-dependent RNA polymerase, that chordate virus spread by receptor-mediated entry does not carry small RNAs along with it as plant cell-to-cell movement does, and that such a polymerase cannot be expressed in mammalian somatic cells without triggering innate immunity. The correlative evidence for incompatibility, the divergent handling of long double-stranded RNA in stem versus somatic cells and the reciprocal inhibition between RISC and interferon signaling, is consistent with the hypothesis but does not establish that incompatibility caused the evolutionary loss. The article is careful to frame what would follow if the hypothesis holds, namely that the loss of RNA interference in chordates may have everything to do with polymerase biology.", "Conceptual or technical advance": "The article's contribution is framing. By reading defense systems against the changing composition of the virome rather than in isolation, it makes the sequence of systems look like responses to a sequence of problems, and it identifies the guide plus nuclease pairing as the recurring solution across domains that share no common defense ancestry.\n\nThe sharper contribution is the incompatibility hypothesis, which converts a descriptive observation, that chordates have interferon and appear not to use antiviral RNA interference, into a mechanistic proposal with an identified molecular obstacle. Because that obstacle is the innate immune consequence of RNA-dependent RNA polymerase expression, the proposal is addressable experimentally, and the article points to a test already performed in the author's own laboratory, that engineering around the polymerase requirement allows RNA interference to substitute for the interferon response in mammalian cells.", "Relationship to the broader research program": "This Perspective sets the conceptual frame for a line of work in the tenOever laboratory on whether mammals use, or could be made to use, RNA-based antiviral defense. Two of the laboratory's own papers carry weight in the argument. Benitez and colleagues in 2015 reported that engineered mammalian RNA interference can provide antiviral protection that negates the requirement for the interferon response, and Aguado and colleagues in 2015 reported that microRNA function in the acute response to virus infection is limited to cytokine control. The 2013 opinion piece co-written with Cullen and Cherry is the laboratory's earlier position on the same controversy.\n\nCategory 3 synthesis. Placed beside the laboratory's engineering papers, in which RNA viruses were built to produce microRNAs and then used to deliver silencing libraries, this Perspective supplies the reason those experiments are informative rather than merely technical. The recurring question across those papers, why RNA viruses do not naturally exploit the host small RNA machinery and why mammals do not use it against them, is here given an evolutionary answer in terms of incompatibility with interferon. That connection is visible when the papers are read together and is not asserted by any one of them.", "Related publications": "- Benitez and colleagues, 2015, Engineered Mammalian RNAi Can Elicit Antiviral Protection that Negates the Requirement for the Interferon Response, from the tenOever laboratory and cited here. Predecessor supplying evidence for the central hypothesis.\n- Aguado and colleagues, 2015, microRNA Function Is Limited to Cytokine Control in the Acute Response to Virus Infection, from the tenOever laboratory and cited here. Predecessor.\n- Cullen, Cherry and tenOever, 2013, Is RNA interference a physiologically relevant innate antiviral immune response in mammals, cited here. Review or synthesis, the earlier statement of the position developed in this Perspective.\n- Varble and colleagues, 2010, Engineered RNA viral synthesis of microRNAs, and Langlois and colleagues, 2012, In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs. Conceptual extension in the reverse direction, since this Perspective supplies the evolutionary reading of why RNA viruses do not naturally make microRNAs.\n- Varble and colleagues, 2013, An In Vivo RNAi Screening Approach to Identify Host Determinants of Virus Replication. Application, using engineered small RNA delivery as a tool in the interferon-competent host discussed here.\n- Koonin and Krupovic, 2015, on the evolution of adaptive immunity from transposable elements combined with innate immune systems, from another group and a principal source for the closing argument. Conceptual foundation.", "Limitations and boundaries": "The article is a Perspective by a single author, and it is not evidence that the tenOever laboratory performed the work it describes. Almost all of the primary findings summarized here belong to other groups, above all the comparative genomics of virus and defense system evolution developed by Koonin and colleagues, the CRISPR-Cas literature, the prokaryotic Argonaute and piRNA literatures, and the mammalian RNA interference work from the Ding, Voinnet, Cullen, Pfeffer and Sullivan laboratories. Only the Benitez, Aguado and Cullen, Cherry and tenOever citations are from the author's own group.\n\nThe evolutionary reconstructions are inferences from conservation under parsimony, which the article itself states is probabilistic and not universally agreed upon, and it describes the proposed trajectory for each defense system as necessarily speculative. No experiment, dataset or analysis is presented, so nothing here is independently verifiable from the article. The exact phylogeny of virus evolution is stated to be unachievable, so the ordering of parasite types that motivates the ordering of defenses is itself uncertain.\n\nThe central hypothesis about chordates is explicitly labeled as one attractive hypothesis among possible explanations, and the article says the evolutionary cause of the transformation cannot be determined. The evidence assembled for incompatibility between RNA interference and interferon is correlative, drawn from cell culture comparisons of stem and somatic cells and from reciprocal inhibition experiments, and the question of whether chordates retain antiviral RNA interference is presented as unresolved rather than settled. The article allows that any residual use may be confined to specialized pluripotent cells. Coverage is also acknowledged to be incomplete, since the author apologizes for original work that could not be cited for reasons of space, and whole areas including plant defense, programmed cell death and dormancy, and viral counter-defense are treated only briefly.", "Audience summaries": "### 25 words\n\nDefenses against viruses, from bacterial antisense RNA to antibodies, reuse a few designs. This Perspective asks why vertebrates traded RNA silencing for interferon, and proposes incompatibility.\n\n### 75 words\n\nAcross bacteria, archaea and eukaryotes, defenses against genetic parasites repeatedly pair a sequence-specific guide with a nuclease, from antisense RNA and restriction enzymes to Argonaute, CRISPR-Cas, piRNAs and RNA interference. Vertebrates instead use pattern recognition receptors, interferons and antibodies. This single-author Perspective, synthesizing work from many laboratories, argues that vertebrates could not keep RNA interference because systemic small RNA defense needs a viral-type polymerase, and expressing one triggers innate immunity.\n\n### 150 words\n\nThis Perspective reads the succession of antiviral defense systems against the changing composition of the virome, arguing that early cells faced mobile DNA and could answer only with antisense transcription, that DNA phages drove sequence-specific endonucleases and then restriction and modification, and that Argonaute, CRISPR-Cas, piRNAs and RNA interference are successive fusions of a nucleic acid guide with a nuclease. Chordates instead built pattern recognition receptors from Toll-like proteins and coupled them to secreted cytokines, with tumor necrosis factor among the earliest and interferons and recombinatorial adaptive immunity following. The article proposes that RNA interference was not merely made redundant but was incompatible, since protecting a large organism would require an RNA-dependent RNA polymerase to amplify and circulate small RNAs, and expressing such a polymerase in mammalian somatic cells triggers innate immunity. Supporting observations come from many groups, including two studies from the author's own laboratory. The evolutionary argument is presented as hypothesis." }, "discoveries": [ "claim-06" ], "relationships": [ { "from": "2016-tenoever-the-evolution-of-antiviral-defense", "to": "2015-aguado-microrna-function-is-limited-to-cy", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2016-tenoever-the-evolution-of-antiviral-defense" }, { "from": "2016-tenoever-the-evolution-of-antiviral-defense", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "review or synthesis", "evidence": "stated in the Related publications section of 2016-tenoever-the-evolution-of-antiviral-defense" }, { "from": "2016-tenoever-the-evolution-of-antiviral-defense", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2016-tenoever-the-evolution-of-antiviral-defense" } ], "canonical_url": "https://tenoeverlab.us/publications/2016-tenoever-the-evolution-of-antiviral-defense/", "controlled_vocabulary": { "pathogens": [ "bacteriophage", "dna-viruses", "rna-viruses" ], "technologies": [ "phylogenetics", "comparative-sequence-analysis" ] } }, { "id": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "slug": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "url": "/publications/2017-aguado-rnase-iii-nucleases-from-diverse-k/", "title": "RNase III nucleases from diverse kingdoms serve as antiviral effectors", "authors": [ "Lauren C. Aguado", "Sonja Schmid", "Jared May", "Leah R. Sabin", "Maryline Panis", "Daniel Blanco-Melo", "Jaehee V. Shim", "David Sachs", "Sara Cherry", "Anne E. Simon", "Jean-Pierre Levraud", "Benjamin R. tenOever" ], "author_count": 12, "first_author": "Lauren C. Aguado", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 12, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2017, "journal": "Nature", "volume": "547", "issue": "7661", "pages": "114-117", "doi": "10.1038/nature22990", "doi_url": "https://doi.org/10.1038/nature22990", "pmid": "28658212", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/28658212/", "pmcid": "PMC5846625", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846625/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846625/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense" ], "themes": [ "rnase-iii-antiviral-effectors", "evolution-of-antiviral-defense" ], "pathogens": [ "Sindbis virus", "Ross River virus", "Langat virus", "influenza A virus", "Sendai virus", "Drosophila C virus", "turnip crinkle virus" ], "viral_families": [ "Togaviridae", "Flaviviridae", "Orthomyxoviridae", "Paramyxoviridae", "Dicistroviridae", "Tombusviridae" ], "host_species": [ "human", "mouse", "Drosophila melanogaster", "zebrafish", "Arabidopsis thaliana" ], "technologies": [ "CRISPR Cas9 gene disruption", "SELEX", "electrophoretic mobility shift assay", "immunoprecipitation", "RNA sequencing", "small RNA sequencing", "Sindbis replicon and luciferase reporters", "in vitro minus-strand synthesis assay", "morpholino knockdown", "northern blot", "western blot" ], "biological_systems": [ "HEK293T cells", "NoDice Dicer-deficient HEK293T cells", "Drosha and Dicer double knockout HEK293T cells", "primary conditional Drosha mouse lung fibroblasts", "Drosophila DL1 cells", "zebrafish embryos", "Arabidopsis protoplasts", "in vitro replicase assays" ], "key_concepts": [ "RNase III nucleases", "Drosha", "Dicer", "microRNA-independent antiviral activity", "RNA stem loop recognition", "steric hindrance of RNA-dependent RNA polymerase", "cytoplasmic translocation of Drosha", "positive-strand RNA virus specificity", "interferon-independent defense", "evolution of antiviral systems" ], "keywords": [ "Drosha", "RNase III", "antiviral defense", "Sindbis virus", "positive-strand RNA viruses", "RNA hairpin", "SELEX", "RNA-dependent RNA polymerase", "microRNA-independent" ], "one_sentence_contribution": "RNase III nucleases, including human Drosha and homologues from bacteria, archaea, yeast and a urochordate, restrict positive-strand RNA viruses by recognizing unbranched RNA stem loops and impairing the viral polymerase, separably from microRNA biogenesis, catalysis and interferon.", "summary_25": "Drosha restricts positive-strand RNA viruses by gripping structured RNA rather than cutting it, an activity shared by RNase III enzymes from bacteria, archaea, yeast, and animals.", "summary_75": "Drosha is known for making microRNAs, but it also leaves the nucleus during infection. Removing it from cells that already lacked microRNAs let positive-strand RNA viruses grow much better, and a version of Drosha that cannot cut RNA or make microRNAs still blocked them. The protein binds hairpin structures in viral genomes and cuts polymerase output roughly in half. Related enzymes from bacteria through yeast did the same, suggesting a very old defensive capability.", "summary_150": "Using human cells engineered to lack both Dicer and Drosha, and therefore all mature microRNAs, this work shows that Drosha restricts positive-strand RNA viruses through a route independent of microRNA biogenesis, of catalysis, of its partner DGCR8, and of interferon. Sindbis, Ross River, and Langat viruses replicated better without Drosha while influenza and Sendai viruses did not. SELEX against the RNA-binding mutant enriched unbranched stem loops carrying no shared sequence, and the same protein bound a hairpin within the first 200 nucleotides of the Sindbis genome. Replicon experiments localized the defect to RNA synthesis rather than decay or translation, and a reconstituted minus-strand assay showed Drosha reducing polymerase output by nearly half, which the authors interpret as steric hindrance by an antiviral clamp. RNase III proteins from bacteria, archaea, yeast, and a urochordate reproduced the activity, and cytoplasmic RNase III function was detected in Drosophila, zebrafish, and Arabidopsis.", "citation": "Aguado LC, Schmid S, May J, Sabin LR, Panis M, Blanco-Melo D, Shim JV, Sachs D, Cherry S, Simon AE, Levraud JP, tenOever BR. RNase III nucleases from diverse kingdoms serve as antiviral effectors. Nature. 2017. 547(7661), 114-117. DOI 10.1038/nature22990. PMID 28658212. PMCID PMC5846625. Lauren C. Aguado and Sonja Schmid are marked in the paper as having contributed equally.", "sections": { "Citation": "Aguado LC, Schmid S, May J, Sabin LR, Panis M, Blanco-Melo D, Shim JV, Sachs D, Cherry S, Simon AE, Levraud JP, tenOever BR. RNase III nucleases from diverse kingdoms serve as antiviral effectors. Nature. 2017. 547(7661), 114-117.\n\nDOI 10.1038/nature22990. PMID 28658212. PMCID PMC5846625.\n\nLauren C. Aguado and Sonja Schmid are marked in the paper as having contributed equally.", "One-sentence contribution": "RNase III nucleases, including human Drosha and homologues from bacteria, archaea, yeast and a urochordate, restrict positive-strand RNA viruses by recognizing unbranched RNA stem loops and impairing the viral polymerase, separably from microRNA biogenesis, catalysis and interferon.", "Executive summary": "Drosha and Dicer are the two catalytically active RNase III enzymes in humans and are usually considered in terms of microRNA biogenesis. Earlier work had reported that Drosha leaves the nucleus after virus infection, which raised the question of what it does there. Disrupting Drosha in an existing Dicer-deficient human cell line produced cells with no mature microRNAs at all, and in those cells Sindbis virus replicated markedly better. The effect was specific to positive-strand RNA viruses, since Ross River virus and Langat virus were also enhanced while influenza A virus and Sendai virus were not, and it was reproduced in primary conditional Drosha mouse fibroblasts. Structure and function mapping showed that a Drosha variant that is catalytically inactive, cannot process primary microRNAs, and does not bind DGCR8 still restricted the virus, while Dicer did not, so the activity does not run through microRNA production. SELEX with the RNA-binding mutant enriched unbranched hairpins, and the same protein bound a hairpin in the first 200 nucleotides of the Sindbis genome. A replicon system showed increased genomic RNA, subgenomic RNA, antigenome, and luciferase in knockout cells without detectable changes in RNA decay or translation, and an in vitro minus-strand synthesis assay showed Drosha reducing polymerase output by nearly half. Cytoplasmic processing of virus-encoded microRNA hairpins in zebrafish embryos and Arabidopsis protoplasts, and antiviral activity of RNase III proteins from three domains of life, extend the phenomenon beyond mammals.", "Scientific context": "The introduction frames antiviral defense in evolutionary terms. Prokaryotes contend mostly with DNA viruses and use systems such as CRISPR, while eukaryotic compartmentalization allowed RNA viruses to expand, creating pressure for RNA-targeting defense, met in plants and invertebrates by antiviral RNA interference and in vertebrates largely by the interferon system. Drosha and Dicer are presented as retained components of that older machinery, with prior work cited indicating that Drosha is more closely related than Dicer to the ancestral bacterial RNase III and that in Drosophila the antiviral Dicer-2 has diverged sharply from the microRNA-dedicated Dicer-1. Three prior reports, two from this laboratory, had found that Drosha translocates to the cytoplasm after infection, and a 2014 paper from the laboratory had already described Drosha as an interferon-independent antiviral factor. What was missing was an account of what cytoplasmic Drosha recognizes and how that recognition restricts a virus, separable from its role in the microprocessor.", "Central question": "Does Drosha possess an antiviral activity that is independent of microRNA biogenesis and of the interferon system, and if so, what does it recognize in viral RNA and by what means does that recognition limit replication, and is the activity restricted to Drosha or shared across the RNase III family?", "Experimental strategy": "The genetic starting point removes the usual confound. Working in a published Dicer-deficient human line in which RNA virus replication was unaltered, the authors disrupted Drosha as well, so that the resulting cells lack mature microRNAs entirely and any antiviral phenotype cannot be attributed to loss of a particular microRNA. Virus panel selection then tests polarity as a variable, with two alphaviruses and a flavivirus against an orthomyxovirus and a paramyxovirus. Separation of function is pursued through mutants rather than inference, using a panel of six Drosha variants all incapable of processing primary microRNAs, including a catalytic mutant and an RNA-binding mutant that fails to associate with DGCR8, and using a phosphorylation variant that sits exclusively in the cytoplasm to ask whether the cytoplasmic pool alone suffices. To find the ligand without assuming it, SELEX is run against the RNA-binding mutant and the enriched structures are then sought in the viral genome and tested directly by mobility shift. To decide among stability, translation, and polymerase inhibition as mechanisms, each is tested separately, with a temperature-sensitive polymerase mutant for decay, an in vitro luciferase construct for translation, and a reconstituted minus-strand replicase assay for polymerase output. Finally, conservation is examined across taxa by expressing RNase III proteins from bacteria, archaea, yeast and a urochordate, and by using virus-encoded microRNA hairpins as reporters of cytoplasmic RNase III activity in zebrafish and plants.", "Key findings": "1. Cells lacking both Dicer and Drosha have no detectable mature microRNAs with or without Sindbis infection, and restoring miR-93 requires both proteins (Fig. 1a and Extended Data Fig. 1b). This establishes the genetic background for everything that follows.\n2. Sindbis virus capsid production and kinetics were strongly increased in the double knockout cells (Fig. 1b). Ross River virus and Langat virus were likewise enhanced, while influenza A virus and Sendai virus were not (Fig. 1c to 1f), so enhancement tracked with positive genome polarity.\n3. Baseline transcriptome comparison showed induction of primary microRNAs and of DGCR8, both expected consequences of losing Drosha, and reconstitution of Drosha restored the transcriptome (Fig. 1g and Extended Data Fig. 1c and 1d). The double-stranded RNA response showed little differential expression (Fig. 1h) and no defects in the type I interferon system were observed (Extended Data Fig. 2a and 2b), which the authors use to exclude an indirect interferon explanation.\n4. The phenotype was reproduced in primary conditional Drosha mouse lung fibroblasts (Extended Data Fig. 2c to 2e), showing it is not an artifact of one engineered human line.\n5. A green fluorescent protein-tagged Drosha carrying serine to alanine substitutions at positions 300 and 302 localized exclusively to the cytoplasm, retained enzymatic activity, and reduced Sindbis titers by roughly one log (Extended Data Fig. 3). The authors read this as implicating the cytoplasmic pool of Drosha directly.\n6. Among six Drosha variants unable to process primary microRNAs, wild-type Drosha reconstituted antiviral activity while Dicer did not, and the RNA-binding mutant that is catalytically inactive and fails to associate with DGCR8 still suppressed the virus (Fig. 2b to 2f). The antiviral activity therefore requires RNA binding and does not require catalysis, DGCR8, or microRNA processing.\n7. Five rounds of SELEX with the RNA-binding mutant enriched RNAs with no conserved sequence that fold into unbranched, microRNA-like hairpins (Fig. 3a), and a recombinant Drosha RNA-binding fragment bound an enriched hairpin by mobility shift with no other host factors present (Fig. 3c and 3d). Recognition is thus structural rather than sequence-specific in this assay.\n8. In Sindbis-infected cells, immunoprecipitated Drosha RNA-binding mutant complexes were enriched about one log for viral RNA relative to a Sendai nucleoprotein control (Extended Data Fig. 4a and 4b), and mobility shift confirmed binding to a hairpin within the first 200 nucleotides of the genome (Extended Data Fig. 4c and 4d).\n9. A Sindbis replicon produced more genomic RNA, subgenomic RNA, antigenome, and luciferase in the knockout cells (Fig. 3e to 3g). Among the candidate explanations, RNA decay was excluded using a temperature-sensitive polymerase at the nonpermissive temperature (Extended Data Fig. 5c) and translation was excluded using an in vitro luciferase-encoding construct (Extended Data Fig. 5d and 5e).\n10. In a vaccinia-based reconstitution of minus-strand replicase complexes, fractions containing both Sindbis replicase and Drosha showed polymerase output reduced by almost 50 percent (Fig. 3h and 3i). This is the direct biochemical support for polymerase impairment.\n11. RNase III proteins from bacteria, archaea, yeast, and Ciona intestinalis conferred antiviral activity against positive-strand but not negative-strand viruses when expressed in the knockout cells (Fig. 4c and 4d and Extended Data Fig. 7a and 7b), indicating the activity is a general property of the domain rather than of Drosha specifically.\n12. Cytoplasmic RNase III activity outside mammals was inferred from processing of virus-delivered microRNA hairpins, Drosha-dependent processing of a Sindbis-encoded artificial microRNA in zebrafish embryos (Fig. 4a) and processing of pri-miR-124 delivered by turnip crinkle virus in Arabidopsis protoplasts (Fig. 4b).\n13. In Drosophila DL1 cells, knockdown of Drosha increased Sindbis capsid expression and increased Drosophila C virus replication (Extended Data Fig. 7c, Fig. 4e and 4f). Small RNA sequencing showed more virus-derived small RNAs in Drosha-depleted cells but indistinguishable alignment contours, which the authors use to argue against an RNA interference explanation.", "Mechanistic model": "The model proposed is that cytoplasmic Drosha, and by extension other RNase III proteins, binds unbranched stem loops in the genomes of positive-strand RNA viruses and acts as a clamp, physically obstructing the viral RNA-dependent RNA polymerase rather than cleaving the RNA. Several components are demonstrated. Binding is to structure and requires only the RNA-binding region, catalysis is dispensable, decay and translation are excluded as the affected step in the systems tested, and polymerase output falls by nearly half in vitro when Drosha is present in the replicase-containing fraction. The clamp language is the authors' interpretation of that set of results and is not itself observed, since no structural or single-molecule evidence for steric occlusion is presented, and the study does not establish which hairpin engagement events in an infected cell produce the replication defect. The restriction of the phenotype to positive-strand viruses is consistent with the model, given that these genomes serve directly as polymerase templates in the cytoplasm, but the paper does not test why negative-strand genomes, which are encapsidated, escape. The evolutionary account in the discussion, in which an ancestral RNase III clamp preceded RNA interference and later gave rise to the sensors of the interferon system, is explicitly offered as speculation by the authors and should be read as category 2 at best.", "Conceptual or technical advance": "The work separates an antiviral function of Drosha from everything Drosha is normally studied for. Because the experiments are done in cells with no microRNAs at all and with mutants that cannot process, cannot cleave, and cannot bind DGCR8, the activity cannot be explained by loss of a regulatory microRNA, and the absence of interferon defects removes the other common indirect route. That leaves direct RNA binding as the operative property, and SELEX supplies an unbiased description of what is bound, namely unbranched stem loops of the kind that positive-strand genomes require and cannot easily discard. The demonstration that RNase III proteins from bacteria through yeast to a urochordate share the activity converts a Drosha observation into a statement about a protein domain, and it makes testable the idea that structured RNA recognition by an ancient nuclease fold can function as defense without any downstream silencing pathway.", "Relationship to the broader research program": "This is the mechanistic extension of a line the laboratory had been building for several years. Prior laboratory work had shown noncanonical cytoplasmic processing of viral microRNAs, presented evidence for a cytoplasmic microprocessor, and identified Drosha as an interferon-independent antiviral factor, all cited here. The engineering of viruses to carry microRNA hairpins, developed in the laboratory for delivery and for targeting, is used here in a different role, as a reporter that reveals cytoplasmic RNase III activity in a zebrafish embryo or a plant protoplast. The evolutionary framing connects to the senior author's review on the evolution of antiviral defense systems, also cited. The recurring question across these papers, namely what the relationship is between the small RNA machinery and antiviral defense in vertebrates, is addressed here by decoupling the two, and that answer sits directly against the position taken in the 2013 Minireview in this corpus, where the somatic-cell case for antiviral RNA interference was judged unproven. Reading those two together is category 3 synthesis, and it belongs to central assembly rather than to either record alone.", "Related publications": "- Shapiro et al. 2014, Drosha as an interferon-independent antiviral factor. Predecessor from the tenOever laboratory, the direct antecedent of the antiviral activity characterized here.\n- Shapiro et al. 2012, evidence for a cytoplasmic microprocessor of pri-miRNAs. Predecessor from the same laboratory, source of the cytoplasmic translocation premise.\n- Shapiro et al. 2010, noncanonical cytoplasmic processing of viral microRNAs. Methodological foundation, source of the virus-encoded hairpin reporters used in zebrafish and plants.\n- tenOever 2016, the evolution of antiviral defense systems. Review or synthesis from the same laboratory, the framework the discussion argues within.\n- tenOever 2013, RNA viruses and the host microRNA machinery. Review or synthesis from the same laboratory, cited for the relationship between microRNA machinery and RNA interference.\n- Bogerd et al. 2014, derivation and characterization of Dicer- and microRNA-deficient human cells. Methodological foundation from the Cullen group, the parental cell line used here.\n- Sabin et al. 2013, Dicer-2 processes diverse viral RNA species. Methodological foundation from the Cherry group, the source of the Drosophila knockdown and small RNA sequencing approach, with the same first author contributing the fly data here.\n- Cullen, Cherry and tenOever 2013, is RNA interference a physiologically relevant innate antiviral immune response in mammals. Conceptual extension, two of its authors appear here and the present work bears on the question that review left open.", "Limitations and boundaries": "The enhancement phenotype is established for a small panel of viruses, and the positive-strand specificity rests on two alphaviruses and one flavivirus against one orthomyxovirus and one paramyxovirus, all in cultured cells. Most of the mammalian mechanism is worked out in one engineered HEK293T-derived line and in transfection or reconstitution settings, with the primary mouse fibroblast experiment serving as the only independent mammalian confirmation, and no whole-animal mammalian infection is reported. The polymerase inhibition result is a roughly 50 percent reduction in an in vitro assay using fractions that contain both replicase and Drosha, which shows interference with output but does not show direct contact with the polymerase or exclude an intermediary in the fraction. The clamp model is an interpretation and no structural data accompany it. Cross-species evidence outside Drosophila is indirect, since zebrafish and plant experiments measure processing of an engineered hairpin as a proxy for cytoplasmic RNase III activity rather than measuring virus restriction in those hosts. Heterologous expression of bacterial, archaeal, and yeast RNase III proteins in human cells shows what those domains can do in that setting and not what they do in their own organisms. The evolutionary narrative in the discussion is labeled speculative by the authors and is not supported by experiment.", "Audience summaries": "### 25 words\n\nDrosha restricts positive-strand RNA viruses by gripping structured RNA rather than cutting it, an activity shared by RNase III enzymes from bacteria, archaea, yeast, and animals.\n\n### 75 words\n\nDrosha is known for making microRNAs, but it also leaves the nucleus during infection. Removing it from cells that already lacked microRNAs let positive-strand RNA viruses grow much better, and a version of Drosha that cannot cut RNA or make microRNAs still blocked them. The protein binds hairpin structures in viral genomes and cuts polymerase output roughly in half. Related enzymes from bacteria through yeast did the same, suggesting a very old defensive capability.\n\n### 150 words\n\nUsing human cells engineered to lack both Dicer and Drosha, and therefore all mature microRNAs, this work shows that Drosha restricts positive-strand RNA viruses through a route independent of microRNA biogenesis, of catalysis, of its partner DGCR8, and of interferon. Sindbis, Ross River, and Langat viruses replicated better without Drosha while influenza and Sendai viruses did not. SELEX against the RNA-binding mutant enriched unbranched stem loops carrying no shared sequence, and the same protein bound a hairpin within the first 200 nucleotides of the Sindbis genome. Replicon experiments localized the defect to RNA synthesis rather than decay or translation, and a reconstituted minus-strand assay showed Drosha reducing polymerase output by nearly half, which the authors interpret as steric hindrance by an antiviral clamp. RNase III proteins from bacteria, archaea, yeast, and a urochordate reproduced the activity, and cytoplasmic RNase III function was detected in Drosophila, zebrafish, and Arabidopsis." }, "discoveries": [ "claim-07" ], "relationships": [ { "from": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "to": "2014-shapiro-drosha-as-an-interferon-independen", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2017-aguado-rnase-iii-nucleases-from-diverse-k" }, { "from": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "to": "2012-shapiro-evidence-for-a-cytoplasmic-micropr", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2017-aguado-rnase-iii-nucleases-from-diverse-k" }, { "from": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2017-aguado-rnase-iii-nucleases-from-diverse-k" }, { "from": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "to": "2013-cullen-is-rna-interference-a-physiologica", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2017-aguado-rnase-iii-nucleases-from-diverse-k" }, { "from": "2018-aguado-homologous-recombination-is-an-int", "to": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2018-aguado-homologous-recombination-is-an-int" } ], "canonical_url": "https://tenoeverlab.us/publications/2017-aguado-rnase-iii-nucleases-from-diverse-k/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sindbis-virus", "sendai-virus", "drosophila-c-virus", "langat-virus", "ross-river-virus", "turnip-crinkle-virus" ], "technologies": [ "bulk-rna-seq", "small-rna-seq", "immunoblotting", "northern-blot", "emsa", "in-vitro-reconstitution", "co-ip", "crispr-knockout", "minigenome-assay", "morpholino-knockdown", "selex" ] } }, { "id": "2017-morales-sars-cov-encoded-small-rnas-contri", "slug": "2017-morales-sars-cov-encoded-small-rnas-contri", "url": "/publications/2017-morales-sars-cov-encoded-small-rnas-contri/", "title": "SARS-CoV-Encoded Small RNAs Contribute to Infection-Associated Lung Pathology", "authors": [ "Lucía Morales", "Juan Carlos Oliveros", "Raúl Fernandez-Delgado", "Benjamin Robert tenOever", "Luis Enjuanes", "Isabel Sola" ], "author_count": 6, "first_author": "Lucía Morales", "senior_authors": [ "Luis Enjuanes", "Isabel Sola" ], "corresponding_authors": [ "Luis Enjuanes" ], "tenoever_position": 4, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2017, "journal": "Cell Host & Microbe", "volume": "21", "issue": "3", "pages": "344-355", "doi": "10.1016/j.chom.2017.01.015", "doi_url": "https://doi.org/10.1016/j.chom.2017.01.015", "pmid": "28216251", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/28216251/", "pmcid": "PMC5662013", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662013/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662013/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "influenza-genome-regulation", "small-rna-antiviral-defense" ], "themes": [ "noncanonical-microrna-biogenesis", "small-viral-rnas" ], "pathogens": [ "SARS-CoV", "severe acute respiratory syndrome coronavirus" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "mouse", "human" ], "technologies": [ "small RNA deep sequencing", "small RNA RT-qPCR", "locked nucleic acid antagomir inhibition", "luciferase 3-prime UTR reporter assay", "intranasal mouse infection", "lung histopathology scoring", "immunohistochemistry" ], "biological_systems": [ "BALB/c mouse lung", "DBT-mACE2 cells", "Calu-3 2B4 cells", "293T cells", "Drosha and Dicer deficient 293T cells" ], "key_concepts": [ "small viral RNA", "noncanonical small RNA biogenesis", "RNase III independence", "post-transcriptional silencing", "proinflammatory cytokine induction", "lung immunopathology", "virulence independent of replication", "antagomir antiviral strategy" ], "keywords": [ "SARS-CoV", "svRNA", "svRNA-N", "nsp3", "nucleocapsid", "LNA antagomir", "lung pathology", "CCL2", "IL-6", "CXCL10" ], "one_sentence_contribution": "SARS-CoV generates three discrete small viral RNAs during infection of mouse lung, and blocking the one derived from the nucleocapsid gene with a locked nucleic acid antagomir reduces pulmonary inflammation, tissue damage and proinflammatory cytokine expression without measurably lowering lung virus titres.", "summary_25": "SARS-CoV makes three short RNAs during infection. Blocking the one from its nucleocapsid gene eased lung inflammation and damage in mice without reducing virus levels.", "summary_75": "Sequencing small RNAs from the lungs of infected mice showed that SARS-CoV produces three discrete short RNAs from its own genome, two from the nsp3 region and one from the nucleocapsid gene. Their production does not need the cell's usual microRNA processing enzymes. Each can silence a matched reporter. Giving mice a chemical inhibitor of the nucleocapsid-derived RNA before infection reduced lung inflammation, tissue damage and inflammatory cytokines while lung virus titres stayed the same.", "summary_150": "Severe SARS-CoV disease is driven substantially by a dysregulated inflammatory response rather than by viral burden alone. Deep sequencing of small RNAs from the lungs of mice infected with mouse-adapted SARS-CoV identified three abundant species of 18 to 22 nucleotides, two from the glutamate-rich domain of nsp3 and one from the nucleocapsid gene. Their abundance scaled with replication rather than with virulence, and they were still produced in cells lacking both Drosha and Dicer, so canonical microRNA biogenesis is not required. Synthetic mimics of all three silenced reporters bearing their complements, but only the nucleocapsid-derived species did so when supplied by the virus during infection. A locked nucleic acid inhibitor given intranasally before infection cut that species to ten percent, significantly reduced gross and histological lung pathology, and lowered CCL2, interleukin 6 and CXCL10, while lung titres were unchanged. No host target was identified, so the mechanism remains open.", "citation": "Morales L, Oliveros JC, Fernandez-Delgado R, tenOever BR, Enjuanes L, Sola I. SARS-CoV-Encoded Small RNAs Contribute to Infection-Associated Lung Pathology. Cell Host & Microbe. 2017. Volume 21, issue 3, pages 344-355. DOI 10.1016/j.chom.2017.01.015. PMID 28216251. PMCID PMC5662013.", "sections": { "Citation": "Morales L, Oliveros JC, Fernandez-Delgado R, tenOever BR, Enjuanes L, Sola I. SARS-CoV-Encoded Small RNAs Contribute to Infection-Associated Lung Pathology. Cell Host & Microbe. 2017. Volume 21, issue 3, pages 344-355.\n\nDOI 10.1016/j.chom.2017.01.015. PMID 28216251. PMCID PMC5662013.", "One-sentence contribution": "SARS-CoV generates three discrete small viral RNAs during infection of mouse lung, and blocking the one derived from the nucleocapsid gene with a locked nucleic acid antagomir reduces pulmonary inflammation, tissue damage and proinflammatory cytokine expression without measurably lowering lung virus titres.", "Executive summary": "Severe acute respiratory syndrome coronavirus causes lethal disease characterised by an excessive inflammatory response and extensive lung damage, and the prevailing view is that much of this pathology is driven by host responses rather than by viral burden alone. Several RNA viruses had been shown to generate small noncoding RNAs from their genomes, but it was not known whether coronaviruses do so or whether such RNAs matter for disease. Deep sequencing of small RNAs from the lungs of mice infected with a mouse-adapted SARS-CoV identified three abundant species of 18 to 22 nucleotides, two from the region encoding the glutamate-rich domain of nsp3 and one from the nucleocapsid gene. Their abundance tracked viral replication rather than virulence, they were positive sense like the genome, and they were produced in cells lacking both Drosha and Dicer, so canonical microRNA processing is not required. All three repressed a luciferase reporter carrying the complementary sequence in its 3 prime untranslated region, though only the nucleocapsid-derived species did so when supplied by the virus during infection. Inhibiting that species in mice with an intranasally delivered locked nucleic acid reduced gross lung pathology, histological inflammation and the expression of CCL2, interleukin 6 and CXCL10, while leaving lung titres unchanged, which the authors present as a contribution of the small RNA to immunopathology that is separable from replication.", "Scientific context": "DNA viruses and nuclear-replicating RNA viruses were already known to encode microRNAs, and improving sequencing sensitivity had extended the catalogue of small viral RNAs to cytoplasmic RNA viruses including influenza virus, enterovirus 71, hepatitis A virus and several flaviviruses. Two functional roles had been documented for such species in other systems, regulation of the viral life cycle and contribution to pathogenesis. A theoretical objection had been raised in the field, that excising a small RNA from a cytoplasmic RNA virus genome would damage that genome, but engineered viruses producing functional microRNAs without a replication penalty had weakened that argument. Against this background no coronavirus small RNA had been described. Separately, SARS-CoV pathology was already understood to involve dysregulated proinflammatory cytokine expression, with the envelope protein established as a virulence factor acting through several routes including nuclear factor kappa B activation, inflammasome activation and p38 signalling, and with an envelope-deleted virus attenuated in vivo. The study sits at the junction of these two lines and asks whether a viral small RNA is another contributor to that inflammatory pathology.", "Central question": "Does SARS-CoV generate small noncoding RNAs during infection, and if so do they contribute to the inflammatory lung pathology that characterises severe disease.", "Experimental strategy": "Discovery was carried out in infected animals rather than in culture, on the reasoning that small noncoding RNA populations are cell specific and that lung tissue provides the context in which pathology arises. Two viruses were compared, a virulent mouse-adapted strain that reproduces severe human disease and an attenuated envelope-deleted derivative, at two time points, which allows small RNA abundance to be separated from virulence and attached instead to replication. A viral sequence yielding no reads was designated as a negative control species and carried through the subsequent assays, which controls for the specificity of the RT-qPCR detection. Cell line work then tested whether production depends on host species or cell type and whether it requires the canonical microRNA machinery, using 293T cells and a matched line lacking both RNase III enzymes made permissive by transient receptor expression. Whether the viral sequences could be processed as microRNA precursors in the absence of infection was tested by placing flanking genomic sequence into introns of reporter plasmids. Function as a silencing guide was assessed with luciferase reporters bearing perfect complementary targets in the 3 prime untranslated region, first with synthetic mimics in uninfected cells and then with the reporter delivered into infected cells so that the virus itself supplies the small RNA, with a cellular microRNA reporter included to confirm that infection does not disable the silencing machinery. Finally the species that performed in both settings was inhibited in mice with a chemically modified locked nucleic acid given intranasally before infection, with readouts spanning weight, viral genomic RNA, subgenomic messenger RNA, lung titre, blinded gross and histological pathology scoring, cytokine and interferon-stimulated gene expression, and immunohistochemistry for viral antigen distribution.", "Key findings": "1. Small RNA sequencing from infected mouse lung at two and four days recovered viral reads at under 0.1 percent of the total, distributed broadly across the genome as expected for breakdown products but with distinct peaks. Three species accounted for roughly 18 percent of viral reads, two mapping within the nsp3 glutamate-rich domain coding region at positions 3,052 and 3,184 and one within the nucleocapsid gene at position 28,461, all positive sense and 18 to 22 nucleotides (Figure 1B, Table 1). Read counts were highest for the virulent virus at two days, matching the titre peak (Figure 1A).\n2. RT-qPCR confirmed the three species and the absence of the control sequence in lung, and detected them to differing degrees in serum, where the second nsp3 species predominated while the other two were near mock levels despite comparable lung abundance (Figures 1C and 1D). The authors offer differential stability or active transport to blood as alternative explanations and do not distinguish between them.\n3. Production was much higher in mouse DBT cells expressing the receptor than in human Calu-3 2B4 cells, in proportion to the titres reached in each, and scaled with genomic RNA and infectious titre over time (Figures 2A and 2B, Figure S1). An envelope ion channel mutant that grows like wild type produced comparable svRNA levels (Figure S2B). The authors conclude that biogenesis is not cell or species restricted but depends on the extent of replication.\n4. Genomic sequences flanking the small RNAs, placed in introns of reporter plasmids, were not processed into the mature species in uninfected cells (Figure S4), indicating a requirement for viral factors or for host factors induced during infection.\n5. In 293T cells lacking Drosha and Dicer, genomic RNA accumulated as in the parental line and titres were slightly lower, and svRNA production was not overtly impaired (Figures 3A and 3B). Canonical microRNA processing is therefore dispensable.\n6. Synthetic mimics of all three species repressed a matched luciferase reporter to 40 to 60 percent of control, comparable to a cellular microRNA mimic, and antisense inhibitors restored activity partially for the first nsp3 species and fully for the other two (Figure 4A). A cellular microRNA reporter was silenced to under 3 percent in both mock and infected cells, showing that infection leaves the silencing machinery functional at the time point tested (Figure 4B). When the reporter was introduced into infected cells so that the virus supplied the small RNA, only the nucleocapsid-derived species produced significant silencing, and less than its mimic did (Figure 4C).\n7. Antisense inhibition in cell culture reduced the nucleocapsid-derived species to about five percent of control and had no significant effect on nsp3 or nucleocapsid protein levels (Figure 2C, Figures S3B and S3C). Titres fell modestly, most clearly for that species with more than one log at seventy-two hours (Figure 2D). The authors note that the RT-qPCR readout of inhibition can give false positives through interference between the oligonucleotide and the assay and therefore treat the functional assays as the confirmation.\n8. Intranasal locked nucleic acid given a day before infection reduced the nucleocapsid-derived species to about ten percent of control at two and four days without adverse effects over ten days (Figure 5A). Weight loss was modestly and persistently reduced but not prevented (Figure 5B). Genomic RNA and nucleocapsid subgenomic messenger RNA fell roughly two-fold at two days while lung titres were not significantly changed (Figures 5C to 5E).\n9. Blinded scoring showed significantly reduced gross lung pathology and reduced histological inflammation, with less alveolar and bronchiolar infiltration and edema, in treated animals (Figures 6A to 6D). Expression of CCL2, interleukin 6 and CXCL10 fell significantly, while ISG15 and MX1 rose and interferon gamma was unchanged (Figure 6E). Immunohistochemistry showed the same tissue distribution of viral antigen in both groups (Figure S5).\n10. The authors state that the genomic sequences giving rise to these species are conserved in the human Urbani strain and in several SARS-related bat coronaviruses but not in MERS-CoV, with the nucleocapsid-derived sequence fully conserved among the SARS-related bat viruses. This is a sequence comparison rather than an experimental demonstration that human SARS-CoV produces the same species.", "Mechanistic model": "The study does not establish a mechanism. It shows that three small viral RNAs exist during infection, that they can act as silencing guides against perfectly complementary targets, and that removing one of them reduces inflammatory pathology in mice. It does not identify how they are generated, what host transcripts they act on, or by what route the nucleocapsid-derived species influences the inflammatory response.\n\nOn biogenesis, the data exclude canonical Drosha and Dicer processing and exclude processing of the flanking genomic sequence as a precursor in uninfected cells. The authors offer two candidate routes without testing either, an argonaute 2-dependent route of the kind described for miR-451, and cleavage by a viral enzyme, with the coronavirus endoribonuclease nsp15 named as a possibility. They also note the precedent of flavivirus subgenomic RNA generated by the cellular exoribonuclease XRN1 stalling at structured RNA.\n\nOn function, the model proposed is that the nucleocapsid-derived species contributes to lung pathology by silencing host messenger RNAs that normally restrain the inflammatory response, acting through RNA interference. The evidence offered is that the species silences a reporter carrying its complement and that inhibiting it lowers cytokines. No endogenous target is identified, and the authors state that it is not known whether these species engage targets through a seed sequence, which they give as the reason target prediction is difficult. They also raise RNA to protein interactions as an alternative mode of action. The rise in ISG15 and MX1 on inhibition is interpreted as those genes acting as immunomodulators limiting proinflammatory output rather than as a direct antiviral effect, an interpretation supported by analogy to published Chikungunya virus work rather than by experiment here.\n\nThe paper argues from the absence of effects on nsp3 and nucleocapsid protein levels, and the absence of any titre increase on inhibition, that producing these small RNAs does not meaningfully damage the genome and that they do not silence the negative-sense replication intermediate to which they are complementary. That is an inference from negative results.", "Conceptual or technical advance": "The work extends the catalogue of virus-derived small RNAs to coronaviruses and does so from infected tissue rather than cell culture, which is what allows a pathology endpoint to be attached to the finding. It separates two things that are usually confounded in virology, the amount of virus and the severity of the inflammatory response, by showing a reagent that lowers the second without lowering the first in the lung. That makes the small RNA a candidate virulence determinant of a kind that would be missed by any screen scored on replication. Practically, it puts forward an antagomir directed at a viral rather than a host sequence as an intervention aimed at immunopathology, and it provides the sequences, detection assays and inhibitors needed to test the idea in other coronaviruses.", "Relationship to the broader research program": "The tenOever contribution here is stated in the author contributions as reagents, conceptual advice and manuscript writing, with the project conceived and the experiments designed by the Enjuanes and Sola group at the National Center of Biotechnology in Madrid. The connection to the tenOever laboratory's own program runs through the small viral RNA concept and the tools used to test it. The paper repeatedly frames itself against the influenza small viral RNA described by the tenOever group in 2010, adopting the same term, the same argument that non-uniform genomic distribution distinguishes regulatory species from degradation products, and the same locked nucleic acid inhibition strategy, while noting the contrasts, that influenza small viral RNAs come from noncoding segment ends and act on the viral life cycle whereas these come from coding regions and act on host pathology. It also uses the intronic reporter assay for testing whether viral sequence can be processed as a microRNA precursor, a method from the same laboratory's engineered-virus work. Setting this paper beside the 2010 influenza study and the laboratory's later coronavirus work indicates that virus-derived small RNAs serve different purposes in different families, which is a category 3 synthesis available only from reading those papers together.", "Related publications": "- Perez and colleagues, 2010, conceptual and methodological foundation, from the tenOever laboratory. Introduced influenza small viral RNA, the term svRNA, the hotspot argument distinguishing regulatory species from breakdown products, and the locked nucleic acid inhibition approach, all of which this paper adopts and explicitly compares itself to.\n- Varble and colleagues, 2010, methodological foundation, from the tenOever laboratory. Source of the intronic reporter constructs used to test whether the viral sequences can be processed as microRNA precursors in the absence of infection, and cited for the demonstration that engineered small RNAs can be excised from a cytoplasmic virus genome without self-silencing.\n- Shapiro and colleagues, 2010, predecessor, from the tenOever laboratory. Cited among the demonstrations that cytoplasmic RNA viruses can produce functional small RNAs.\n- Benitez and colleagues, 2015, methodological foundation. Source of the Drosha and Dicer deficient cells used to test biogenesis requirements.\n- DeDiego and colleagues, 2007 and 2014, Nieto-Torres and colleagues, 2014, and Jimenez-Guardeño and colleagues, 2014, predecessor, from the Enjuanes laboratory. Establish the envelope protein as a SARS-CoV virulence factor driving inflammatory pathology, and supply the attenuated viruses used here.\n- Roberts and colleagues, 2007, methodological foundation, from another laboratory. Source of the mouse-adapted MA15 virus that underlies the animal model.\n- Parameswaran and colleagues, 2010, Weng and colleagues, 2014, Shi and colleagues, 2014, Bidet and colleagues, 2014, and Roby and colleagues, 2014, predecessor, from other laboratories. The prior reports of small RNAs from cytoplasmic RNA viruses against which the coronavirus findings are positioned.", "Limitations and boundaries": "No endogenous target of any of the three small RNAs is identified, so the proposed silencing of host inflammatory regulators is a hypothesis rather than a result, and the authors state that the absence of information about seed-based targeting makes prediction difficult. The mechanism of biogenesis is unknown and the candidates offered are untested. Silencing activity in the context of infection was seen only for the nucleocapsid-derived species, and the authors note that the two nsp3 species may be compartmentalised away from plasmid-derived targets or competitively blocked, which is an explanation rather than a demonstration. Inhibition of the second nsp3 species could not be confirmed by RT-qPCR at all. In vivo the effect on pathology is measured after a single intranasal dose given before infection, the reduction in weight loss is modest, and the treatment did not improve survival, which the authors attribute to an unoptimised dose and schedule. Several statistical claims rest on a p value threshold of 0.1 rather than 0.05, including the effects on genomic RNA and on cytokine expression, and the histopathology used three animals per condition. All in vivo work uses one inbred mouse strain and a mouse-adapted virus, and human relevance rests on sequence conservation rather than on demonstration that human SARS-CoV infection of human tissue produces these species at comparable levels, with the human lung cell line tested here producing them only at low levels alongside low titres. The dissociation between pathology and titre is established for lung titre at the days sampled and does not exclude effects on replication elsewhere or at other times. Finally, the observed rise in ISG15 and MX1 on inhibition is interpreted through analogy to another virus system rather than tested.", "Audience summaries": "### 25 words\n\nSARS-CoV makes three short RNAs during infection. Blocking the one from its nucleocapsid gene eased lung inflammation and damage in mice without reducing virus levels.\n\n### 75 words\n\nSequencing small RNAs from the lungs of infected mice showed that SARS-CoV produces three discrete short RNAs from its own genome, two from the nsp3 region and one from the nucleocapsid gene. Their production does not need the cell's usual microRNA processing enzymes. Each can silence a matched reporter. Giving mice a chemical inhibitor of the nucleocapsid-derived RNA before infection reduced lung inflammation, tissue damage and inflammatory cytokines while lung virus titres stayed the same.\n\n### 150 words\n\nSevere SARS-CoV disease is driven substantially by a dysregulated inflammatory response rather than by viral burden alone. Deep sequencing of small RNAs from the lungs of mice infected with mouse-adapted SARS-CoV identified three abundant species of 18 to 22 nucleotides, two from the glutamate-rich domain of nsp3 and one from the nucleocapsid gene. Their abundance scaled with replication rather than with virulence, and they were still produced in cells lacking both Drosha and Dicer, so canonical microRNA biogenesis is not required. Synthetic mimics of all three silenced reporters bearing their complements, but only the nucleocapsid-derived species did so when supplied by the virus during infection. A locked nucleic acid inhibitor given intranasally before infection cut that species to ten percent, significantly reduced gross and histological lung pathology, and lowered CCL2, interleukin 6 and CXCL10, while lung titres were unchanged. No host target was identified, so the mechanism remains open." }, "discoveries": [], "relationships": [ { "from": "2017-morales-sars-cov-encoded-small-rnas-contri", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2017-morales-sars-cov-encoded-small-rnas-contri" }, { "from": "2017-morales-sars-cov-encoded-small-rnas-contri", "to": "2010-shapiro-noncanonical-cytoplasmic-processin", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2017-morales-sars-cov-encoded-small-rnas-contri" } ], "canonical_url": "https://tenoeverlab.us/publications/2017-morales-sars-cov-encoded-small-rnas-contri/", "controlled_vocabulary": { "pathogens": [ "sars-cov" ], "technologies": [ "small-rna-seq", "in-vivo-infection-route", "histopathology", "immunohistochemistry", "silencing-reporter-assay", "lna-antisense-inhibition", "small-rna-rt-qpcr" ] } }, { "id": "2018-aguado-homologous-recombination-is-an-int", "slug": "2018-aguado-homologous-recombination-is-an-int", "url": "/publications/2018-aguado-homologous-recombination-is-an-int/", "title": "Homologous recombination is an intrinsic defense against antiviral RNA interference", "authors": [ "Lauren C. Aguado", "Tristan X. Jordan", "Emily Hsieh", "Daniel Blanco-Melo", "John Heard", "Maryline Panis", "Marco Vignuzzi", "Benjamin R. tenOever" ], "author_count": 8, "first_author": "Lauren C. Aguado", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 8, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2018, "journal": "Proceedings of the National Academy of Sciences", "volume": "115", "issue": "39", "pages": null, "doi": "10.1073/pnas.1810229115", "doi_url": "https://doi.org/10.1073/pnas.1810229115", "pmid": "30209219", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/30209219/", "pmcid": "PMC6166822", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166822/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166822/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense", "viral-populations-evolution" ], "themes": [ "reconstructing-antiviral-rnai", "recombination-and-escape" ], "pathogens": [ "Sendai virus", "influenza A virus", "Sindbis virus", "Semliki Forest virus", "poliovirus", "vesicular stomatitis virus" ], "viral_families": [ "Paramyxoviridae", "Orthomyxoviridae", "Togaviridae", "Picornaviridae" ], "host_species": [ "mouse", "human" ], "technologies": [ "reverse genetics", "microRNA target cassette engineering", "serial passage", "deep sequencing of virus populations", "genome-wide CRISPR knockout screening", "flow cytometry", "immunofluorescence microscopy", "TCID50 titration" ], "biological_systems": [ "mouse embryonic fibroblasts", "RNase III deficient fibroblasts", "Dicer knockout fibroblasts", "Argonaute knockout fibroblasts", "A549 cells" ], "key_concepts": [ "antiviral RNA interference", "microRNA-mediated targeting", "Argonaute 2 slicing", "genome polarity", "homologous recombination", "template switching", "escape variant selection", "encapsidated genome", "virus population dynamics" ], "keywords": [ "RNA interference", "homologous recombination", "positive-strand RNA virus", "negative-strand RNA virus", "microRNA targeting", "poliovirus", "Sindbis virus", "Sendai virus", "escape mutant", "virus evolution" ], "one_sentence_contribution": "Applying one uniform small RNA-based selective pressure to four virus families in vertebrate cells shows that the ability to escape it tracks with the capacity for polymerase template switching rather than with genome polarity as such, since positive-strand viruses excise the targeted sequence while negative-strand viruses are cleared and a recombination-defective poliovirus cannot escape.", "summary_25": "Faced with the same engineered small RNA attack, positive-strand RNA viruses cut the targeted sequence out of their genomes, while negative-strand viruses, unable to recombine, were eliminated.", "summary_75": "RNA interference is the main antiviral defence in plants and insects, and vertebrate viruses have no reason to resist it. The authors built such a defence in mammalian cells by giving viruses perfectly matched binding sites for five common host microRNAs. Sendai and influenza viruses were wiped out. Sindbis, Semliki Forest and polioviruses recovered by precisely deleting the targeted sequence. A poliovirus unable to recombine could not delete it and was cleared.", "summary_150": "A cassette of perfectly complementary sites for five ubiquitous host microRNAs converts endogenous Argonaute 2 into a slicing antiviral defence, with a reverse-orientation cassette as a sequence-matched control and RNase III deficient fibroblasts as the silencing-off condition. Genetic tests and a genome-wide CRISPR screen confirmed that the pressure runs through the microRNA machinery, implicating Drosha, Dicer, DGCR8, Argonaute 2, TP53, miR-21 and XPO5, with no interferon genes. Placed in essential transcripts, the cassette cleared Sendai virus and influenza A virus by more than five logs. Sindbis virus, a Semliki Forest virus chimera and poliovirus were suppressed initially but recovered by passage through precise excision of the cassette that preserved downstream promoter elements, with heterogeneous populations at early passage consistent with complementation. A poliovirus carrying the recombination-defective D79H polymerase substitution could not excise the cassette and became undetectable, establishing template switching rather than polarity itself as the escape requirement.", "citation": "Aguado LC, Jordan TX, Hsieh E, Blanco-Melo D, Heard J, Panis M, Vignuzzi M, tenOever BR. Homologous recombination is an intrinsic defense against antiviral RNA interference. *Proceedings of the National Academy of Sciences* 2018, volume 115, issue 39, pages E9211 to E9219. DOI 10.1073/pnas.1810229115. PMID 30209219. PMCID PMC6166822.", "sections": { "Citation": "Aguado LC, Jordan TX, Hsieh E, Blanco-Melo D, Heard J, Panis M, Vignuzzi M, tenOever BR. Homologous recombination is an intrinsic defense against antiviral RNA interference. *Proceedings of the National Academy of Sciences* 2018, volume 115, issue 39, pages E9211 to E9219.\n\nDOI 10.1073/pnas.1810229115. PMID 30209219. PMCID PMC6166822.", "One-sentence contribution": "Applying one uniform small RNA-based selective pressure to four virus families in vertebrate cells shows that the ability to escape it tracks with the capacity for polymerase template switching rather than with genome polarity as such, since positive-strand viruses excise the targeted sequence while negative-strand viruses are cleared and a recombination-defective poliovirus cannot escape.", "Executive summary": "RNA interference is the dominant antiviral defence in plants and invertebrates, so the ability to evade it should have shaped which viruses persist in those hosts. Testing that idea directly is difficult because most viruses that face RNA interference also encode antagonists of it. The authors sidestep this by rebuilding an RNA interference-like pressure in vertebrate cells, where viruses have no reason to carry such an antagonist. A cassette carrying perfectly complementary sites for five ubiquitously expressed host microRNAs was inserted into viral genomes, alongside a control cassette containing the same sequence in reverse orientation. Because perfect complementarity licenses Argonaute 2 cleavage, the cassette converts endogenous microRNAs into a slicing defence. The system was validated genetically, with silencing lost in cells lacking Dicer or Argonaute 2 but retained in cells lacking the other Argonautes, and with a genome-wide CRISPR screen recovering the canonical microRNA machinery and no interferon genes. Applied to essential transcripts, the pressure eliminated Sendai virus and influenza A virus, both negative-strand, with more than five logs of titre loss and near-total loss of viral reads. Sindbis virus, Semliki Forest virus and poliovirus, all positive-strand, were initially suppressed but recovered within passages through precise excision of the cassette. A poliovirus carrying a polymerase mutation that blocks homologous recombination could not excise the cassette and was undetectable by passage four, and about half of its reads at that codon had reverted to wild type.", "Scientific context": "Different domains of life meet viruses with different defences. Prokaryotes use restriction enzymes and CRISPR systems, plants and invertebrates use RNA interference, and vertebrates use the protein-based type I and type III interferon systems. All of these acquire or exploit information about the invader to direct an otherwise nonspecific activity. In vertebrates, some sequencing studies have suggested a low-level RNA interference-like activity, but functional work has found RNA interference and interferon to be mutually incompatible, which the paper reads as evidence that RNA interference plays a minor physiological role in reducing viral replication in these hosts. The small RNA machinery itself remains, repurposed for microRNAs, and among the vertebrate Argonautes only Argonaute 2 has retained cleavage activity, which can be recruited to a transcript by inserting a perfectly complementary site. The broader question motivating the study is whether host defences bias which kinds of viruses flourish, noting the general observation that DNA viruses dominate in prokaryotes while RNA viruses dominate in eukaryotes.", "Central question": "Do intrinsic features of a virus replication strategy, in the absence of any dedicated antagonist, determine whether a virus can evade an RNA interference-like defence, and if so which feature is decisive?", "Experimental strategy": "The design rests on making the pressure identical across otherwise incomparable viruses. Five microRNAs expressed ubiquitously in mammalian cells were selected, and a cassette of perfectly complementary target sites for all five was built. Perfect complementarity is essential, because ordinary microRNA pairing produces only subtle repression, whereas full complementarity licenses Argonaute 2 slicing and therefore reproduces the destructive character of invertebrate and plant RNA interference. The reverse-orientation cassette is the key control, since it has identical base composition and length but cannot be bound, so any difference between the two constructs is attributable to targeting rather than to the insertion itself. Two classes of cell are used, wild-type fibroblasts as the silencing-enabled setting and RNase III deficient fibroblasts as the silencing-deficient setting, which lets the same virus pair be compared with the pressure on and off. Placement matters, and the cassette was moved from a reporter to the untranslated region of an essential transcript in each virus so the pressure would be lethal rather than cosmetic. Four families were chosen to vary both polarity and gene expression strategy, namely a nonsegmented negative-strand paramyxovirus, a segmented negative-strand orthomyxovirus, two positive-strand alphaviruses with separate nonstructural and structural messages, and a monocistronic positive-strand picornavirus. Serial passage with deep sequencing of the whole population, rather than endpoint titre alone, is what allows escape events to be seen as excisions and mapped precisely. The final test is causal rather than correlative, using a poliovirus polymerase point mutation previously shown to prevent homologous recombination.", "Key findings": "1. Each of the five microRNA mimics individually silenced a reporter carrying the cassette in silencing-deficient cells, while an unrepresented microRNA did not, and the cassette was also silenced by endogenous microRNAs in wild-type cells.\n2. Expressed from Sendai virus, the cassette suppressed the reporter in wild-type and Argonaute 1, 3 and 4 knockout fibroblasts but not in cells lacking Dicer or Argonaute 2, while the reverse-orientation control was expressed in all genotypes (Figure 1, B and C). Silencing therefore requires the slicing-competent Argonaute.\n3. A genome-wide CRISPR knockout screen selecting cells that had lost silencing recovered Drosha, Dicer, Argonaute 2 and DGCR8, along with TP53, miR-21 and XPO5, all of which act on the available cytoplasmic microRNA pool. No interferon signature genes were implicated, which the authors take as evidence that the pressure is RNA interference activity alone (Figure 1E).\n4. With the cassette in the untranslated region of the Sendai virus nucleoprotein transcript, the targeted and control viruses were indistinguishable in silencing-deficient cells, while in silencing-enabled cells the targeted virus lost nucleoprotein entirely, fell more than five logs in titre within one passage and was undetectable by passage four (Figure 2, B through D). Sequencing of the targeted virus in silencing-enabled cells recovered only two reads aligning to the reporter open reading frame (Figure 2H).\n5. The same cassette in the untranslated region of influenza A virus segment five produced no difference in silencing-deficient cells but abolished nucleoprotein production and reduced infectious units by about five logs in silencing-enabled cells, with no evidence from sequencing that the cassette had been excised.\n6. Sindbis virus carrying the cassette between the nonstructural and structural regions showed only an initial delay in capsid expression under pressure and reached comparable levels by six hours after infection (Figure 3C). A separate construct expressing the targeted reporter from an extra promoter confirmed that silencing was active during Sindbis virus infection despite this replication.\n7. Serial passage with deep sequencing showed the control Sindbis virus stable over 96 hours, while the targeted virus gave rise by passage four to a single dominant species that had excised the cassette while retaining a functional subgenomic promoter (Figure 3, D and E). Three amino acid substitutions in nonstructural proteins, K232T, N370K and G595V, rose in frequency across three independent experiments. The paper does not determine whether these substitutions contribute to escape.\n8. At passage zero the Sindbis virus population was already heterogeneous, with roughly 90 percent intact genome and three distinct excision events making up the remainder. The authors interpret this as complementation within the population, in which intact genomes supply structural messages while excised genomes supply untargeted nonstructural messages.\n9. A Semliki Forest virus chimera replicating more slowly than Sindbis virus was undetectable under pressure through 36 hours but recovered by 48 hours, and passage produced a dominant genome with a precise excision that left the downstream subgenomic promoter intact. Replication kinetics therefore do not explain the failure of the negative-strand viruses to escape. The reported composition of the early heterogeneous population, given as about 65 percent wild-type genome and 45 percent excision events, sums to more than 100 percent in the text as extracted, so those specific proportions should be checked against the figure.\n10. Poliovirus, which makes all its proteins from one RNA, lost about two logs under pressure at first but escaped by excising the cassette, demonstrating that escape does not depend on a multi-transcript genome organisation (Figure 4).\n11. Introducing the D79H substitution into the poliovirus polymerase, previously characterised as blocking homologous recombination, left titres comparable to the parent in silencing-deficient cells but rendered the virus undetectable by passage four under pressure. Sequencing showed no excision, poor genomic coverage, and reversion of roughly half the reads at that codon to the wild-type sequence (Figure 4, B through D). This is the causal evidence that recombination, rather than polarity itself, is the escape mechanism.", "Mechanistic model": "The data support a specific and limited model. When a perfectly complementary target is placed in an essential viral transcript, Argonaute 2 cleaves it, and a virus survives only if it can remove the target from its genome. Removal occurs by precise excision, and the requirement for polymerase-mediated template switching is established by the recombination-defective poliovirus, which cannot excise and is cleared. Negative-strand viruses are cleared because they do not perform this reaction efficiently, which the authors attribute, following other work, to their genomes being encapsidated and therefore less accessible to the polymerase for template jumping. That structural explanation is cited reasoning rather than a result of this study. Several further statements in the discussion are labelled by the authors as speculation or inference and should not be read as demonstrated. These include the proposal that inefficient homologous recombination is directly responsible for the lower representation of negative-strand RNA viruses across the tree of life, the suggestion that exclusive polymerase control over the first message gives negative-strand viruses compensating advantages in controlling RNA structures and avoiding detection, and the framing of defective interfering particle formation as mechanistically distinct from template jumping. The study also does not establish that the same excision route would operate against naturally occurring antiviral RNA interference, and the authors state that excision of targeted genomic material would not be as straightforward in a physiological setting.", "Conceptual or technical advance": "The principal advance is a comparative assay. By reconstructing a slicing-competent small RNA defence out of endogenous vertebrate microRNAs, the authors create a selective pressure that is genuinely the same across four virus families and that no tested virus has evolved to antagonise, which removes the usual confound in comparing evasion capacity. Pairing the targeted cassette with a reverse-orientation control of identical sequence isolates targeting from insertion burden, and pairing silencing-enabled with RNase III deficient cells isolates the pressure from everything else about the infection. Reading escape by whole-population deep sequencing rather than titre turns evasion into a mapped genomic event. Biologically, the work reframes the polarity difference in RNA interference susceptibility as a consequence of recombination capacity, a proposition made testable and then tested with a single polymerase substitution. It also has a practical implication for microRNA-based attenuation strategies, since it predicts which virus classes will regain fitness by excision.", "Relationship to the broader research program": "The study grows out of a sustained line in the laboratory on the relationship between small RNA silencing and vertebrate antiviral immunity. Its reference list cites the group's own earlier findings that the mammalian response to virus infection is independent of small RNA silencing, that RNase III nucleases from diverse kingdoms can serve as antiviral effectors, that engineered mammalian RNA interference can provide antiviral protection that removes the requirement for the interferon response, and a review on the evolution of antiviral defence systems. The cassette design itself is attributed to the engineered mammalian RNA interference work. Situating this paper against the laboratory's later work on virus population structure and on host transcriptional responses would be category 3 synthesis and is not attempted here.", "Related publications": "- Benitez and colleagues, 2015, methodological foundation. Cited as the source of the microRNA silencing cassette design used throughout.\n- Aguado and colleagues, 2017, predecessor. The laboratory's report that RNase III nucleases from diverse kingdoms act as antiviral effectors, cited as the origin of the RNase III deficient cells used as the silencing-deficient setting.\n- Backes and colleagues, 2014, predecessor. Cited for the finding that the mammalian response to virus infection is independent of small RNA silencing, part of the argument that vertebrate viruses lack RNA interference antagonists.\n- tenOever, 2016, review or synthesis. The laboratory's review on the evolution of antiviral defence systems, which supplies the comparative framing.\n- tenOever, 2013, review or synthesis. Cited for exploitation of the host microRNA machinery by engineering perfect target sites.\n- Pham, Langlois and tenOever, 2012, application. Cited among prior studies that used microRNA targeting to restrict a positive-strand RNA virus, with complete silencing achieved.", "Limitations and boundaries": "The defence tested here is engineered rather than natural. It uses five fixed target sites placed at one chosen location in each genome, so the results describe escape from a single concentrated target region and not from the dispersed, regenerating small RNA population that a plant or invertebrate would produce, a limitation the authors state directly when they note that excision of naturally targeted material would not be as straightforward. All work is in cultured fibroblast and epithelial cell lines, with no animal infection, and the host cells are vertebrate cells that do not normally mount this defence. The comparison covers four families and six viruses, with only two negative-strand representatives, so the polarity generalisation rests on a small sample even though the poliovirus experiment supplies a mechanism. The recombination requirement is demonstrated by one polymerase substitution in one virus, and that mutant reverted under pressure, which complicates interpretation of its failure to persist. The escape-associated amino acid substitutions seen in Sindbis virus are correlative and untested. Read proportions reported for the early Semliki Forest virus population are internally inconsistent in the extracted text. The evolutionary claims about the representation of negative-strand viruses in nature are inference from the assay and are flagged as speculative by the authors themselves.", "Audience summaries": "### 25 words\n\nFaced with the same engineered small RNA attack, positive-strand RNA viruses cut the targeted sequence out of their genomes, while negative-strand viruses, unable to recombine, were eliminated.\n\n### 75 words\n\nRNA interference is the main antiviral defence in plants and insects, and vertebrate viruses have no reason to resist it. The authors built such a defence in mammalian cells by giving viruses perfectly matched binding sites for five common host microRNAs. Sendai and influenza viruses were wiped out. Sindbis, Semliki Forest and polioviruses recovered by precisely deleting the targeted sequence. A poliovirus unable to recombine could not delete it and was cleared.\n\n### 150 words\n\nA cassette of perfectly complementary sites for five ubiquitous host microRNAs converts endogenous Argonaute 2 into a slicing antiviral defence, with a reverse-orientation cassette as a sequence-matched control and RNase III deficient fibroblasts as the silencing-off condition. Genetic tests and a genome-wide CRISPR screen confirmed that the pressure runs through the microRNA machinery, implicating Drosha, Dicer, DGCR8, Argonaute 2, TP53, miR-21 and XPO5, with no interferon genes. Placed in essential transcripts, the cassette cleared Sendai virus and influenza A virus by more than five logs. Sindbis virus, a Semliki Forest virus chimera and poliovirus were suppressed initially but recovered by passage through precise excision of the cassette that preserved downstream promoter elements, with heterogeneous populations at early passage consistent with complementation. A poliovirus carrying the recombination-defective D79H polymerase substitution could not excise the cassette and became undetectable, establishing template switching rather than polarity itself as the escape requirement." }, "discoveries": [ "claim-09" ], "relationships": [ { "from": "2018-aguado-homologous-recombination-is-an-int", "to": "2017-aguado-rnase-iii-nucleases-from-diverse-k", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2018-aguado-homologous-recombination-is-an-int" }, { "from": "2018-aguado-homologous-recombination-is-an-int", "to": "2014-backes-the-mammalian-response-to-virus-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2018-aguado-homologous-recombination-is-an-int" }, { "from": "2018-aguado-homologous-recombination-is-an-int", "to": "2012-pham-replication-in-cells-of-hematopoie", "relationship": "application", "evidence": "stated in the Related publications section of 2018-aguado-homologous-recombination-is-an-int" } ], "canonical_url": "https://tenoeverlab.us/publications/2018-aguado-homologous-recombination-is-an-int/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "vsv", "sindbis-virus", "sendai-virus", "poliovirus", "semliki-forest-virus" ], "technologies": [ "plaque-assay", "reverse-genetics", "immunofluorescence-microscopy", "flow-cytometry", "mirna-target-site-insertion", "viral-population-deep-sequencing", "genome-wide-crispr-screen", "serial-passage" ] } }, { "id": "2018-han-genome-wide-crispr-cas9-screen-ide", "slug": "2018-han-genome-wide-crispr-cas9-screen-ide", "url": "/publications/2018-han-genome-wide-crispr-cas9-screen-ide/", "title": "Genome-wide CRISPR/Cas9 Screen Identifies Host Factors Essential for Influenza Virus Replication", "authors": [ "Julianna Han", "Jasmine T. Perez", "Cindy Chen", "Yan Li", "Asiel Benitez", "Matheswaran Kandasamy", "Yoontae Lee", "Jorge Andrade", "Benjamin tenOever", "Balaji Manicassamy" ], "author_count": 10, "first_author": "Julianna Han", "senior_authors": [ "Balaji Manicassamy" ], "corresponding_authors": [ "Balaji Manicassamy" ], "tenoever_position": 9, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2018, "journal": "Cell Reports", "volume": "23", "issue": "2", "pages": "596-607", "doi": "10.1016/j.celrep.2018.03.045", "doi_url": "https://doi.org/10.1016/j.celrep.2018.03.045", "pmid": "29642015", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/29642015/", "pmcid": "PMC5939577", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939577/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939577/pdf/", "publication_type": "methods/resource", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling", "programmable-virology" ], "themes": [ "homeostatic-repression-of-isgs", "in-vivo-screening-through-fitness" ], "pathogens": [ "influenza A virus", "vesicular stomatitis virus", "Zika virus", "encephalomyocarditis virus" ], "viral_families": [ "Orthomyxoviridae", "Rhabdoviridae", "Flaviviridae", "Picornaviridae" ], "host_species": [ "human" ], "technologies": [ "genome-wide CRISPR knockout screening", "GeCKO library", "lentiviral transduction", "MAGeCK analysis", "deep sequencing", "beta-lactamase virus-like particle entry assay", "lectin staining", "flow cytometry", "luciferase promoter reporter assay", "quantitative RT-PCR", "cDNA complementation" ], "biological_systems": [ "A549 cells", "Cas9-expressing A549 clonal line", "CRISPR knockout clonal lines" ], "key_concepts": [ "host factor discovery", "positive selection survival screen", "sialic acid biosynthesis", "CMP-sialic acid transport", "viral receptor expression", "cell-intrinsic immunity", "transcriptional repression of interferon-stimulated genes", "capicua and ATXN1 corepressor", "pan-proviral versus virus-specific factors" ], "keywords": [ "CRISPR screen", "GeCKO", "influenza A virus", "SLC35A1", "capicua", "CIC", "JAK2", "PIAS3", "sialic acid", "H5N1" ], "one_sentence_contribution": "A survival-based genome-wide CRISPR knockout screen in human lung epithelial cells selected with an avian H5N1 isolate recovers sialic acid biosynthesis and transport as the dominant requirement for influenza entry, with the CMP-sialic acid transporter SLC35A1 as the top hit, and identifies the transcriptional repressor capicua as a negative regulator of cell-intrinsic immunity.", "summary_25": "Deleting genes across the human genome and selecting cells that survive influenza infection pointed to sialic acid supply for viral entry and to capicua for antiviral control.", "summary_75": "A pooled CRISPR knockout library in human lung cells was put through repeated lethal infection with an avian H5N1 isolate, so that cells missing a required host gene survive. Guides against sialic acid biosynthesis, transport and glycan processing were most enriched, with the CMP-sialic acid transporter SLC35A1 ranked first and its loss removing the viral receptor. The screen also found capicua, a transcriptional repressor whose loss raises antiviral gene expression and restricts viruses from four families.", "summary_150": "Prior genome-wide screens for influenza host factors overlapped poorly with one another, motivating a different perturbation and readout. A genome-scale CRISPR knockout library was established in human lung epithelial cells and subjected to repeated lethal infection with a human isolate of a low-pathogenic avian H5N1 strain, under two selection regimes whose intersection defined the candidate set. Sialic acid biosynthesis, nucleotide sugar transport, glycan processing and GPI anchor synthesis were enriched along with vacuolar ATPase subunits found in earlier screens. The top hit, SLC35A1, was required for surface sialic acid in both linkages, for hemagglutinin binding and for entry, and its loss did not affect vesicular stomatitis virus. Capicua, a DNA-binding transcriptional repressor previously linked to cancer and neuropathology, restricted influenza together with vesicular stomatitis, Zika and encephalomyocarditis viruses when deleted, raised antiviral gene expression, and repressed interferon-stimulated gene promoters with its corepressor. Most hits rest on a single guide RNA.", "citation": "Han J, Perez JT, Chen C, Li Y, Benitez A, Kandasamy M, Lee Y, Andrade J, tenOever B, Manicassamy B. Genome-wide CRISPR/Cas9 Screen Identifies Host Factors Essential for Influenza Virus Replication. Cell Reports. 2018. Volume 23, issue 2, pages 596-607. DOI 10.1016/j.celrep.2018.03.045. PMID 29642015. PMCID PMC5939577.", "sections": { "Citation": "Han J, Perez JT, Chen C, Li Y, Benitez A, Kandasamy M, Lee Y, Andrade J, tenOever B, Manicassamy B. Genome-wide CRISPR/Cas9 Screen Identifies Host Factors Essential for Influenza Virus Replication. Cell Reports. 2018. Volume 23, issue 2, pages 596-607.\n\nDOI 10.1016/j.celrep.2018.03.045. PMID 29642015. PMCID PMC5939577.", "One-sentence contribution": "A survival-based genome-wide CRISPR knockout screen in human lung epithelial cells selected with an avian H5N1 isolate recovers sialic acid biosynthesis and transport as the dominant requirement for influenza entry, with the CMP-sialic acid transporter SLC35A1 as the top hit, and identifies the transcriptional repressor capicua as a negative regulator of cell-intrinsic immunity.", "Executive summary": "Influenza A virus emerges repeatedly from animal reservoirs, seasonal vaccines do not cover zoonotic strains, and drug resistance arises quickly, which has motivated interest in host-directed intervention. Multiple genome-wide screens using interfering RNA, proteomics and insertional mutagenesis had been performed, but meta-analyses found little overlap between them. This study applied a pooled CRISPR knockout library to human lung epithelial cells and selected survivors through repeated lethal infection with a human isolate of a low-pathogenic avian H5N1 virus, which enriches for cells that cannot support the virus rather than scoring a reporter. Two selection regimes were run, one with minimal expansion between rounds and one allowing expansion, and their overlap was used to prioritise candidates. Guide RNAs targeting sialic acid biosynthesis, nucleotide sugar transport, glycan processing and GPI anchor synthesis were enriched, along with vacuolar ATPase subunits that previous screens had also found. Eleven candidates were knocked out individually and seven taken to clonal lines. The top hit, SLC35A1, proved essential for surface sialic acid, hemagglutinin binding and viral entry, and its loss did not affect vesicular stomatitis virus. Capicua, a DNA-binding transcriptional repressor not previously connected to antiviral immunity, restricted influenza together with three unrelated viruses when deleted, raised baseline and infection-induced antiviral gene expression, and repressed interferon-stimulated gene promoters when expressed with its corepressor.", "Scientific context": "Host-directed antiviral strategies require knowing which cellular genes influenza depends on, and by 2018 at least nine genome-wide screens had addressed that question using small interfering RNA, proteomic and insertional mutagenesis approaches. The paper notes that these efforts converged on relatively few common hits, most consistently members of the vacuolar ATPase family, and that meta-analyses found little overlap otherwise, which the authors attribute to differences in virus strain, time point and functional readout. CRISPR/Cas9 had recently made genome-scale gene disruption feasible in mammalian cells, and pooled knockout libraries had begun to be applied to virus-host questions in other systems. The rationale given for adding another screen is that a different perturbation modality with a different readout should both surface new factors and independently test previously reported ones. Separately, capicua was known as a conserved DNA-binding transcriptional repressor acting with the corepressor ATXN1 or its paralogue, implicated in cancer, neuropathology and autoimmunity, with one prior report of elevated proinflammatory cytokines in mice deficient for its long isoform, but it had not been connected to cell-intrinsic antiviral immunity.", "Central question": "Which human genes are required for influenza A virus replication in lung epithelial cells, as revealed by a positive selection screen in which cells lacking a required factor survive lethal infection.", "Experimental strategy": "The design rests on survival as the selective pressure, which is what distinguishes it from reporter-based or knockdown-based screens and biases the output towards factors acting early. A clonal Cas9-expressing A549 line was transduced with a pooled library of 65,383 guide RNAs against 19,050 protein-coding genes and 1,864 microRNA precursors at low multiplicity so that most cells carry one guide, and puromycin selection for fourteen days both established the library and removed cells whose disrupted gene is required for viability, so that hits are not simply essential genes. Library composition was verified by sequencing before selection. Two selection schemes were then run in parallel, a stringent one with five consecutive rounds of lethal infection and minimal expansion, and a less stringent sequential one in duplicate that allows expansion between rounds and permits sgRNA representation to be tracked round by round. Running both allows the intersection to be used as the prioritised candidate set, which partly compensates for the replicate divergence that stringent selection produces. Candidate ranking used MAGeCK. Validation moved from polyclonal knockout pools to clonal lines with sequenced target sites, because incomplete disruption in pools understates effects. Specificity was addressed on three axes, across influenza subtypes including H1N1 and H3N2, against an unrelated virus to separate influenza-specific from broadly proviral factors, and by cDNA complementation to exclude off-target explanations. Mechanism was then assigned by stage, using synchronised high multiplicity infection to test single-cycle competence, beta-lactamase virus-like particles bearing either influenza glycoproteins or the vesicular stomatitis virus glycoprotein to isolate entry and fusion, strand-specific quantitative RT-PCR at three and six hours to separate primary transcription from genome replication, and antiviral gene expression under mock and infected conditions.", "Key findings": "1. The library was established at roughly 140-fold coverage with 62,659 guides recovered, and 4.2 percent of guides lost during selection, which the authors read as removal of a non-viable population (Figures S1A and S1B).\n2. In the sequential screen, guide representation was unchanged after one round and robustly enriched from round two onwards, so selection of a less permissive population requires two rounds of lethal infection (Figure 1B). Replicates correlated at 0.92 at round one and 0.25 or below thereafter, meaning the two replicates diverged at the same point enrichment began, though a common enriched set was still recovered (Figures S1D and S1E).\n3. MAGeCK identified 798 enriched genes at round two and 501 at round five. Genes covered by two or more independent guides fell from 161 to 16 across those rounds, which the authors interpret as stringent selection favouring individual guides rather than genes (Figure 1C). The preliminary consecutive screen gave 119 positively selected genes with SLC35A1 ranked first and represented by all three of its guides.\n4. Comparison with nine published influenza screens found 33 of 453 round five hits in common, with vacuolar ATPase subunits recovered as in six prior screens, leaving more than 400 genes not previously reported (Figure 1D, Table S2). Pathway analysis returned proton transport and vacuolar acidification alongside N-acetylneuraminate metabolism, GPI anchor biosynthesis, COPII coating, autophagy and JAK-STAT and MAPK signalling (Figure 1E).\n5. Of eleven candidates taken forward from the 63 genes shared between screens, eight polyclonal knockouts reduced H5N1 titre by more than 60 percent, and clonal knockouts gave roughly five logs of reduction for SLC35A1 and capicua and more than 80 percent for the rest (Figures 2B and 2C). The gap between polyclonal and clonal results indicates that complete disruption was needed to see the full effect.\n6. Across strains, SLC35A1 loss cost more than five logs and capicua loss more than three logs for H1N1 and H3N2 (Figure 2C). Vesicular stomatitis virus was unaffected in SLC35A1 and PIGN knockouts but reduced by more than two logs in capicua knockouts, which the authors use to classify SLC35A1 and PIGN as influenza-specific and capicua, JAK2, PIAS3, C2CD4C and TRIM23 as broadly proviral. PIAS3 showed strain specificity, affecting H1N1 and H5N1 but not H3N2.\n7. Virus-like particle assays placed SLC35A1 at entry or fusion, with only 3.6 percent of knockout cells positive for influenza particles, and implicated PIAS3 more modestly at 25 percent, while particles bearing the vesicular stomatitis virus glycoprotein entered all lines normally (Figure 3B).\n8. Capicua knockouts took up normal levels of input genomic RNA but showed more than 90 percent reduction in primary nucleoprotein transcription at three hours and about 80 percent reduction in both genomic RNA and messenger RNA at six hours, placing the block between fusion and primary transcription (Figure 3C).\n9. SLC35A1 knockouts lost binding of lectins specific for both 2-3 and 2-6 linked sialic acid and failed to bind recombinant H5 hemagglutinin (Figures 4B to 4D). Complementation with SLC35A1 cDNA restored influenza replication without affecting vesicular stomatitis virus, and overexpression in wild-type cells did not increase replication (Figure 4G, Figure S4B).\n10. Chemical inhibition of downstream sialyltransferases with a CMP-sialic acid analogue restricted H1N1 and H3N2 but left H5N1 unchanged despite near-complete loss of lectin binding (Figures 4E and 4F). The authors do not resolve this discrepancy.\n11. Capicua knockouts made with an independent guide reproduced the phenotype, showed reduced ATXN1L protein and elevated expression of the known capicua-regulated gene ETV4, and restricted H5N1, H1N1, H3N2, vesicular stomatitis virus, Zika virus and encephalomyocarditis virus (Figure 5A, Figures S5B and S5C). Antiviral gene expression was elevated under both mock and infected conditions by RT-PCR and immunoblot (Figures 5B to 5D).\n12. Co-expression of capicua with ATXN1 reduced RIG-I-stimulated IFIT1 reporter activity by up to 50 percent and MxA reporter activity by about 40 percent, more than either protein alone (Figure 5E).\n13. Capicua protein declined between forty and sixty minutes after H1N1 infection and its messenger RNA fell by about half by sixteen hours (Figures 5F and 5G). The authors present this as consistent with capicua being downregulated to permit antiviral gene induction, and speculate about MAPK-dependent degradation and about viral usurpation of the COP9 signalosome, both explicitly as speculation.", "Mechanistic model": "For SLC35A1 the mechanism is well supported and simple. The transporter delivers CMP-sialic acid into the Golgi, its loss removes sialic acid from cell surface glycans in both linkage types, hemagglutinin cannot bind, and the virus does not enter. Loss of entry accounts for the downstream absence of input genome and transcription, complementation restores the phenotype, and an unrelated virus that uses a different receptor is unaffected.\n\nFor capicua the study does not establish a mechanism and the paper is careful about this. The data show that capicua loss raises antiviral gene expression at baseline and during infection, restricts four unrelated viruses, and blocks influenza between fusion and primary transcription, and that ectopic capicua with its corepressor suppresses two interferon-stimulated gene promoters under RIG-I stimulation. The authors write that it is possible that capicua suppresses antiviral gene expression through its transcriptional repressor activity, which is the natural reading but is not demonstrated by occupancy at those promoters or by separation of repressor function from the observed phenotype. Whether the antiviral state is the sole cause of the influenza block, and in particular why primary transcription specifically is impaired, is not resolved. The downregulation of capicua during infection is observed, and the routes proposed for it, MAPK-driven degradation and interference through the COP9 signalosome, are labelled by the authors as speculation and left to future work.\n\nFor JAK2 and PIAS3 the paper reports discordant observations without resolving them. JAK2 had been implicated in entry in a prior screen, but no entry defect was seen here while genome replication fell, and the authors suggest that raised antiviral gene expression may account for it. PIAS3 shows both an entry defect and increased antiviral gene expression only upon infection, and possible roles in cytoskeletal regulation through SUMOylation and in negative regulation of STAT signalling are offered as untested candidates.", "Conceptual or technical advance": "The study shows that a survival-based CRISPR knockout screen returns a different and largely non-overlapping slice of the influenza host factor landscape than the interfering RNA screens that preceded it, recovering the sialic acid biosynthesis, transport and glycan processing pathway as a coherent block rather than as isolated hits. That coherence is itself informative, since it is the pathway rather than any single gene that the screen resolves. The screen also promotes SLC35A1 from a marginal hit in one earlier study to the top-ranked factor with a five log effect, which illustrates how the readout shapes what a screen can see. Independently of influenza, the identification of capicua as a repressor whose loss raises the antiviral set point and restricts viruses from four families opens a line on transcriptional restraint of cell-intrinsic immunity that is not virus specific. The paired stringent and expansion-permitting selection schemes, with their intersection used for prioritisation, are a practical contribution to how such screens can be run given that stringent selection drives replicate divergence.", "Relationship to the broader research program": "This is a collaborative paper led by the Manicassamy laboratory at the University of Chicago, with the tenOever laboratory represented by Asiel Benitez in the author list and no author contributions statement in the article to apportion roles further. The point of contact with the tenOever program is the parallel interest in unbiased genetic identification of influenza host factors, and the paper cites the tenOever laboratory's own in vivo RNAi screen that identified MDA5 as a contributor to cellular defence against influenza A virus. Both studies approach the same question with a loss-of-function library and arrive at regulators of cell-intrinsic immunity rather than only at replication machinery, which is a category 3 synthesis visible when the two are read together rather than a claim made in either. The influenza reverse genetics and virology context here also overlaps with the strains and tools used across the corpus.", "Related publications": "- Benitez and colleagues, 2015, companion in approach, from the tenOever laboratory. An in vivo RNAi screen for influenza host factors that identified MDA5, cited here among the prior genome-wide screening efforts, and the source of the RNase III deficient cell resources used elsewhere in this corpus.\n- Shalem and colleagues, 2014, Sanjana and colleagues, 2014, and Wang and colleagues, 2014, methodological foundation, from other laboratories. Supply the GeCKO library and the pooled screening procedure followed here.\n- Li and colleagues, 2014, methodological foundation, from another laboratory. The MAGeCK analysis used to rank enriched genes.\n- Brass and colleagues, 2009, Hao and colleagues, 2008, Karlas and colleagues, 2010, König and colleagues, 2010, Shapira and colleagues, 2009, Sui and colleagues, 2009, Ward and colleagues, 2012, Su and colleagues, 2013, and Watanabe and colleagues, 2014, predecessor, from other laboratories. The nine prior genome-wide influenza screens against which the hit list is compared, and the source of the earlier and weaker SLC35A1 and JAK2 observations.\n- Tscherne and colleagues, 2010, methodological foundation, from another laboratory. Source of the beta-lactamase virus-like particle entry assay.\n- Kim and colleagues, 2015, and Jiménez and colleagues, 2012, predecessor, from other laboratories. Establish capicua as a transcriptional repressor acting with ATXN1 and report elevated proinflammatory cytokines in mice lacking its long isoform, the prior observation most consistent with the immune phenotype found here.", "Limitations and boundaries": "The two screen replicates diverged sharply once selection took effect, with correlation falling from 0.92 to 0.25 or below, and the number of genes supported by more than one guide fell from 161 at round two to 16 at round five, so most reported hits rest on a single guide RNA and the screen output should be treated as a candidate list rather than a validated gene set. Only eleven of 63 shared candidates were tested individually and seven taken to clonal lines. One of those clonal capicua lines retained a wild-type allele, which the authors report. Off-target risk was assessed computationally by mismatch analysis rather than experimentally for most lines, though complementation was performed for SLC35A1 and JAK2. All work is in one immortalised human lung epithelial cell line with no primary cells, no differentiated airway model and no animal infection, and a survival-based design is acknowledged by the authors to favour factors acting at early steps, so requirements for assembly, egress or spread would not be recovered. The sialyltransferase inhibitor restricted H1N1 and H3N2 but not H5N1 despite loss of detectable lectin binding, an internal inconsistency the paper notes but does not explain. For capicua no promoter occupancy or direct target is shown, the connection between the raised antiviral state and the specific block at primary transcription is not established, and the proposed mechanisms for its downregulation during infection are labelled speculative. The JAK2 entry result conflicts with a prior report and is left unresolved.", "Audience summaries": "### 25 words\n\nDeleting genes across the human genome and selecting cells that survive influenza infection pointed to sialic acid supply for viral entry and to capicua for antiviral control.\n\n### 75 words\n\nA pooled CRISPR knockout library in human lung cells was put through repeated lethal infection with an avian H5N1 isolate, so that cells missing a required host gene survive. Guides against sialic acid biosynthesis, transport and glycan processing were most enriched, with the CMP-sialic acid transporter SLC35A1 ranked first and its loss removing the viral receptor. The screen also found capicua, a transcriptional repressor whose loss raises antiviral gene expression and restricts viruses from four families.\n\n### 150 words\n\nPrior genome-wide screens for influenza host factors overlapped poorly with one another, motivating a different perturbation and readout. A genome-scale CRISPR knockout library was established in human lung epithelial cells and subjected to repeated lethal infection with a human isolate of a low-pathogenic avian H5N1 strain, under two selection regimes whose intersection defined the candidate set. Sialic acid biosynthesis, nucleotide sugar transport, glycan processing and GPI anchor synthesis were enriched along with vacuolar ATPase subunits found in earlier screens. The top hit, SLC35A1, was required for surface sialic acid in both linkages, for hemagglutinin binding and for entry, and its loss did not affect vesicular stomatitis virus. Capicua, a DNA-binding transcriptional repressor previously linked to cancer and neuropathology, restricted influenza together with vesicular stomatitis, Zika and encephalomyocarditis viruses when deleted, raised antiviral gene expression, and repressed interferon-stimulated gene promoters with its corepressor. Most hits rest on a single guide RNA." }, "discoveries": [ "claim-05" ], "relationships": [ { "from": "2025-manivasagam-transcriptional-repressor-capicua-", "to": "2018-han-genome-wide-crispr-cas9-screen-ide", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2025-manivasagam-transcriptional-repressor-capicua-" } ], "canonical_url": "https://tenoeverlab.us/publications/2018-han-genome-wide-crispr-cas9-screen-ide/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "vsv", "emcv", "zika-virus" ], "technologies": [ "rt-qpcr", "flow-cytometry", "luciferase-promoter-reporter", "viral-population-deep-sequencing", "lentiviral-transduction", "pseudotyped-entry-reporter", "genome-wide-crispr-screen", "genetic-complementation", "lectin-staining" ] } }, { "id": "2018-m-ller-mirna-mediated-targeting-of-human-", "slug": "2018-m-ller-mirna-mediated-targeting-of-human-", "url": "/publications/2018-m-ller-mirna-mediated-targeting-of-human-/", "title": "miRNA-mediated targeting of human cytomegalovirus reveals biological host and viral targets of IE2", "authors": [ "Rasmus Møller", "Toni M. Schwarz", "Vanessa M. Noriega", "Maryline Panis", "David Sachs", "Domenico Tortorella", "Benjamin R. tenOever" ], "author_count": 7, "first_author": "Rasmus Møller", "senior_authors": [ "Domenico Tortorella", "Benjamin R. tenOever" ], "corresponding_authors": [ "Domenico Tortorella", "Benjamin R. tenOever" ], "tenoever_position": 7, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2018, "journal": "Proceedings of the National Academy of Sciences", "volume": "115", "issue": "5", "pages": "1069-1074", "doi": "10.1073/pnas.1719036115", "doi_url": "https://doi.org/10.1073/pnas.1719036115", "pmid": "29339472", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/29339472/", "pmcid": "PMC5798380", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798380/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798380/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "programmable-virology" ], "themes": [ "cell-type-restriction-as-a-tool" ], "pathogens": [ "human cytomegalovirus" ], "viral_families": [ "Herpesviridae" ], "host_species": [ "human" ], "technologies": [ "galK one-step BAC recombineering", "microRNA target site insertion", "lentiviral microRNA transduction", "small RNA Northern blot", "RNA sequencing", "differential expression analysis with DESeq2", "gene ontology enrichment analysis", "multicycle growth curves", "plaque and TCID50 titration", "quantitative RT-PCR" ], "biological_systems": [ "MRC-5 fibroblasts", "THP-1-derived macrophages", "miR-122-expressing MRC-5 fibroblasts", "miR-142-expressing MRC-5 fibroblasts", "TB40/E bacterial artificial chromosome" ], "key_concepts": [ "cell-type-specific conditional knockdown", "hematopoietic-specific microRNA targeting", "immediate early gene circuitry", "IE2 autorepression through the cis-repression sequence", "essential gene function in myeloid cells", "herpesvirus latency models", "viral transcriptional cascade", "host transcriptome remodelling", "engineered viral vectors" ], "keywords": [ "human cytomegalovirus", "IE2", "IE1", "miR-142", "myeloid cells", "macrophages", "recombineering", "conditional knockout", "latency", "microRNA targeting" ], "one_sentence_contribution": "A one-step recombineering strategy that inserts hematopoietic-specific miR-142 target sites into the untranslated region of the human cytomegalovirus IE2 transcript permits virus rescue in fibroblasts while silencing IE2 selectively in myeloid cells, revealing that IE2 loss raises rather than abolishes replication in macrophages.", "summary_25": "Placing blood-cell-specific microRNA targets in a cytomegalovirus gene lets the virus be grown normally in fibroblasts while the gene is switched off only in macrophages.", "summary_75": "Cytomegalovirus mutants must be grown in fibroblasts, which blocks study of genes essential there but interesting elsewhere. Inserting target sites for the blood-lineage microRNA miR-142 into the IE2 transcript silenced IE2 only in macrophages while leaving fibroblast growth intact. Loss of IE2 raised IE1 sharply and sustained virus titres in macrophages instead of abolishing them, and it reshaped both viral and host gene expression far more in macrophages than in fibroblasts.", "summary_150": "Human cytomegalovirus genetics is limited by the requirement to rescue recombinant virus in fibroblasts, which precludes deleting genes essential during acute fibroblast infection. Møller and colleagues inserted four perfect target sites for the hematopoietic-restricted microRNA miR-142 into the noncoding 3 prime untranslated region of IE2 in the TB40/E bacterial artificial chromosome using a single galK and loxP recombination step. The resulting virus replicated indistinguishably from control in fibroblasts, which lack miR-142, while in THP-1-derived macrophages IE2 protein was silenced, IE1 rose through relief of cis-repression, and titres were sustained two to three logs above a control that declined over ten days. RNA sequencing showed extensive viral transcriptome remodelling and more than 750 host gene changes in macrophages against roughly fifty in miR-142-expressing fibroblasts. The authors note that residual IE2 may still support an early essential role, so the system represents a strong lineage-restricted knockdown rather than a null.", "citation": "Møller R, Schwarz TM, Noriega VM, Panis M, Sachs D, Tortorella D, tenOever BR. miRNA-mediated targeting of human cytomegalovirus reveals biological host and viral targets of IE2. Proceedings of the National Academy of Sciences. 2018. Volume 115, issue 5, pages 1069-1074. DOI 10.1073/pnas.1719036115. PMID 29339472. PMCID PMC5798380.", "sections": { "Citation": "Møller R, Schwarz TM, Noriega VM, Panis M, Sachs D, Tortorella D, tenOever BR. miRNA-mediated targeting of human cytomegalovirus reveals biological host and viral targets of IE2. Proceedings of the National Academy of Sciences. 2018. Volume 115, issue 5, pages 1069-1074.\n\nDOI 10.1073/pnas.1719036115. PMID 29339472. PMCID PMC5798380.", "One-sentence contribution": "A one-step recombineering strategy that inserts hematopoietic-specific miR-142 target sites into the untranslated region of the human cytomegalovirus IE2 transcript permits virus rescue in fibroblasts while silencing IE2 selectively in myeloid cells, revealing that IE2 loss raises rather than abolishes replication in macrophages.", "Executive summary": "Human cytomegalovirus persists in a large fraction of the human population and causes serious disease in neonates and immunocompromised people, yet the biology of its latent phase in myeloid cells remains poorly defined. A central technical obstacle is that viral mutants must be rescued in fibroblasts, which means genes essential for acute fibroblast infection cannot be deleted even when their function in myeloid lineages is the question of interest. The authors address this by making the silencing conditional on cell lineage rather than on temperature or a small molecule. Four perfectly complementary target sites for the hematopoietic-restricted microRNA miR-142 were inserted into the noncoding 3 prime untranslated region of IE2 in the TB40/E bacterial artificial chromosome using a single galK-based recombination step with loxP-flanked selection. Because fibroblasts lack miR-142, the recombinant virus grew normally during rescue and in multicycle growth curves. In THP-1-derived macrophages, IE2 protein was silenced, derepression of the cis-repression sequence produced a large excess of IE1, and virus titres were sustained at levels roughly two to three logs above the untargeted control, which declined over ten days. RNA sequencing showed that IE2 loss reshaped both the viral and the host transcriptome in a cell-type-dependent way, with more than 750 host genes differentially expressed in macrophages against roughly fifty in miR-142-expressing fibroblasts. The work supplies a general method for lineage-restricted study of essential herpesvirus genes.", "Scientific context": "Herpesvirus genetics depends on bacterial artificial chromosomes or temperature-sensitive mutants, systems that permit rescue only in particular cells or at particular temperatures. This constrains what can be asked about genes that are essential during acute fibroblast infection but may serve different roles in the myeloid compartment where cytomegalovirus establishes latency. Existing conditional approaches require additional stimuli such as small compounds or temperature shifts and are described by the authors as laborious. On the biology side, IE1 and IE2 arise from a shared major immediate early promoter and enhancer and share the first three exons. IE2 had been characterised as both a transactivator of early and late genes and an autorepressor that binds the 14 base pair cis-repression sequence as a dimer, damping transcription from the promoter and thereby limiting both IE1 and itself. IE1 was known to be dispensable for replication while IE2 was considered essential, a conclusion drawn from fibroblast studies. The function of these proteins in shaping viral and cellular gene expression during infection of myeloid cells had not been characterised. Recent work from other groups had implicated valosin-containing protein as required for IE2 function and had shown IE2 to have a broad transcriptional footprint on viral genes, and had reported UL144 as a gene whose expression rises in the absence of IE2.", "Central question": "Can a lineage-restricted cellular microRNA be used to silence an essential cytomegalovirus gene only in the cells where its function is in question, and if so, what does selective loss of IE2 do to viral replication, to the viral transcriptional cascade, and to the host transcriptome in myeloid cells as opposed to fibroblasts?", "Experimental strategy": "The strategy converts a genetic problem into a post-transcriptional one. Rather than deleting or mutating the IE2 open reading frame, four perfect miR-142 target sites were placed in the noncoding 3 prime untranslated region of IE2 in the TB40/E clone, so the coding capacity of the gene is untouched and silencing is imposed only where miR-142 is present. Recombineering was simplified to a single step by combining a galK positive selection marker with flanking loxP sites, allowing Cre excision during virus rescue and removing the need for a counterselection round. The cellular logic rests on the restriction of miR-142 to the hematopoietic lineage, which the authors confirmed by small RNA Northern blot against ubiquitous miR-93 in MRC-5 fibroblasts and THP-1-derived macrophages, including during infection. Two comparisons were then run in parallel. Untargeted and targeted viruses were compared in fibroblasts, where the targeted virus should behave as wild type, and in macrophages, where IE2 should be silenced. To separate effects of IE2 loss from effects of cell type, the authors built fibroblast populations transduced to express either miR-142 or the hepatocyte-specific miR-122, giving a fibroblast background in which IE2 could be silenced and a matched control in which it could not. Readouts combined immunoblotting for IE1 and IE2, multicycle growth curves, and RNA sequencing of both viral and host transcripts at nine days post-infection with differential expression called by DESeq2.", "Key findings": "1. Insertion of the four miR-142 target sites plus loxP-flanked galK into the IE2 3 prime untranslated region by one round of homologous recombination produced the targeted virus TB40/E142T, and multicycle growth curves in fibroblasts showed no discernible difference from the untargeted control, with peak titres near 1 times 10 to the sixth plaque-forming units per millilitre (Figure 1A and Results).\n\n2. Small RNA Northern blot confirmed that miR-142 is absent from MRC-5 fibroblasts and abundant in THP-1-derived macrophages, that miR-93 is present in both, and that infection did not alter either profile after two days (Figure 1B).\n\n3. At twenty-four hours post-infection, IE1 and IE2 protein levels were comparable between the control and targeted viruses in fibroblasts, while in macrophages IE2 was silenced and IE1 was strongly overproduced (Figure 1C). The authors attribute the IE1 excess to relief of IE2-mediated repression at the cis-repression element, which follows from established IE2 autorepression but is not separately tested here.\n\n4. RNA sequencing of infected macrophages at twenty-four hours showed IE1-specific exon 4 reads six times higher with miR-142 targeting, representing 1.2 percent of the cytomegalovirus transcriptome against 0.2 percent for the control, with IE2 reads inversely affected, and at this early time point only IE1 and IE2 expression was changed (Figure 1D and Figure S1).\n\n5. Across twenty-four, forty-eight and seventy-two hours, fibroblasts showed no difference in IE1 or IE2 between the two viruses, while macrophages showed sustained loss of IE2 and elevated IE1 that remained detectable at seventy-two hours when IE1 was undetectable in control infections (Figures 2A and 2B).\n\n6. At nine days post-infection, RNA sequencing of fibroblasts lacking miR-142 showed no significant viral gene expression changes between the two viruses, whereas macrophages showed extensive remodelling of the viral transcriptome including UL144, IE1, RNA5.0, vIL-10, US29, US30, US31 and US32 (Figures 2C and 2D, Figure S2, Table S1). The authors note that many of these genes lie near putative cis-repression elements and that some may nonetheless be indirect targets of IE2.\n\n7. Replication diverged by cell type. In fibroblasts the two viruses reached comparable peak titres near 10 to the sixth plaque-forming units per millilitre over ten days. In macrophages the control virus declined steadily to as low as 10 plaque-forming units per millilitre while the targeted virus was maintained between 1,000 and 10,000 (Figures 3A and 3B). The abstract states this as a greater than 100-fold increase in titre in myeloid cells.\n\n8. In fibroblasts engineered to express miR-142, the targeted virus reproduced the IE1 and IE2 pattern seen in macrophages, although knockdown was less complete than with endogenous miR-142, while miR-122-expressing fibroblasts showed no effect (Figures 4A and 4B). Cytopathic effect and viral read counts were comparable between the viruses in this system (Table S2).\n\n9. Viral genes lost in the absence of IE2 in both miR-142-expressing fibroblasts and macrophages included IE2 itself, UL24, UL25, UL69, UL71, UL94, UL95, UL97, pp65 and pp71, while vIL-10, US29 and UL145 were induced in the fibroblast setting (Figure 4C, Figure S2C, Tables S1 and S2). RNA5.0 rose sharply in macrophages without significant change in fibroblasts, which the authors read as evidence of cell-specific viral gene function.\n\n10. Host transcriptional consequences were markedly cell-type-dependent. Roughly fifty host genes changed in miR-142-expressing fibroblasts, enriched for cellular metabolism, and included upregulation of the interferon-stimulated gene IFIT2, which the authors suggest may indicate a role for IE2 in damping innate defences as proposed by others. More than 750 host genes changed in macrophages, validated in part by quantitative PCR, with about 20 percent overlap with the fibroblast set, additional enrichment for immune system processes, and loss of genes involved in cell-to-cell communication (Figure 4D, Figures S4 and S5, Tables S3 and S4).", "Mechanistic model": "The study does not establish a definitive mechanism for how IE2 controls the genes whose expression changes, and the authors state that many of the macrophage changes are likely not direct consequences of transcriptional repression by the viral product. What the data support is a layered account. Silencing is imposed post-transcriptionally through perfect complementarity between miR-142 and sites in the IE2 untranslated region, so it occurs only where that microRNA is expressed. Loss of IE2 protein removes occupancy of the cis-repression sequence and thereby raises IE1, consistent with the established negative feedback architecture of the major immediate early locus. Elevated IE1 accompanies sustained rather than abolished replication in macrophages, and the authors propose that high IE1 supports continued virus production in this lineage. On why the virus survives at all without a gene deemed essential, the authors offer two non-exclusive readings. Myeloid cells may supply compensatory factors that substitute for IE2 regulatory functions, or the residual near-undetectable IE2 that microRNA targeting cannot eliminate may suffice for an early essential step, since silencing efficiency falls as target abundance falls. The data do not distinguish these. The assignment of individual differentially expressed viral genes to direct IE2 repression is supported only by proximity to putative cis-repression motifs identified by sequence search, which is suggestive rather than demonstrative.", "Conceptual or technical advance": "The method decouples the cell type in which a recombinant herpesvirus must be produced from the cell type in which a gene is to be studied, removing the fibroblast bottleneck that had prevented direct interrogation of essential genes in the myeloid compartment. Because the open reading frame is untouched and the trigger is an endogenous lineage-restricted microRNA, no exogenous stimulus, temperature shift, or complementing cell line is required, and the single galK plus loxP recombination step lowers the labour of construction. Conceptually, the work makes visible that essentiality determined in fibroblasts does not transfer to myeloid cells, and it supplies a transcriptome-level map of what IE2 loss does in each lineage. The authors also point to tunable control of gene expression by lineage as relevant to the use of cytomegalovirus as a vaccine vector.", "Relationship to the broader research program": "The paper applies a targeting principle the tenOever laboratory had developed and reviewed previously, namely that inserting perfectly complementary microRNA binding sites into a viral transcript silences it potently without altering coding capacity, and that the tissue restriction of the chosen microRNA sets where silencing occurs. Earlier work in the corpus applied that principle to attenuate RNA viruses in a species- or tissue-restricted manner. Here the same logic is turned from attenuation into a genetics tool and extended from RNA viruses to a large DNA virus manipulated through a bacterial artificial chromosome. The use of miR-142 to confine activity to the hematopoietic lineage and miR-122 as a non-hematopoietic control reflects the same design vocabulary. Marked as category 3 synthesis, the recurring thread across these papers is the use of the host microRNA machinery as a programmable cell-type switch for viral gene expression rather than as an object of study in its own right, a shift from asking what microRNAs do to viruses toward using them to ask what viral genes do.", "Related publications": "- tenOever 2013 (Nature Reviews Microbiology), review or synthesis. The author's own review of RNA viruses and the host microRNA machinery, cited here as the basis for the perfect-complementarity silencing approach.\n- Perez et al. 2009 and related engineered microRNA-targeting work from the same laboratory, methodological foundation. Establishes microRNA target insertion as a means of restricting virus replication by cell or species, the principle repurposed here as a conditional knockdown.\n- Noriega et al. 2014 (Viruses), methodological foundation. Source of the cytomegalovirus BAC electroporation and rescue procedure used here, from the co-senior author's group.\n- Lin et al. 2017 (PLoS Pathogens), predecessor. Reported the broad transcriptional footprint of IE2 on viral genes and the response of UL144 to IE2 loss, predictions the present work tested in macrophages.", "Limitations and boundaries": "Silencing by microRNA targeting is incomplete by design, and the authors state that residual low-level IE2 cannot be excluded as sufficient for an early essential function, which means the macrophage phenotype is a strong knockdown rather than a null. Spliced IE2 variants would also carry the inserted target sites, and while these products were not detected by immunoblot, the authors say they cannot rule out that part of the phenotype derives from their loss. The myeloid model is THP-1-derived macrophages treated with phorbol ester rather than primary monocytes or a physiological latency system, and the fibroblast model is a single primary line, MRC-5, with cells passaged fewer than twenty times. No animal work, primary hematopoietic progenitors, or latency and reactivation assays are included, so conclusions about latency itself are framed by the authors as prospective rather than demonstrated. Only one viral gene, IE2, and one microRNA, miR-142, were used, so the generality of the approach across genes and lineages is asserted rather than tested. The host and viral transcriptome comparisons rest on a single late time point of nine days at low multiplicity, with the differential expression sets validated only in part by quantitative PCR. Assignment of differentially expressed genes to direct IE2 action is based on proximity to consensus cis-repression motifs found by sequence search. Finally, the greater titres of the targeted virus in macrophages are measured by plaque assay on fibroblasts, so they report production of fibroblast-infectious particles rather than any myeloid-specific outcome.", "Audience summaries": "### 25 words\n\nPlacing blood-cell-specific microRNA targets in a cytomegalovirus gene lets the virus be grown normally in fibroblasts while the gene is switched off only in macrophages.\n\n### 75 words\n\nCytomegalovirus mutants must be grown in fibroblasts, which blocks study of genes essential there but interesting elsewhere. Inserting target sites for the blood-lineage microRNA miR-142 into the IE2 transcript silenced IE2 only in macrophages while leaving fibroblast growth intact. Loss of IE2 raised IE1 sharply and sustained virus titres in macrophages instead of abolishing them, and it reshaped both viral and host gene expression far more in macrophages than in fibroblasts.\n\n### 150 words\n\nHuman cytomegalovirus genetics is limited by the requirement to rescue recombinant virus in fibroblasts, which precludes deleting genes essential during acute fibroblast infection. Møller and colleagues inserted four perfect target sites for the hematopoietic-restricted microRNA miR-142 into the noncoding 3 prime untranslated region of IE2 in the TB40/E bacterial artificial chromosome using a single galK and loxP recombination step. The resulting virus replicated indistinguishably from control in fibroblasts, which lack miR-142, while in THP-1-derived macrophages IE2 protein was silenced, IE1 rose through relief of cis-repression, and titres were sustained two to three logs above a control that declined over ten days. RNA sequencing showed extensive viral transcriptome remodelling and more than 750 host gene changes in macrophages against roughly fifty in miR-142-expressing fibroblasts. The authors note that residual IE2 may still support an early essential role, so the system represents a strong lineage-restricted knockdown rather than a null." }, "discoveries": [ "claim-10" ], "relationships": [], "canonical_url": "https://tenoeverlab.us/publications/2018-m-ller-mirna-mediated-targeting-of-human-/", "controlled_vocabulary": { "pathogens": [ "hcmv" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "small-rna-northern-blot", "mirna-target-site-insertion", "pathway-enrichment-analysis", "growth-curve", "lentiviral-transduction", "bac-recombineering", "differential-expression-analysis" ] } }, { "id": "2019-eggenberger-type-i-interferon-response-impairs", "slug": "2019-eggenberger-type-i-interferon-response-impairs", "url": "/publications/2019-eggenberger-type-i-interferon-response-impairs/", "title": "Type I interferon response impairs differentiation potential of pluripotent stem cells", "authors": [ "Julie Eggenberger", "Daniel Blanco-Melo", "Maryline Panis", "Kristen J. Brennand", "Benjamin R. tenOever" ], "author_count": 5, "first_author": "Julie Eggenberger", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 5, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2019, "journal": "Proceedings of the National Academy of Sciences", "volume": "116", "issue": "4", "pages": "1384-1393", "doi": "10.1073/pnas.1812449116", "doi_url": "https://doi.org/10.1073/pnas.1812449116", "pmid": "30606801", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/30606801/", "pmcid": "PMC6347712", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347712/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347712/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling" ], "themes": [ "interferon-and-cell-identity" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "human", "mouse" ], "technologies": [ "cellular reprogramming with OCT4 SOX2 KLF4 and c-MYC", "lentiviral doxycycline-inducible expression", "constitutively active IRF7 truncation", "RNA sequencing", "multidimensional scaling", "sparse principal component analysis", "quantitative RT-PCR", "immunoblotting", "hPSC ScoreCard assay", "directed trilineage differentiation", "embryoid body cardiomyocyte differentiation", "influenza A virus lacking NS1" ], "biological_systems": [ "human induced pluripotent stem cells", "human primary foreskin fibroblasts", "rederived fibroblasts", "mouse embryonic stem cells", "mouse embryonic fibroblasts", "HEK293T cells", "embryoid bodies", "iPSC-derived cardiomyocytes" ], "key_concepts": [ "type I interferon response", "interferon-stimulated genes", "pluripotency", "cellular reprogramming", "IRF7 as transactivator of interferon-stimulated response elements", "KLF4-mediated repression of antiviral induction", "differentiation potential", "germ layer specification", "developmental and defence system incompatibility" ], "keywords": [ "pluripotent stem cells", "interferon", "IRF7", "KLF4", "induced pluripotent stem cells", "interferon-stimulated genes", "differentiation", "cardiomyocyte", "influenza A virus", "innate immunity" ], "one_sentence_contribution": "Forcing an interferon-stimulated gene program in human induced pluripotent stem cells with a constitutively active IRF7 produces lasting transcriptional change and impaired germ layer differentiation, supporting the proposal that the canonical type I interferon system and pluripotency are difficult to hold simultaneously.", "summary_25": "Stem cells ignore interferon signals. Forcing the antiviral gene program on anyway leaves thousands of genes altered days later and damages their ability to form tissues.", "summary_75": "Pluripotent stem cells neither make nor respond to type I interferon. Reprogramming factors, KLF4 most strongly, block the transcription factor that drives interferon-stimulated genes. Using a constitutively active IRF7 to switch the program on regardless changed stem cell morphology and gene expression, and left roughly 2,000 genes altered after five days of recovery. Differentiation afterwards was impaired for ectoderm and endoderm and distorted within mesoderm, with cardiomyocytes beating more but transcriptionally abnormal.", "summary_150": "Eggenberger and colleagues compared human foreskin fibroblasts, induced pluripotent stem cells derived from them, and fibroblasts rederived from those stem cells. Only the pluripotent state failed to respond to 5 prime triphosphate RNA, to interferon beta, or to influenza A virus lacking NS1, and mouse embryonic stem cells behaved the same way. Co-expression experiments showed that KLF4, and less potently SOX2 and OCT4, suppress transcription driven by a constitutively active IRF7, apparently by restricting access to interferon-stimulated response elements. A doxycycline-inducible IRF7 delta then allowed the program to be engaged in pluripotent cells directly. A forty-eight hour pulse induced interferon-stimulated genes and altered morphology, and after five days of rest roughly 2,000 genes remained differentially expressed although the direct targets had reset. Subsequent differentiation compromised ectoderm and endoderm and distorted mesoderm sublineage output, with cardiomyocytes showing increased beating alongside roughly 5,000 differentially expressed genes.", "citation": "Eggenberger J, Blanco-Melo D, Panis M, Brennand KJ, tenOever BR. Type I interferon response impairs differentiation potential of pluripotent stem cells. Proceedings of the National Academy of Sciences. 2019. Volume 116, issue 4, pages 1384-1393. DOI 10.1073/pnas.1812449116. PMID 30606801. PMCID PMC6347712.", "sections": { "Citation": "Eggenberger J, Blanco-Melo D, Panis M, Brennand KJ, tenOever BR. Type I interferon response impairs differentiation potential of pluripotent stem cells. Proceedings of the National Academy of Sciences. 2019. Volume 116, issue 4, pages 1384-1393.\n\nDOI 10.1073/pnas.1812449116. PMID 30606801. PMCID PMC6347712.", "One-sentence contribution": "Forcing an interferon-stimulated gene program in human induced pluripotent stem cells with a constitutively active IRF7 produces lasting transcriptional change and impaired germ layer differentiation, supporting the proposal that the canonical type I interferon system and pluripotency are difficult to hold simultaneously.", "Executive summary": "Differentiated vertebrate cells detect viral replication intermediates through pattern recognition receptors and respond by inducing type I interferon and hundreds of interferon-stimulated genes. Pluripotent stem cells do neither, failing to induce interferon in response to viral RNA or infection and responding only weakly to interferon beta itself. Why cells of such developmental importance forgo this defence was unresolved. The authors built a matched primary cell system in which human foreskin fibroblasts were reprogrammed to induced pluripotent stem cells and then redifferentiated to fibroblasts, so pluripotent and differentiated states of one genetic background could be compared directly. Only the pluripotent state failed to respond to 5 prime triphosphate RNA, to interferon beta, or to influenza A virus lacking its interferon antagonist NS1, and the same behaviour was seen in mouse embryonic stem cells. Co-expression of individual reprogramming factors with a constitutively active IRF7 in cells competent to respond showed that KLF4, and to a lesser degree SOX2 and OCT4, suppress induction of interferon-stimulated genes, with KLF4 the most potent. To ask what happens when the defence is nonetheless engaged, the authors placed that active IRF7 under doxycycline control in pluripotent stem cells. A brief pulse induced interferon-stimulated genes and altered morphology, and after five days of rest roughly 2,000 genes remained differentially expressed even though IRF7 and its direct targets had returned to baseline. Differentiation after such a pulse was compromised for ectoderm and endoderm and dysregulated within the mesoderm lineage, with cardiomyocyte cultures showing more beating alongside roughly 5,000 differentially expressed genes.", "Scientific context": "Type I interferon and the interferon-stimulated genes it induces constitute a broadly conserved vertebrate defence capable of fully protecting cells from many viruses. Pluripotent stem cells stand apart. Earlier work by others had established that mouse embryonic stem cells and human pluripotent cells respond poorly to viral and bacterial pathogen-associated molecular patterns, that interferon beta treatment produces little transcriptional output in these cells, and that low pattern recognition receptor levels can account for only part of the picture. Despite this, pluripotent cells are not unusually susceptible to infection, an observation other groups had attributed variously to stem-cell-specific factors, to RNA interference, and to constitutively high baseline expression of a subset of interferon-stimulated genes. What remained unaddressed was why the system is unused rather than simply how it is blocked. The present study reframes the question as one about compatibility, asking what the consequences of switching the defence on would be for the pluripotent state itself.", "Central question": "Why do pluripotent stem cells not use the canonical type I interferon system, and specifically is the maintenance of pluripotency compatible with engagement of an interferon-stimulated gene program?", "Experimental strategy": "Three design choices carry the argument. First, comparison is made within a single genetic background by reprogramming human primary foreskin fibroblasts to induced pluripotent stem cells and then redifferentiating them back to fibroblasts, which controls for clonal selection and loss of genetic material as explanations for unresponsiveness. Second, the block is probed from the direction of the reprogramming factors themselves, co-expressing OCT4, SOX2 and KLF4 individually with a constitutively active IRF7 in cells that normally respond, so that repression can be attributed to a defined factor rather than to the pluripotent state as a whole. The IRF7 construct, IRF7 delta, lacks residues 247 to 467 and therefore bypasses the requirement for phosphorylation by the IKK-related kinases, allowing the interferon-stimulated gene program to be driven without upstream signalling. Third, and centrally, the defence is forced on in pluripotent cells using a doxycycline-inducible lentiviral cassette expressing either IRF7 delta or GFP, which supplies a matched control for the induction procedure itself. Because IRF7 engages degenerate as well as perfect interferon-stimulated response elements, it can drive a wide subset of these genes independently of interferon signalling, which matters in cells where interferon signalling is itself inert. Consequences are then read at three depths. Immediate transcriptional output at forty-eight hours, persistence after a five day rest in stem-cell-maintaining conditions, and functional developmental potential measured by the hPSC ScoreCard panel, by undirected differentiation, by directed differentiation into all three germ layers analysed by sparse principal component analysis, and by a defined embryoid body protocol for cardiomyocytes as a mesoderm sublineage.", "Key findings": "1. Multidimensional scaling of RNA sequencing data placed fibroblasts and rederived fibroblasts close together and both far from the induced pluripotent state, while the pluripotent cells expressed NANOG, SOX2, TRA-1-60 and OCT4 and formed all three germ layers in directed differentiation (Figure 1A and SI Appendix Figure S1).\n\n2. Only the pluripotent cells failed to induce RIG-I and IFIT1 protein after treatment with 5 prime triphosphate RNA or interferon beta, and failed to induce IFNB transcript after 5 prime triphosphate RNA, while both fibroblast populations responded (Figures 1B and 1C). Redifferentiation restored responsiveness, which excludes clonal selection or genetic loss. Small RNA profiles of resting and treated cells were unchanged, which the authors take as evidence that neither microRNAs nor virus-induced small RNAs underlie the phenotype (SI Appendix Table S2).\n\n3. RNA sequencing after interferon beta showed strong enrichment for virus response and interferon signalling genes in fibroblasts and little in pluripotent cells, with IRF7 induced roughly 30-fold, MX1 roughly 300-fold and IFIT1 roughly 200-fold in fibroblasts against no induction, roughly 4-fold and roughly 5-fold respectively in pluripotent cells, corroborated by quantitative RT-PCR (Figure 2A and SI Appendix Figure S2A).\n\n4. Infection with influenza A virus lacking NS1 produced a broad antiviral and non-antiviral transcriptional response in fibroblasts and only a small subset of interferon-stimulated genes in pluripotent cells (Figure 2A). Virus levels in pluripotent cells were nonetheless relatively contained, which the authors attribute to high baseline interferon-stimulated gene expression as reported by others, an attribution taken from the literature rather than established here.\n\n5. Interferon beta pretreatment protected fibroblasts completely from influenza A virus lacking NS1, with STAT1 and IFIT1 induction, while pluripotent cells accumulated viral nucleoprotein and showed neither induction. Phosphorylation of IRF3 at S386 and STAT1 at Y701 was reduced in pluripotent cells, which the authors read as suggesting the presence of a dominant negative factor, a suggestion not tested directly (Figure 2B).\n\n6. Mouse embryonic stem cells reproduced the phenotype, failing to induce IFIT1 or IFIT2 or to control viral nucleoprotein after murine interferon beta, in contrast to mouse embryonic fibroblasts (Figure 2C). This extends the observation across species and beyond cells produced by reprogramming.\n\n7. In cells competent to respond, co-expression of OCT4, SOX2 or KLF4 with IRF7 delta repressed induction of endogenous IFIT1 and ISG15, with KLF4 the most potent (Figures 3A and 3B). RNA sequencing showed that KLF4 alone has its own transcriptional footprint that does not include the canonical interferon-stimulated genes, and that when co-expressed it retains its own signature while neutralising the IRF7 delta output (Figure 3C). The authors interpret this as KLF4 preventing access to interferon-stimulated response elements through chromatin compaction, citing chromatin accessibility work by others, and note in the discussion that since the binding motifs do not overlap the effect is presumably indirect.\n\n8. Doxycycline-inducible IRF7 delta in pluripotent stem cells induced IFNA and IFIT1 within twelve to twenty-four hours together with distinct morphological change, and RNA sequencing at forty-eight hours showed induction of interferon-stimulated genes including IFIT1, IFITM3 and TRIM22 (Figures 4A to 4C). This is the engagement the authors state they could not achieve by any other means.\n\n9. After a forty-eight hour pulse followed by five days of rest in stem-cell-maintaining conditions, roughly 2,000 genes remained differentially expressed relative to the GFP control, while IRF7 delta itself and the canonical interferon-stimulated genes had returned to baseline (Figure 4D and SI Appendix Figures S4D and S4E). A comparable pattern of transient interferon-stimulated gene induction followed by loss of pluripotency markers including KLF4 and changes in chromatin remodelling enzymes was seen in mouse embryonic stem cells using transient transfection of a different IRF-inducing transcript (SI Appendix Table S7).\n\n10. The hPSC ScoreCard panel after pulse and rest showed that pluripotency was not formally lost but that NANOG and IDO1 were dysregulated, along with markers of mesendoderm, ectoderm, mesoderm and endoderm (Figure 5A). Undirected differentiation produced a general tendency toward mesoderm in both populations, with ectoderm and endoderm significantly dysregulated after the IRF7 delta pulse (Figure 5B).\n\n11. Directed trilineage differentiation analysed by sparse principal component analysis showed overlapping positions for pulsed and control cells at rest, tight clustering for mesoderm, partial overlap for ectoderm, and a large departure for endoderm after the IRF7 delta pulse (Figure 5C).\n\n12. Within the mesoderm lineage, pulsed cells showed elevated baseline expression of several cardiomyocyte markers before any directed differentiation, and after embryoid body differentiation with activin A and BMP4 they showed higher TNNT2, SIRPA, MYH6 and MYL7 while MYL2 was reduced relative to control despite high baseline induction (Figure 6A and SI Appendix Figure S6B). Beating was significantly increased at day 20 (Figure 6B), yet RNA sequencing of the resulting cardiomyocytes showed roughly 5,000 differentially expressed genes (Figure 6C). The authors read this as successful but aberrant differentiation.", "Mechanistic model": "The study does not establish a single definitive mechanism, and the authors are explicit that the basis for the incompatibility is likely multifactorial. Two partially separable layers are supported. On the repression side, reprogramming factors and above all KLF4 prevent transcriptional output at interferon-stimulated response elements, and because the KLF4 binding motif does not overlap the response element the authors infer an indirect route through chromatin remodelling, resting their case on published chromatin accessibility data rather than on measurements made here. They also point out that this account does not explain the reduced phosphorylation of IRF3, NF-kappaB and STAT1 that they and others observe, and note that RIG-I protein is low in pluripotent cells despite comparable DDX58 read counts, which they suggest may reflect stem-cell-specific proteases or microRNAs, a suggestion offered as speculation. On the consequence side, the data show that when the program is driven directly by IRF7 delta, bypassing all of this, the pluripotent cell is durably altered. Direct IRF7 targets revert when the stimulus is withdrawn while thousands of other transcripts do not, and subsequent differentiation is skewed and internally inconsistent. The authors frame this as IRF7 priming cells to exit pluripotency, with a bias toward mesoderm, and state that whether the mesoderm bias is specific or reflects the baseline tendency of the particular cell line remains to be determined. The broader proposal, that pluripotency and the interferon system are incompatible, is offered as a hypothesis the data support rather than as a demonstrated mechanism.", "Conceptual or technical advance": "The work supplies a way to switch on an interferon-stimulated gene program inside cells that cannot be induced by interferon, viral RNA, or infection, which converts a descriptive observation about unresponsiveness into a testable manipulation. It identifies KLF4 as a specific and potent repressor of IRF7-driven transcription and shows that a reprogramming factor alone can confer the pluripotent cell's unresponsiveness on a differentiated cell. It also shifts the question from why the defence is absent to what the defence would cost, and shows that the cost is measurable as lasting transcriptional change and compromised germ layer potential after a stimulus as brief as forty-eight hours. That result makes the developmental consequences of interferon exposure in early embryonic cells an experimentally approachable problem and raises a caution for protocols in which pluripotent cultures encounter interferon or infection.", "Relationship to the broader research program": "The paper draws directly on earlier work from the laboratory on the transcriptional architecture of the antiviral response, including redundancy among the transcription factors that induce the antiviral state and mitogen-activated protein kinase-mediated licensing of IRF3 and IRF7, which supply the rationale for using a constitutively active IRF7 as a sufficient driver of interferon-stimulated genes. It also connects to the laboratory's interest in the evolution of antiviral defence systems and in how defence and developmental programs borrow from one another, a theme the discussion develops with examples including Toll-like receptors, RNA interference and microRNA, and RNase III nucleases. Marked as category 3 synthesis, the corpus-level thread here is a recurring use of influenza A virus lacking NS1 as a probe that reports on host sensing capacity, appearing in this stem cell context as a tool rather than a subject. A second synthesis thread is the treatment of the interferon response as a program with costs to the cell beyond its antiviral benefit, an idea that recurs where the laboratory examines how the same response shapes differentiation, tissue state, or disease outcome rather than only virus replication.", "Related publications": "- Schmid et al. 2010 (Journal of Biological Chemistry), methodological foundation. Established transcription factor redundancy in induction of the antiviral state, from the same laboratory, and underpins the use of IRF7 as a sufficient driver.\n- Schmid, Sachs and tenOever 2014 (Journal of Biological Chemistry), methodological foundation. Licensing of IRF3 and IRF7 by mitogen-activated protein kinase signalling, cited here for the properties of IRF7 that make IRF7 delta effective.\n- tenOever 2016 (Cell Host and Microbe), review or synthesis. The author's review of the evolution of antiviral defence systems, cited in the opening framing of defence diversity.\n- Aguado et al. 2017 (Nature), conceptual extension. Work from the same laboratory on RNase III nucleases as antiviral effectors across kingdoms, cited in the discussion of repurposing between developmental and defence systems.\n- Blanco-Melo, Venkatesh and Bieniasz 2016 (Cell Host and Microbe), conceptual extension. Origins and evolution of tetherin, cited by a co-author of the present paper as an example of gene duplication serving both systems.\n- Brennand et al. 2011 (Nature), methodological foundation. Source of the induced pluripotent stem cell modelling approach contributed by the co-author's laboratory.", "Limitations and boundaries": "The central manipulation is artificial. IRF7 delta drives a subset of interferon-stimulated genes directly and bypasses upstream signalling entirely, so what is tested is the consequence of an interferon-like transcriptional program rather than of a physiological infection or of interferon exposure, which pluripotent cells cannot mount in any case. The authors describe the induced response as partial. Dosage and duration are fixed at a single forty-eight hour pulse followed by five days of rest, so the shape of the dose and time relationship is unknown. The main human work rests on a small number of induced pluripotent stem cell clones derived from foreskin fibroblasts and from healthy patient fibroblasts by two different reprogramming routes, and the authors state explicitly that whether the mesoderm bias is a property of the response or of the baseline tendency of their particular line is unresolved. RNA sequencing conditions used two biological replicates, and differentiation readouts are transcriptional and morphological rather than functional in most cases, the beating cardiomyocytes being the exception. The repression mechanism attributed to KLF4 is inferred from published chromatin accessibility data rather than measured here, and the authors note it does not account for the reduced phosphorylation of IRF3, NF-kappaB and STAT1 that they observe. All differentiation is in vitro, with no embryo or animal work, so no claim is made about development in a living organism. Finally, the framing that pluripotency and the interferon system are incompatible is presented by the authors as a hypothesis their data support and as speculation where it extends to evolutionary crosstalk.", "Audience summaries": "### 25 words\n\nStem cells ignore interferon signals. Forcing the antiviral gene program on anyway leaves thousands of genes altered days later and damages their ability to form tissues.\n\n### 75 words\n\nPluripotent stem cells neither make nor respond to type I interferon. Reprogramming factors, KLF4 most strongly, block the transcription factor that drives interferon-stimulated genes. Using a constitutively active IRF7 to switch the program on regardless changed stem cell morphology and gene expression, and left roughly 2,000 genes altered after five days of recovery. Differentiation afterwards was impaired for ectoderm and endoderm and distorted within mesoderm, with cardiomyocytes beating more but transcriptionally abnormal.\n\n### 150 words\n\nEggenberger and colleagues compared human foreskin fibroblasts, induced pluripotent stem cells derived from them, and fibroblasts rederived from those stem cells. Only the pluripotent state failed to respond to 5 prime triphosphate RNA, to interferon beta, or to influenza A virus lacking NS1, and mouse embryonic stem cells behaved the same way. Co-expression experiments showed that KLF4, and less potently SOX2 and OCT4, suppress transcription driven by a constitutively active IRF7, apparently by restricting access to interferon-stimulated response elements. A doxycycline-inducible IRF7 delta then allowed the program to be engaged in pluripotent cells directly. A forty-eight hour pulse induced interferon-stimulated genes and altered morphology, and after five days of rest roughly 2,000 genes remained differentially expressed although the direct targets had reset. Subsequent differentiation compromised ectoderm and endoderm and distorted mesoderm sublineage output, with cardiomyocytes showing increased beating alongside roughly 5,000 differentially expressed genes." }, "discoveries": [ "claim-06" ], "relationships": [ { "from": "2019-eggenberger-type-i-interferon-response-impairs", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2019-eggenberger-type-i-interferon-response-impairs" } ], "canonical_url": "https://tenoeverlab.us/publications/2019-eggenberger-type-i-interferon-response-impairs/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "immunoblotting", "dimensionality-reduction", "hpsc-directed-differentiation", "inducible-expression", "engineered-tf-constructs", "cellular-reprogramming", "delns1-virus" ] } }, { "id": "2019-munoz-moreno-viral-fitness-landscapes-in-divers", "slug": "2019-munoz-moreno-viral-fitness-landscapes-in-divers", "url": "/publications/2019-munoz-moreno-viral-fitness-landscapes-in-divers/", "title": "Viral Fitness Landscapes in Diverse Host Species Reveal Multiple Evolutionary Lines for the NS1 Gene of Influenza A Viruses", "authors": [ "Raquel Muñoz-Moreno", "Carles Martínez-Romero", "Daniel Blanco-Melo", "Christian V. Forst", "Raffael Nachbagauer", "Asiel Arturo Benitez", "Ignacio Mena", "Sadaf Aslam", "Vinod Balasubramaniam", "Ilseob Lee", "Maryline Panis", "Juan Ayllón", "David Sachs", "Man-Seong Park", "Florian Krammer", "Benjamin R. tenOever", "Adolfo García-Sastre" ], "author_count": 17, "first_author": "Raquel Muñoz-Moreno", "senior_authors": [ "Adolfo García-Sastre" ], "corresponding_authors": [ "Adolfo García-Sastre" ], "tenoever_position": 16, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2019, "journal": "Cell Reports", "volume": "29", "issue": "12", "pages": "3997-4009.e5", "doi": "10.1016/j.celrep.2019.11.070", "doi_url": "https://doi.org/10.1016/j.celrep.2019.11.070", "pmid": "31851929", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/31851929/", "pmcid": "PMC7010214", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010214/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010214/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "influenza-genome-regulation", "viral-populations-evolution" ], "themes": [ "ns1-and-interferon-antagonism", "fitness-landscapes" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "mouse", "chicken", "dog", "human" ], "technologies": [ "influenza reverse genetics", "barcoded virus library", "split NS segment design", "Illumina deep sequencing", "phylogenetic analysis", "multidimensional scaling", "network and medoid clustering analysis", "plaque assay" ], "biological_systems": [ "MDCK cells", "A549 cells", "HEK293T cells", "embryonated chicken eggs", "C57BL/6 mouse lung", "Stat1 deficient mice", "Rag1 deficient mice" ], "key_concepts": [ "viral fitness landscape", "NS1 protein", "host tropism", "allele A and allele B NS segments", "interferon antagonism", "within-population competition", "convergent and divergent evolution", "barcoded library competition assay", "STAT1 dependent selection" ], "keywords": [ "influenza A virus", "NS1", "host range", "viral fitness", "barcoded library", "deep sequencing", "allele B", "type I interferon", "STAT1", "pandemic risk assessment" ], "one_sentence_contribution": "A library of 107 barcoded influenza A viruses differing only in their NS1 sequence, competed in dog cells, human cells, chicken eggs and mice, resolves NS1-driven fitness as a set of divergent and partly convergent evolutionary trajectories rather than a single ordered adaptation gradient.", "summary_25": "Influenza NS1 genes were swapped into one identical virus backbone and competed across hosts. Closely related NS1 sequences often behaved very differently, and avian allele B NS1 performed well everywhere.", "summary_75": "The influenza NS1 protein blocks host antiviral responses, and different influenza strains carry very different NS1 sequences. Fifty-six natural NS1 sequences were placed into an otherwise identical virus, each tagged with a genetic barcode, and the whole pool was grown in dog cells, human cells, chicken eggs and mice. Sequencing revealed which versions thrived in which host. Relatedness on the family tree did not reliably predict shared behaviour, and avian allele B versions replicated well in every host tested.", "summary_150": "A pooled library of 107 barcoded influenza A viruses, identical apart from a natural NS1 sequence carried on a split NS segment that leaves NEP unchanged, was used to compete 56 NS1 variants against one another in MDCK cells, A549 cells, embryonated chicken eggs and mice. Relative barcode abundance after replication defined an NS1 fitness landscape for each host. Allele B NS1, largely of avian origin, was overrepresented in every substrate. Human H3N2 NS1 was underrepresented everywhere except in human A549 cells. Network analysis found clusters sharing fitness profiles despite substantial amino acid divergence, indicating that phylogenetic position predicts phenotype poorly for this gene. Loss of STAT1 collapsed much of the spread, while loss of RAG1 did not, placing the selective pressure in early innate immune signalling, though human H3N2 NS1 remained restricted in murine lung even without STAT1. No molecular mechanism for the individual fitness differences is established.", "citation": "Muñoz-Moreno R, Martínez-Romero C, Blanco-Melo D, Forst CV, Nachbagauer R, Benitez AA, Mena I, Aslam S, Balasubramaniam V, Lee I, Panis M, Ayllón J, Sachs D, Park MS, Krammer F, tenOever BR, García-Sastre A. Viral Fitness Landscapes in Diverse Host Species Reveal Multiple Evolutionary Lines for the NS1 Gene of Influenza A Viruses. Cell Reports. 2019. Volume 29, issue 12, pages 3997-4009.e5. DOI 10.1016/j.celrep.2019.11.070. PMID 31851929. PMCID PMC7010214.", "sections": { "Citation": "Muñoz-Moreno R, Martínez-Romero C, Blanco-Melo D, Forst CV, Nachbagauer R, Benitez AA, Mena I, Aslam S, Balasubramaniam V, Lee I, Panis M, Ayllón J, Sachs D, Park MS, Krammer F, tenOever BR, García-Sastre A. Viral Fitness Landscapes in Diverse Host Species Reveal Multiple Evolutionary Lines for the NS1 Gene of Influenza A Viruses. Cell Reports. 2019. Volume 29, issue 12, pages 3997-4009.e5.\n\nDOI 10.1016/j.celrep.2019.11.070. PMID 31851929. PMCID PMC7010214.", "One-sentence contribution": "A library of 107 barcoded influenza A viruses differing only in their NS1 sequence, competed in dog cells, human cells, chicken eggs and mice, resolves NS1-driven fitness as a set of divergent and partly convergent evolutionary trajectories rather than a single ordered adaptation gradient.", "Executive summary": "The influenza A virus NS1 protein antagonises the type I interferon response through many distinct mechanisms, several of which are strain and host specific. Because NS1 sequences are phylogenetically diverse and because host interaction partners differ between species, NS1 is a plausible determinant of which hosts a given influenza strain can replicate in, but previous work assessed NS1 function mostly one virus at a time. This study places 56 natural NS1 sequences, spanning both allele A and allele B and spanning human, avian, swine, equine, canine, camel and marine mammal isolates from 1954 to 2013, into a common A/Puerto Rico/8/1934 backbone using a split NS segment that separates the NS1 and NEP reading frames and carries a neutral 22-nucleotide barcode. Most sequences were represented by two independently barcoded viruses. The pooled library was used to infect MDCK cells, A549 cells, embryonated chicken eggs and mice, and relative barcode abundance after replication was compared with the input. Fitness varied widely across the library and varied by host. Viruses carrying allele B NS1 were overrepresented in every substrate tested, human H3N2 NS1 viruses were underrepresented everywhere except in human A549 cells, and network analysis identified groups of viruses with similar fitness but substantially different NS1 sequence. Infection of Stat1 deficient mice removed the advantage of several groups, while Rag1 deficiency did not change the profile.", "Scientific context": "NS1 is encoded on segment 8 alongside NEP and is a major contributor to influenza A virus pathogenicity. The paper summarises the known repertoire of NS1 activities, including sequestration of double-stranded RNA away from OAS, RIG-I like receptors, PKR and MDA5, direct inhibition of RIG-I and PKR, and interference with host messenger RNA maturation and nuclear export through CPSF30. Several of these activities are strain specific, and not every influenza strain retains all of them, which the authors attribute to functional redundancy. Host interaction partners of NS1 are not identically conserved across species, which opens the possibility that a given NS1 supports replication better in some host species than in others. NS segments fall into two gene pools, allele A and allele B, with sequence identity above ninety percent within a pool and around sixty percent between pools. Allele B is largely avian in origin, and work by Turnbull and colleagues in 2016 had reported that allele B segment 8 does not impose mammalian host restriction, although the individual contributions of NS1 and NEP were not separated in that study. The unresolved question the paper sets up is how NS1 evolution has distributed function across this sequence space and what that implies for host preference.", "Central question": "How has the influenza A virus NS1 gene evolved across its phylogenetic lineages with respect to host tropism, and does phylogenetic relatedness among NS1 sequences predict comparable replicative fitness in a given host.", "Experimental strategy": "The design converts a question about sequence space into a competition experiment readable by sequencing. Holding the entire viral genome constant except NS1 isolates the contribution of that one protein, and pooling all the viruses into a single inoculum means every variant experiences the same host environment and the same inoculating conditions, which removes the between-experiment variability that limits one-virus-at-a-time comparisons. A modified NS segment separates the NS1 and NEP reading frames so that NS1 can be replaced without altering NEP, which is what allows the fitness readout to be attributed to NS1 rather than to segment 8 as a whole. A neutral 22-nucleotide barcode in the intervening non-coding region gives each virus an identity readable by amplicon sequencing, and representing most NS1 sequences with two distinct barcodes provides an internal control for barcode-specific amplification bias and for rescue-associated variability.\n\nSequence selection was structured rather than opportunistic, drawing on sequence feature variant type annotation as well as on phylogenetic distance, host, country and year, so that the library samples the observed NS1 sequence space rather than a convenience set. Four host substrates spanning three species plus two cell lines of different species and tissue origin allow host dependence to be separated from intrinsic replicative capacity. Validation layers were built in around the core competition. A four-virus pilot including an RNA-binding-deficient NS1 mutant established that the barcode readout tracks expected fitness. Single-virus infections of three library members tested whether library behaviour is reproduced outside competition. Repeating three of those viruses in an A/Vietnam/1203/04 HALo backbone, phylogenetically distant from H1N1, tested whether the observed differences depend on the rest of the viral genome. Finally, running the library in Stat1 deficient and Rag1 deficient mice partitions the selective pressure between interferon signalling and adaptive immunity.", "Key findings": "1. The barcoded library reports fitness as intended. In a four-virus pilot, barcode reads for the RNA-binding-deficient PR8-R38A/K41A NS1 virus fell sharply relative to input and A/Udorn/1972 NS1 fell to a lesser extent, while wild-type PR8 NS1 rose and A/Shanghai/02/2013 NS1 followed (Figure S1C). Profiles were similar at 50 and 100 plaque-forming units per virus, and 100 was used thereafter.\n\n2. Viruses carrying phylogenetically related NS1 behaved similarly across MDCK cells, eggs and mouse lung, and duplicate-barcoded versions of the same NS1 gave concordant abundances (Figure 2A). Two independently prepared libraries gave highly correlated profiles in mouse lung and MDCK cells at 48 hours (Data S1).\n\n3. Fitness is host dependent in a manner specific to NS1 clade. Avian H5N1 NS1 viruses replicated poorly in mouse lung relative to MDCK supernatant and allantoic fluid, human H3N2 NS1 viruses were underrepresented in all three, and NS1 from non-human mammalian H3N8 isolates was overrepresented in MDCK supernatant and allantoic fluid (Figure 2A).\n\n4. Allele B NS1 viruses were overrepresented in every substrate tested, including mouse lung, MDCK cells, allantoic fluid and human A549 cells (Figures 2A, 4B, 4D, 4F, and S2). The authors read this as evidence that allele B NS1, despite avian origin, supports efficient replication across a range of hosts, and note that their split NS design attributes this to NS1 specifically rather than to segment 8 as a whole.\n\n5. Human A549 cells were the only substrate in which human H3N2 NS1 viruses were not underrepresented (Figure S2), which the authors interpret as specific adaptation of human H3N2 NS1 to human cells. Human H1N1 NS1 did not show the same pattern. The authors state in the discussion that their design cannot separate species origin of a cell line from its tissue origin or passage history.\n\n6. Sequence distance and fitness distance are not proportional. Network analysis in mouse lung, MDCK cells and eggs identified clusters of viruses with similar fitness profiles but significantly different NS1 amino acid sequence (Figure 2B to 2D). The authors read this as possible convergent evolution of some NS1 subsets toward host-specific factors.\n\n7. Library ranking is reproduced outside competition, but only in one substrate. Single-virus infection with A/Moscow/2/2007, A/Tasmania/277/2007 or A/Udorn/1972 NS1 recombinants showed no statistically significant replication differences in eggs or MDCK cells, while mouse lung replication matched the library ranking and correlated with differences in morbidity and mortality (Figure 3B to 3E). The authors propose that eggs and MDCK cells offer effectively unlimited replicative resource, so differences emerge only under competition.\n\n8. The observed differences are driven by host rather than by the rest of the viral genome. The same three NS1 sequences in an A/Vietnam/1203/04 HALo H5N1 backbone reproduced the direction of the differences seen in the PR8 library, with differences in eggs and MDCK cells more apparent than in the PR8 context (Figure S3).\n\n9. Selection acts largely through innate immune signalling. Infection of Stat1 deficient mice flattened differences within the library relative to wild-type mice, and the enrichment of allele B NS1 viruses was lost in the absence of STAT1 although those viruses still outperformed the rest of the library (Figures 5 and S5A). H7N9, H9N2 and H10N4 NS1 viruses also gained fitness in Stat1 deficient mice. Human H3N2 NS1 viruses remained underrepresented even without STAT1, which the authors interpret as an NS1-regulated restriction that is independent of interferon. Rag1 deficiency, which removes adaptive immunity, did not alter the library profile (Figures S5B and S6).\n\n10. Fitness trajectories over time fall into distinct patterns. Medoid-based clustering of barcode profiles from days 2 to 5 in wild-type mice grouped phylogenetically related viruses together, with human H3N2 NS1 viruses declining over time and a heterogeneous cluster containing avian H7N9, H9N2, H10N7 and H10N4 NS1 grouping with pandemic H1N1 NS1 viruses and increasing over time (Figure 6A, Data S2). Among the fittest viruses two distinct dynamics were seen, with allele B NS1 viruses high from early on and A/Shanghai/02/2013 and A/Chicken/Rizhao/651/2013 NS1 viruses rising sharply after day 3 to reach comparable levels by day 5 (Figure 6B). The authors propose that this second group confers enhanced fitness under the high inflammatory conditions that develop in mice after day 3, which is offered as a proposal rather than as a demonstrated mechanism.", "Mechanistic model": "The study does not establish a molecular mechanism for any of the fitness differences it measures. It is a phenotypic mapping exercise, and the authors are explicit that the appropriate next step is protein interaction work.\n\nWhat the data do constrain is the level at which selection operates. Because only NS1 varies across the library and because the phenotype is preserved when three representative NS1 sequences are moved into a distantly related H5N1 backbone, the differences are attributable to NS1 rather than to segment 8 as a whole or to interaction with a particular genome constellation. Because the STAT1 deficiency collapses much of the spread but does not eliminate the underrepresentation of human H3N2 NS1, the selective pressure is partly but not entirely interferon signalling, and the authors conclude that an NS1-regulated host factor independent of interferon restricts those viruses in murine lung. Because Rag1 deficiency has no effect at these timescales, adaptive immunity is not the relevant filter.\n\nThe interpretive framework the authors propose is that NS1 tropism differences reflect adaptation to particular host factors among the many known NS1 interactors, with NS1 proteins that engage highly conserved partners such as CPSF30 being permissive across hosts and NS1 variants that instead target more sequence-divergent partners becoming host specialised. They offer TRIM25 as an example of such a divergent target. This is presented as a proposal consistent with the fitness data, and the authors caution in the same passage that focusing on a single domain or a set of amino acid changes may not be the right way to explain the phenotypes they observed.", "Conceptual or technical advance": "Placing natural sequence variation of a single viral gene into an isogenic backbone and competing the whole set in one inoculum converts a comparative virology question into a quantitative one, and the split NS segment makes that possible for segment 8 by decoupling NS1 from NEP. The result is a fitness landscape for NS1 across several hosts rather than a ranking of a few strains, and it shows that phylogenetic proximity is a poor predictor of shared phenotype in this gene. Practically, the authors present the approach as a way to flag influenza strains whose NS1 supports broad host tropism, which bears on pandemic risk assessment, and note that the same barcoded-library strategy generalises to other viral genes and other viruses with high sequence diversity.", "Relationship to the broader research program": "The barcoded influenza library used here descends directly from the method developed in Varble and colleagues in 2014, a tenOever laboratory paper on transmission bottlenecks, and the barcode design, the split NS segment and the sequencing analysis parameters are all cited to that work. The tenOever contribution to the present study sits in that methodological lineage and in supervision. The authors of the contributions statement list tenOever under supervision and under writing review and editing, and the corresponding author and lead contact is García-Sastre, so the work is led by another laboratory with the tenOever laboratory supplying one component.\n\nCategory 3 synthesis. The corpus contains other work using barcoded or otherwise individually trackable influenza populations to read selection within a host, including Varble and colleagues in 2014 on transmission bottlenecks. Reading this paper together with those would allow a statement about how within-host population composition is shaped by route, host and gene identity, but that comparison requires those records and is not established here.", "Related publications": "- Varble and colleagues, 2014, methodological foundation. The barcoded NS segment design, the barcode generation strategy and the sequencing analysis pipeline used here are taken from that tenOever laboratory study of influenza transmission bottlenecks.\n- Benitez and colleagues, 2015, predecessor. Cited here for in vivo RNAi screening identifying MDA5 as a contributor to cellular defence against influenza A virus, work from the tenOever laboratory that bears on the innate sensing pathways NS1 antagonises.\n- Turnbull and colleagues, 2016, predecessor. Cited as the report that allele B segment 8 does not restrict mammalian host range, a claim this study revisits at the level of NS1 alone.\n- Noronha and colleagues, 2012, methodological foundation. The sequence feature variant type framework used to structure NS1 sequence selection for the library.", "Limitations and boundaries": "All viruses share a single genetic backbone, A/Puerto Rico/8/1934, with a partial check in an A/Vietnam/1203/04 HALo backbone for three NS1 sequences only, so the generality of the fitness ranking across the full diversity of influenza genome constellations is untested. Fitness is measured as relative barcode abundance under competition, which is not the same quantity as replicative capacity in isolation, and indeed single-virus infections reproduced the library ranking only in mouse lung and not in eggs or MDCK cells. The authors state explicitly that they cannot distinguish fitness effects arising from the species origin of a substrate from those arising from tissue origin or passage history, using the MDCK and A549 comparison as the example. Host coverage is limited to mouse, chicken embryo, and two cell lines, with no ferret, swine or primary human airway model, and human data come only from an adenocarcinoma cell line. The main competition readout is taken at 48 hours, with time course data in wild-type mice extending only to day 5. Five of the 56 NS1 sequences are represented by a single barcode rather than two, and 107 of 112 intended viruses were rescued. Duplicate-barcode measurements identified as significantly different at a threshold of 0.13 were treated as outliers and excluded from cluster analysis. The study measures fitness phenotype only and does not test any of the candidate molecular explanations the discussion offers, including the CPSF30 and TRIM25 proposals. Weight loss and mortality data are reported for a small selection of viruses rather than across the library.", "Audience summaries": "### 25 words\n\nInfluenza NS1 genes were swapped into one identical virus backbone and competed across hosts. Closely related NS1 sequences often behaved very differently, and avian allele B NS1 performed well everywhere.\n\n### 75 words\n\nThe influenza NS1 protein blocks host antiviral responses, and different influenza strains carry very different NS1 sequences. Fifty-six natural NS1 sequences were placed into an otherwise identical virus, each tagged with a genetic barcode, and the whole pool was grown in dog cells, human cells, chicken eggs and mice. Sequencing revealed which versions thrived in which host. Relatedness on the family tree did not reliably predict shared behaviour, and avian allele B versions replicated well in every host tested.\n\n### 150 words\n\nA pooled library of 107 barcoded influenza A viruses, identical apart from a natural NS1 sequence carried on a split NS segment that leaves NEP unchanged, was used to compete 56 NS1 variants against one another in MDCK cells, A549 cells, embryonated chicken eggs and mice. Relative barcode abundance after replication defined an NS1 fitness landscape for each host. Allele B NS1, largely of avian origin, was overrepresented in every substrate. Human H3N2 NS1 was underrepresented everywhere except in human A549 cells. Network analysis found clusters sharing fitness profiles despite substantial amino acid divergence, indicating that phylogenetic position predicts phenotype poorly for this gene. Loss of STAT1 collapsed much of the spread, while loss of RAG1 did not, placing the selective pressure in early innate immune signalling, though human H3N2 NS1 remained restricted in murine lung even without STAT1. No molecular mechanism for the individual fitness differences is established." }, "discoveries": [ "claim-11" ], "relationships": [], "canonical_url": "https://tenoeverlab.us/publications/2019-munoz-moreno-viral-fitness-landscapes-in-divers/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "plaque-assay", "reverse-genetics", "barcoded-virus-library", "phylogenetics", "dimensionality-reduction", "short-read-sequencing-platform", "split-ns-segment" ] } }, { "id": "2019-tenoever-synthetic-virology-building-viruse", "slug": "2019-tenoever-synthetic-virology-building-viruse", "url": "/publications/2019-tenoever-synthetic-virology-building-viruse/", "title": "Synthetic Virology: Building Viruses to Better Understand Them", "authors": [ "Benjamin R. tenOever" ], "author_count": 1, "first_author": "Benjamin R. tenOever", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 1, "tenoever_role": "sole", "contribution_character": "lab-led", "year": 2019, "journal": "Cold Spring Harbor Perspectives in Medicine", "volume": "10", "issue": "11", "pages": "a038703", "doi": "10.1101/cshperspect.a038703", "doi_url": "https://doi.org/10.1101/cshperspect.a038703", "pmid": "31871242", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/31871242/", "pmcid": "PMC7605229", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605229/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605229/pdf/", "publication_type": "review", "declared_conflicts": null, "research_areas": [ "programmable-virology" ], "themes": [ "synthetic-virology-as-method" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "human", "mouse", "ferret", "chicken" ], "technologies": [ "reverse genetics", "minireplicon systems", "transposon insertional mutagenesis", "deep mutational scanning", "2A peptide polycistronic design", "fluorescent and luciferase reporter viruses", "Cre-LoxP lineage tracing", "RNA affinity tagging", "artificial microRNA expression", "RNA barcoding", "deep sequencing", "in vivo RNAi screening", "small molecule-assisted shutoff" ], "biological_systems": [ "mammalian cell culture", "embryonated chicken eggs", "mouse", "ferret", "LoxP reporter mice", "club cells", "alveolar epithelium" ], "key_concepts": [ "synthetic virology", "viral genetic circuitry", "segment packaging signals", "mutational tolerance", "reporter virus design", "microRNA-mediated species restriction", "biocontainment kill switch", "transmission bottleneck", "lineage tracing of infected cells", "virus-delivered RNA interference", "learning by building" ], "keywords": [ "influenza A virus", "reverse genetics", "virus engineering", "reporter viruses", "microRNA targeting", "Cre recombinase", "barcoded viruses", "transmission bottleneck", "RNAi screening", "segment 8" ], "one_sentence_contribution": "Frames influenza A virus as a compact genetic circuit whose modules can be disrupted, tagged or replaced, and organizes two decades of virus engineering into a design vocabulary of tracking, override, positioning and silencing modules that turns virus construction into a method of inquiry.", "summary_25": "Treating influenza A virus as a compact genetic circuit, this perspective organizes two decades of virus engineering into functional modules for tracking, override, barcoding and host silencing.", "summary_75": "Influenza A virus carries only about ten gene products, which makes it tractable as an engineered system. This perspective organizes the field by what an added element does within the viral circuit, covering fluorescent reporters, Cre recombinase for marking cells that survived infection, microRNA target sites as host-specific kill switches, RNA barcodes that revealed that aerosol transmission passes only two or three virions, and virus-delivered artificial microRNAs used to screen host factors during infection.", "summary_150": "Reverse genetics made it possible to build influenza A virus from DNA, and this perspective argues that building the virus is itself a way of understanding it. Systematic work on packaging signals and on mutational tolerance, largely from other laboratories, established that the genome has little room for random insertion, that packaging sequences extend into coding regions, and that hemagglutinin and NS1 are unusually permissive. Against that background the article sorts engineered viruses by circuit function. Reporters track location but usually attenuate, except when placed in PB2 or in an engineered intergenic space in segment 8. Cre recombinase combined with LoxP reporter mice marks cells that survived infection and identified club cells as clearing the virus. MicroRNA target sites act as species-restricted or lineage-restricted kill switches without altering coding material or fitness. RNA barcodes resolved transmission bottlenecks, and virus-delivered artificial microRNAs support in vivo host factor screening.", "citation": "tenOever BR. Synthetic Virology. Building Viruses to Better Understand Them. Cold Spring Harbor Perspectives in Medicine, 2019, volume 10, issue 11, article a038703. DOI 10.1101/cshperspect.a038703. PMID 31871242. PMCID PMC7605229.", "sections": { "Citation": "tenOever BR. Synthetic Virology. Building Viruses to Better Understand Them. Cold Spring Harbor Perspectives in Medicine, 2019, volume 10, issue 11, article a038703.\n\nDOI 10.1101/cshperspect.a038703. PMID 31871242. PMCID PMC7605229.", "One-sentence contribution": "Frames influenza A virus as a compact genetic circuit whose modules can be disrupted, tagged or replaced, and organizes two decades of virus engineering into a design vocabulary of tracking, override, positioning and silencing modules that turns virus construction into a method of inquiry.", "Executive summary": "Influenza A virus carries eight negative-sense RNA segments encoding roughly ten major products, and the review treats this compactness as an invitation to read the virus as a genetic circuit with discrete functional modules for entry, nuclear import, transcription, the switch to replication, nuclear export and egress. The organizing argument is that understanding a circuit of this kind comes from rebuilding it, an approach the article labels learning by building. The review traces the enabling technology from minireplicon systems that defined the RNA sequences the viral polymerase recognizes, through complete reverse genetics systems that permit rescue of virus entirely from DNA, to the systematic mapping of packaging signals and of mutational tolerance across viral proteins. It then organizes the applications as engineering modules. A tracking module covers fluorescent and luciferase reporter viruses and the persistent problem that most insertions attenuate. A browser history module uses virus-delivered Cre recombinase with LoxP reporter mice to mark cells that survived infection, which identified club cells as a population that clears the virus. A genetic override module covers microRNA target site insertion and drug-controlled degron tags as kill switches and biocontainment devices. A positioning module uses engineered intergenic space in segment 8 to carry RNA barcodes, which established that aerosol transmission passes only two to three virions while contact transmission passes much more. An RNA interference module has the virus deliver artificial microRNAs in an in vivo screen. Throughout, the review distinguishes designs that attenuate from the few that leave fitness unchanged.", "Scientific context": "Reverse genetics for positive-strand RNA viruses came early, since their genomes launch without accompanying viral proteins. Negative-strand RNA viruses were harder, because rescue requires an RNA with precise termini correctly bound to nucleoprotein in the presence of its cognate polymerase. The review credits the intermediate step to minireplicon systems, DNA-based platforms using small genomic mimics together with nucleoprotein to study polymerase biology, work it attributes to Hsu, Luytjes, Enami and colleagues in the Palese laboratory and to the broader field summarized by Neumann and Kawaoka. Complete rescue of influenza A virus from cloned DNA followed, credited to Fodor and colleagues and to Neumann and colleagues, and the review notes that the same laboratories that built the minireplicon and reverse genetics systems were the first to apply them to influenza RNA biology. What remained unresolved at the start of that period was how much of the viral genome is available for modification, which is the practical question the review treats as prior to every application that follows.", "Central question": "If influenza A virus is treated as a genetic circuit, which of its modules can be disrupted, tagged, replaced or added to without destroying the circuit, and what can be learned about virus and host biology by building the resulting recombinants and observing them.", "Experimental strategy": "Not applicable in the experimental sense. This is a single-author review and presents no new data. Its organizing strategy is a design taxonomy. Rather than surveying the literature chronologically or by virus, the article sorts engineered influenza viruses by the function the added element performs within the circuit, borrowing vocabulary from engineering. The taxonomy does real work, because it separates the questions that each class of design can answer. A tracking module reports where the virus is now. A lineage-marking module reports where it has been, including in cells that survived. An override module controls whether the program runs at all, in a host-specific or drug-dependent way. A barcode module reports the identity and number of founding virions without altering their behavior. A silencing module turns the virus into a delivery vehicle for a perturbation of the host. Against each class the review applies a common criterion, whether the design preserves viral fitness, since an attenuated recombinant reports on a different virus than the one under study.", "Key findings": "This is a review. Entries below identify the laboratory that produced each result, and mark claims that belong to the review's own argument rather than to any cited study.\n\n1. Reported by many laboratories. Packaging signals for each of the eight segments extend beyond the terminal promoter elements and into the open reading frames, with a different length of required material for each segment. The review credits the initial demonstration that foreign RNA could be packaged to Luytjes and colleagues and to Fujii and colleagues, and the systematic mapping to Duhaut and Dimmock, Fujii, Liang, Marsh, Hutchinson, Essere and Gavazzi and colleagues. The idea that packaging is mediated by RNA to RNA interactions is described as a hypothesis, corroborated by more recent work from Dadonaite and colleagues and Majarian and colleagues. Gao and Palese used the same knowledge to swap packaging material between reading frames and build a virus incapable of reassortment with natural influenza.\n\n2. Reported by the Palese laboratory. Genome-wide insertional mutagenesis using bacteriophage Mu transposase, leaving a 15 nucleotide insertion, found most amino acid insertions were not tolerated, with hemagglutinin and NS1 as the exceptions, reported by Heaton and colleagues. The review interprets that exception as reflecting the flexibility those two proteins need in order to adapt to differing host immune defences.\n\n3. Reported by other laboratories. Two complementary mutagenesis approaches refined the picture. Error-prone PCR on segments 4 and 8 mapped residues involved in receptor binding, receptor structure and NS1 antagonism, reported by Wu and colleagues. Codon-based library construction and deep mutational scanning of nucleoprotein and hemagglutinin was reported by Bloom and colleagues. The generalization drawn, that influenza A virus is a highly optimized circuit with little tolerance for random insertion and that the consensus strain generally represents the optimal composition for its host, is the review's synthesis of these datasets.\n\n4. Reported by other laboratories. Affinity tags inserted at the RNA level allow purification of replication intermediates. A 25 nucleotide PP7 hairpin placed in the neuraminidase stalk region in the complementary RNA orientation allowed immunoprecipitation of complementary ribonucleoproteins with the PP7 coat protein, which supported structural and functional characterisation of that intermediate and found it organized as a filamentous double helix, reported by York and colleagues. A parallel protein-level approach generated eight Flag-tagged recombinants, successful for all major products except M1 and nucleoprotein, used to map a virus to host proteome interaction network, reported by Heaton and colleagues.\n\n5. Reported by other laboratories. Early reporter viruses came at a fitness cost. NS1 truncated and fused to green fluorescent protein gives a reporter that is attenuated in any interferon-competent model, from Kittel and colleagues. Replacing the hemagglutinin and neuraminidase entry system with the single rhabdovirus glycoprotein freed a segment for green fluorescent protein and showed the circuit to be partly modular, from Watanabe and colleagues. Use of 2A ribosomal skipping to build polycistronic segments produced replication-competent but attenuated and unstable reporters in segment 8, from Manicassamy and colleagues, and a stable but less replicative neuraminidase-GFP virus from Li and colleagues.\n\n6. Reported by other laboratories. The finding that considerable foreign material can be added to PB2, the largest segment, came from Dos Santos Afonso and colleagues, and led to a suite of split green fluorescent protein and luciferase reporters described as both stable and showing little to no attenuation, from Avilov, Heaton, Tran and Karlsson and colleagues. Serial passage in mice of the NS1 fusion design recovered a variant with improved stability that remained less pathogenic than wild type, from Fukuyama and colleagues. The review reads the recurrence of attenuation across these designs as evidence that the virus occupies an optimal fitness space in which any major change is detrimental.\n\n7. Reported by the tenOever laboratory with a collaborating group, and by another laboratory independently. Delivering bacteriophage P1 Cre recombinase from PB2 was well tolerated, reducing the lethal dose by only one log, whereas expression from NS1 via a 2A site attenuated substantially, reported by Heaton and colleagues with tenOever as a senior author and by Reuther and colleagues in the Schwemmle laboratory. Combined with a LoxP tdTomato reporter mouse, this permits marking of cells that were infected at any point rather than only cells currently infected. Monitoring non-immune tdTomato-positive cells over the infection showed that although alveolar cells succumb, a subset of club cells clears the virus.\n\n8. Reported by other laboratories, following that observation. Club cells that survive infection were characterized as inherently more resistant, with genetic changes altering their response to virus and to interferon for weeks after clearance, from Hamilton and colleagues, and the basis of the resistance was later attributed to unusually strong antiviral gene induction together with evasion of CD8-mediated clearance, from Chambers and colleagues and Fiege and colleagues.\n\n9. Reported by the tenOever laboratory. MicroRNA-mediated targeting was the first described genetic override for influenza A virus, reported by Perez and colleagues. Two target sites for miR-93, which is absent from the chicken egg but ubiquitous in mammalian cells, placed in the nucleoprotein reading frame gave a virus unable to establish productive infection in mammalian cells while growing to wild-type titres in eggs. The same approach was extended to restrict replication to ferrets as a molecular biocontainment device and to restrict it in haematopoietic cells as an immunological tool, reported by Langlois and colleagues. Because no coding material is altered, these designs give stably engineered viruses with unchanged fitness, supported by Benitez and colleagues and Aguado and colleagues.\n\n10. Reported by the tenOever laboratory. As an alternative to host-supplied silencing, influenza A virus can be engineered to express an artificial microRNA processed into a small interfering RNA against a structurally constrained and conserved part of its own genome, producing a self-inactivating virus, reported by Benitez and colleagues.\n\n11. Reported by another laboratory. A drug-dependent override was built by attaching the small molecule-assisted shutoff tag, composed of the hepatitis C virus NS3 protease followed by part of NS4a, to the PA subunit, reported by Fay and colleagues using the tag described by Chung and colleagues. In the presence of asunaprevir, NS3 cleavage is blocked and the retained NS4a peptide directs degradation. The approach reduced titres by one to two logs and prevented disease in animals, and selective mutation of the target site was observed.\n\n12. Reported by the tenOever laboratory. Destroying the natural 3 prime splice acceptor for NEP and duplicating that region downstream of the NS1 stop codon creates an intergenic region in segment 8 while retaining the splicing dynamics of the segment, and does so without loss of fitness, reported by Chua and colleagues. This space accommodates modules that act only at the RNA level.\n\n13. Reported by the tenOever laboratory. Inserting 22 nucleotide barcodes with no function beyond identity produced a library of more than 100 tagged viruses at roughly equal proportion, which allowed population dynamics to be read out by deep sequencing at single-virion resolution, reported by Varble and colleagues. Applied to transmission, contact transmission transferred up to half the viral population whereas aerosol transmission produced founder populations of only two to three virions. The review notes independent support from reassortment work by Fonville and colleagues and Jacobs and colleagues, and related observations from Brooke and colleagues and Russell and colleagues that infections frequently fail to deliver all eight segments, so co-infection is often required.\n\n14. Reported by the tenOever laboratory. The same intergenic space supports microRNA production from a replication-competent virus, reported by Varble and colleagues, and because hairpin processing depends on structure rather than sequence the stem can be rewritten to silence a chosen host factor, reported by Benitez and colleagues. A library in which each virus silenced a different host factor was followed over an in vivo infection, and the greatest enrichment was seen for viruses targeting Ddx58, Tlr7 and Ifih1, Irf1, Irf7 and Stat1, and Adar and Rnasel. The review presents that enrichment as suggestive rather than as a demonstration, and notes the module is constrained by kinetics, since the virus can silence targets only from the point of infection onward and cannot affect protein made earlier.", "Mechanistic model": "This article does not establish a mechanism and does not attempt one. Its argument is architectural rather than mechanistic. Influenza A virus is presented as a genetic circuit of interacting modules, with each viral product assigned to a stage of the cycle and NS1 assigned not to the circuit itself but to protecting it from host interference. The proposition that follows is methodological, that a circuit understood well enough can be reprogrammed to run additional modules concurrently with infection, and that the success or failure of a build is itself informative about the circuit.\n\nTwo general claims in the article are the author's interpretation of assembled results rather than demonstrated conclusions. The first is that influenza A virus occupies an optimal fitness space, inferred from the recurrence of attenuation across independent reporter designs and from the mutational tolerance datasets. The second is that the consensus sequence of a strain generally represents the optimal composition for the host it came from, inferred from deep mutational scanning rather than measured directly. Both should be read as the framing the review adopts and not as settled results.", "Conceptual or technical advance": "The article's contribution is organizational and pedagogical rather than empirical. By sorting engineered viruses according to the circuit function of the added element, it makes visible which questions each design class can answer and which it cannot, and it applies a consistent criterion of preserved fitness that separates designs usable for studying wild-type biology from those that necessarily study an attenuated variant. Several specific lessons are made portable in the process, including the observation that insertions into PB2 and into engineered intergenic space in segment 8 are better tolerated than insertions that alter coding material, and that modules acting purely at the RNA level avoid the fitness cost that protein-level modifications incur. The Cre and LoxP application illustrates a broader point, that engineering can convert a virus into a reagent for a host question, in this case the identity of cells that survive infection, which is not addressable by any assay that only detects cells currently infected. The review closes by extending the framing toward therapeutics, naming oncolytic use, biologics production and RNA editing as directions, with work attributed to Schmid, Pizzuto and Hamilton and colleagues.", "Relationship to the broader research program": "This article is the clearest statement in the corpus of the premise underlying much of the tenOever laboratory's work, that building a virus is a way of understanding it. Most of the designs the review credits to that laboratory were produced in service of a host or population question rather than as tools in themselves, including microRNA targeting to restrict replication to defined species or lineages, barcoding to measure transmission bottlenecks, and virus-delivered artificial microRNAs to screen host factors in vivo.\n\nCategory 3 synthesis, visible only across papers. The review's engineering premise is the direct continuation of the argument in the 2013 Nature Reviews Microbiology article, which established why the chordate microRNA machinery is available for this kind of exploitation, and the 2013 review is cited here for exactly that purpose. The in vivo RNAi screen result, where enrichment fell on pattern recognition receptors, interferon-pathway transcription factors and effectors including Adar, connects to the treatment of the interferon-stimulated gene set as a structured output in the 2007 Science report, though neither paper cites the other and the connection is synthesis rather than a claimed lineage.", "Related publications": "- tenOever 2013, Nature Reviews Microbiology, predecessor and cited here. It supplies the argument that chordate microRNAs are not engaged by RNA viruses and are therefore available for engineering.\n- Perez et al. 2009, Nature Biotechnology, application from the tenOever laboratory, microRNA-mediated species-specific attenuation, cited as the first described genetic override for influenza A virus.\n- Langlois et al. 2012, PNAS, and Langlois et al. 2013, Nature Biotechnology, applications from the tenOever laboratory on lineage-restricted and host-restricted targeting.\n- Chua et al. 2013, Cell Reports, methodological foundation from the tenOever laboratory for the segment 8 intergenic design used by several later modules.\n- Varble et al. 2010, PNAS, and Varble et al. 2014, Cell Host Microbe, methodological foundation and application from the tenOever laboratory for virus-encoded microRNAs and for barcoded transmission studies.\n- Benitez et al. 2015, Cell Reports, two reports, application from the tenOever laboratory for self-targeting virus and for in vivo RNAi screening.\n- Heaton et al. 2014, Journal of Experimental Medicine, application with tenOever as a senior author, Cre-expressing virus and club cell survival.\n- Aguado et al. 2018, PNAS, from the tenOever laboratory, cited in support of stable engineered designs with unchanged fitness.\n- Neumann et al. 1999, PNAS, and Fodor et al. 1999, Journal of Virology, methodological foundation from other laboratories for influenza reverse genetics.", "Limitations and boundaries": "The article is a review with no new data, so nothing in it is demonstrated here. Its scope is almost entirely influenza A virus, and the circuit framing is presented for a segmented negative-strand virus with roughly ten products, so it does not transfer automatically to other families. Several designs the review describes work only in heavily laboratory-adapted strains, a qualification the article states explicitly for the segment 8 intergenic modification. Many reporter designs carry attenuation, so results obtained with them describe a modified virus, and the review is careful to record which designs preserve fitness and which do not. The transposon mutagenesis approach is noted to select at the level of protein subdomain and to favour surface loops, and the review states that mapping the relative importance of every residue in every gene would require further work. The RNA interference module is limited by kinetics and can only act on transcripts made after infection begins, which restricts it to identifying virus-induced genes that reduce replication. The in vivo RNAi screen result is described as suggesting rather than establishing the role of the enriched targets. The drug-controlled override showed escape by mutation of the target site. Priority and emphasis language in the source, including its praise of certain studies and its description of several as the first of their kind, belongs to the review and is not adopted here. A bibliographic discrepancy should be noted. The inventory records the year as 2019, matching the online publication and PubMed record, while the article itself carries a 2020 copyright line and citation instruction.", "Audience summaries": "### 25 words\n\nTreating influenza A virus as a compact genetic circuit, this perspective organizes two decades of virus engineering into functional modules for tracking, override, barcoding and host silencing.\n\n### 75 words\n\nInfluenza A virus carries only about ten gene products, which makes it tractable as an engineered system. This perspective organizes the field by what an added element does within the viral circuit, covering fluorescent reporters, Cre recombinase for marking cells that survived infection, microRNA target sites as host-specific kill switches, RNA barcodes that revealed that aerosol transmission passes only two or three virions, and virus-delivered artificial microRNAs used to screen host factors during infection.\n\n### 150 words\n\nReverse genetics made it possible to build influenza A virus from DNA, and this perspective argues that building the virus is itself a way of understanding it. Systematic work on packaging signals and on mutational tolerance, largely from other laboratories, established that the genome has little room for random insertion, that packaging sequences extend into coding regions, and that hemagglutinin and NS1 are unusually permissive. Against that background the article sorts engineered viruses by circuit function. Reporters track location but usually attenuate, except when placed in PB2 or in an engineered intergenic space in segment 8. Cre recombinase combined with LoxP reporter mice marks cells that survived infection and identified club cells as clearing the virus. MicroRNA target sites act as species-restricted or lineage-restricted kill switches without altering coding material or fitness. RNA barcodes resolved transmission bottlenecks, and virus-delivered artificial microRNAs support in vivo host factor screening." }, "discoveries": [ "claim-10" ], "relationships": [ { "from": "2019-tenoever-synthetic-virology-building-viruse", "to": "2009-perez-microrna-mediated-species-specific", "relationship": "application", "evidence": "stated in the Related publications section of 2019-tenoever-synthetic-virology-building-viruse" }, { "from": "2019-tenoever-synthetic-virology-building-viruse", "to": "2013-langlois-microrna-based-strategy-to-mitigat", "relationship": "related", "evidence": "stated in the Related publications section of 2019-tenoever-synthetic-virology-building-viruse" }, { "from": "2019-tenoever-synthetic-virology-building-viruse", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2019-tenoever-synthetic-virology-building-viruse" }, { "from": "2019-tenoever-synthetic-virology-building-viruse", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2019-tenoever-synthetic-virology-building-viruse" }, { "from": "2019-tenoever-synthetic-virology-building-viruse", "to": "2014-heaton-long-term-survival-of-influenza-vi", "relationship": "application", "evidence": "stated in the Related publications section of 2019-tenoever-synthetic-virology-building-viruse" } ], "canonical_url": "https://tenoeverlab.us/publications/2019-tenoever-synthetic-virology-building-viruse/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "reverse-genetics", "viral-population-deep-sequencing", "barcoded-virus-library", "artificial-mirna", "in-vivo-rnai-screen", "minigenome-assay", "2a-recoding", "cre-lox-lineage-tracing", "deep-mutational-scanning", "reporter-virus", "rna-affinity-tagging", "degron-shutoff", "transposon-mutagenesis" ] } }, { "id": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "slug": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "url": "/publications/2020-blanco-melo-imbalanced-host-response-to-sars-c/", "title": "Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19", "authors": [ "Daniel Blanco-Melo", "Benjamin E. Nilsson-Payant", "Wen-Chun Liu", "Skyler Uhl", "Daisy Hoagland", "Rasmus Møller", "Tristan X. Jordan", "Kohei Oishi", "Maryline Panis", "David Sachs", "Taia T. Wang", "Robert E. Schwartz", "Jean K. Lim", "Randy A. Albrecht", "Benjamin R. tenOever" ], "author_count": 15, "first_author": "Daniel Blanco-Melo", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Robert E. Schwartz", "Jean K. Lim", "Randy A. Albrecht", "Benjamin R. tenOever" ], "tenoever_position": 15, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2020, "journal": "Cell", "volume": "181", "issue": "5", "pages": "1036-1045.e9", "doi": "10.1016/j.cell.2020.04.026", "doi_url": "https://doi.org/10.1016/j.cell.2020.04.026", "pmid": "32416070", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/32416070/", "pmcid": "PMC7227586", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227586/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227586/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling", "pandemic-host-response" ], "themes": [ "calibration-of-interferon-in-vivo", "imbalanced-host-response" ], "pathogens": [ "SARS-CoV-2", "SARS-CoV-1", "MERS-CoV", "influenza A virus", "human parainfluenza virus 3", "respiratory syncytial virus" ], "viral_families": [ "Coronaviridae", "Orthomyxoviridae", "Paramyxoviridae", "Pneumoviridae" ], "host_species": [ "human", "ferret" ], "technologies": [ "messenger RNA sequencing", "adenoviral vector transduction", "principal component analysis", "gene ontology enrichment", "quantitative PCR", "immunoblotting", "ELISA cytokine profiling", "JAK inhibitor treatment" ], "biological_systems": [ "A549 cells", "A549 cells expressing ACE2", "Calu-3 cells", "primary normal human bronchial epithelial cells", "ferret nasal wash and trachea", "post-mortem human lung", "human serum" ], "key_concepts": [ "type I interferon", "type III interferon", "interferon-stimulated genes", "chemokine induction", "imbalanced host response", "IL-6", "leukocyte recruitment", "viral interferon antagonism", "multiplicity of infection dependence", "COVID-19 pathogenesis" ], "keywords": [ "SARS-CoV-2", "COVID-19", "interferon", "chemokine", "transcriptomics", "ferret model", "bronchial epithelium", "IL-6", "inflammation", "respiratory virus" ], "one_sentence_contribution": "Across cell lines, primary bronchial epithelium, ferrets and patient material, SARS-CoV-2 infection produces a transcriptional response distinguishable from that to other respiratory viruses, combining low type I and type III interferon induction with a moderate interferon-stimulated gene response and strong chemokine and IL-6 expression.", "summary_25": "SARS-CoV-2 provokes unusually little interferon while driving strong chemokine and IL-6 output, a combination seen in cells, primary airway tissue, ferrets and patients, and linked to inflammatory disease.", "summary_75": "Comparing SARS-CoV-2 with five other respiratory viruses in the same systems showed a distinctive host response. Infected cells made little type I or type III interferon and only some interferon-stimulated genes, yet produced abundant chemokines and IL-6. The same pattern appeared in primary airway cells, in infected ferrets and in patient lung and serum. Blocking interferon signalling did not reduce the chemokine output, indicating the inflammatory arm operates independently of interferon.", "summary_150": "Matched transcriptional profiling across respiratory cell lines, primary bronchial epithelium, ferrets and COVID-19 patient material positions the SARS-CoV-2 host response against SARS-CoV-1, MERS-CoV, influenza A virus, parainfluenza virus 3 and respiratory syncytial virus. In A549 cells made permissive by adenoviral ACE2, SARS-CoV-2 replicated to high levels at low multiplicity without activating TBK1 or inducing STAT1 and MX1, while high multiplicity did engage interferon, an observation the authors attribute either to saturation of an unidentified antagonist or to non-physiological pattern formation. The virus was sensitive to interferon beta, and ruxolitinib abolished interferon-stimulated gene induction while leaving chemokine induction largely intact. Primary bronchial cells infected with SARS-CoV-2 shared only eight induced genes with wild-type influenza A virus yet produced a broad chemokine program. Ferrets showed a cytokine response that outlasted viral clearance. Patient lung showed chemokines without detectable interferon, and patient serum showed raised IL-6, IL1RA and multiple chemokines.", "citation": "Blanco-Melo D, Nilsson-Payant BE, Liu W-C, Uhl S, Hoagland D, Møller R, Jordan TX, Oishi K, Panis M, Sachs D, Wang TT, Schwartz RE, Lim JK, Albrecht RA, tenOever BR. Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19. *Cell* 2020, volume 181, issue 5, pages 1036-1045.e9. DOI 10.1016/j.cell.2020.04.026. PMID 32416070. PMCID PMC7227586.", "sections": { "Citation": "Blanco-Melo D, Nilsson-Payant BE, Liu W-C, Uhl S, Hoagland D, Møller R, Jordan TX, Oishi K, Panis M, Sachs D, Wang TT, Schwartz RE, Lim JK, Albrecht RA, tenOever BR. Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19. *Cell* 2020, volume 181, issue 5, pages 1036-1045.e9.\n\nDOI 10.1016/j.cell.2020.04.026. PMID 32416070. PMCID PMC7227586.", "One-sentence contribution": "Across cell lines, primary bronchial epithelium, ferrets and patient material, SARS-CoV-2 infection produces a transcriptional response distinguishable from that to other respiratory viruses, combining low type I and type III interferon induction with a moderate interferon-stimulated gene response and strong chemokine and IL-6 expression.", "Executive summary": "Early in the COVID-19 pandemic there was little information on how host cells respond to SARS-CoV-2 relative to other respiratory viruses. This study profiles the transcriptional response across four levels of system. In respiratory cell lines, SARS-CoV-2, SARS-CoV-1, MERS-CoV, influenza A virus, human parainfluenza virus 3 and respiratory syncytial virus were compared by sequencing, with A549 cells rendered permissive by adenoviral delivery of ACE2. In primary human bronchial epithelial cells, SARS-CoV-2 was compared with wild-type influenza A virus, an influenza A virus lacking its NS1 antagonist and interferon beta treatment. Ferrets were infected and followed longitudinally by nasal wash sequencing. Post-mortem lung from two COVID-19 patients was compared with healthy lung, and serum from 24 patients who tested positive was compared with 24 controls. Across these systems the recurring pattern is low type I and type III interferon with only a subset of interferon-stimulated genes induced, alongside pronounced chemokine and interleukin expression. In primary bronchial cells SARS-CoV-2 and influenza A virus produced responses similar in magnitude but sharing only eight significantly induced genes, and interferon-stimulated gene induction by SARS-CoV-2 was not restored by blocking interferon signalling with ruxolitinib, indicating that the chemokine response is interferon-independent. High multiplicity infection in permissive cells did induce interferons, which the authors read as antagonism being overcome or as artificial pattern generation. Serum from patients showed no detectable interferon beta or lambda but significantly raised IL-6, IL1RA, CCL2, CCL8, CXCL2, CXCL8, CXCL9 and CXCL16.", "Scientific context": "Three highly pathogenic betacoronaviruses had emerged from zoonotic events in two decades, and the newest, SARS-CoV-2, was spreading with an undetermined mortality rate and an incompletely characterised host interaction. The general framework was established, in which intracellular pattern recognition receptors detect aberrant viral RNA, activate interferon regulatory factors and nuclear factor kappa B, and launch two programs, interferon-driven cellular defence and chemokine-driven leukocyte recruitment. It was also known that respiratory viruses encode antagonists of this system, with SARS-CoV-1 antagonism attributed to ORF3B, ORF6 and the nucleocapsid protein plus the host shutoff nuclease nsp1, and influenza A virus antagonism attributed to NS1. Work on SARS-CoV-1 had proposed that clinical severity stems from a dysregulated immune response with delayed type I interferon and improper recruitment of inflammatory monocyte and macrophage populations. What was missing for SARS-CoV-2 was a direct, matched comparison of the host response across systems and against other respiratory viruses.", "Central question": "How does the host transcriptional response to SARS-CoV-2 differ from the response to other respiratory viruses, and does that difference account for the inflammatory character of COVID-19?", "Experimental strategy": "The design is comparative at every level. Rather than characterising SARS-CoV-2 alone, the authors place it alongside two other pathogenic coronaviruses and three common respiratory viruses, using the same cell systems, the same sequencing pipeline and the same differential expression framework, so that the response can be positioned rather than merely described. Because A549 cells express little ACE2 and support poor replication, adenoviral delivery of ACE2 is used to separate the question of permissiveness from the question of response, with an mCherry vector as the matched control, and infections are run at both low and high multiplicity because the two conditions distinguish a virus that fails to trigger detection from one whose antagonist is saturated. Primary bronchial epithelial cells provide a physiological comparison, and the inclusion of an influenza A virus lacking NS1 supplies a positive control for what a full interferon response in those cells looks like when antagonism is removed. Ruxolitinib is used to ask whether the induced genes depend on interferon signalling at all. The ferret study adds time and an intact host with an immune system, sampling the upper respiratory tract repeatedly and the trachea at a fixed day. Human material closes the loop in two ways, transcriptionally in lung tissue and at the protein level in serum, where circulating cytokines can be measured in a larger group than the tissue analysis allows.", "Key findings": "1. Viral read fraction across infections ranged from 0.1 percent to over 50 percent of total reads. A549 cells were relatively non-permissive for SARS-CoV-2 while Calu-3 cells supported roughly 15 percent viral reads, and adenoviral ACE2 raised SARS-CoV-2 in A549 cells to about 54 percent of reads at low multiplicity, corroborated by nucleocapsid immunoblot and by more than three orders of magnitude higher Envelope and nsp14 transcript (Figure 1, A through D).\n2. Despite that high viral load at low multiplicity, there was no TBK1 activation and no induction of STAT1 or MX1. Raising the multiplicity tenfold did engage interferon even though total viral reads at 24 hours were comparable. The authors offer two readings, that an antagonist is overcome at high multiplicity or that high multiplicity generates pathogen patterns that may not form physiologically, and they do not adjudicate between them.\n3. SARS-CoV-2 was sensitive to interferon beta pretreatment. Blocking interferon signalling with ruxolitinib prevented interferon-stimulated gene induction without increasing viral Spike or viral reads appreciably, and had minimal effect on cytokine and chemokine induction. The last point is the direct evidence that the chemokine response is independent of type I and type III interferon signalling.\n4. Principal component analysis placed the response to SARS-CoV-2 under conditions of high replication apart from all other viruses tested, while MERS-CoV clustered with SARS-CoV-1 and influenza A virus in a pattern of overall antiviral repression, and parainfluenza virus 3 and respiratory syncytial virus formed a separate cluster marked by high interferon and interferon-stimulated gene expression (Figure 1, E and F).\n5. In primary bronchial epithelial cells, interferon beta induced 381 genes, most of which were also induced by the NS1-deficient influenza A virus. SARS-CoV-2 and wild-type influenza A virus produced responses similar in magnitude but sharing only eight significantly induced genes, among them IL-6, IRF9, ICAM1 and TNF (Figure 2A).\n6. In those cells type I and type III interferons were undetectable for both SARS-CoV-2 and wild-type influenza A virus with only a small subset of interferon-stimulated genes induced, whereas the NS1-deficient virus induced IFNB and IFNL1 through 3 robustly (Figure 2, C and D). For influenza A virus the muting is attributable to NS1. For SARS-CoV-2 the paper does not identify the responsible viral product.\n7. Despite absent interferon expression, SARS-CoV-2 in primary bronchial cells induced CCL20, CXCL1, IL-1B, IL-6, CXCL3, CXCL5, CXCL6, CXCL2, CXCL16 and TNF, together with enrichment for chemokine signalling and a response to type II interferon (Figure 2, C and E).\n8. In ferrets, SARS-CoV-2 reached 1.2 percent of nasal wash reads at day three, fell to 0.05 percent by day seven and was undetectable at day 14. Transcriptional change was negligible at day one, began at day three with CCL8 and CXCL9, and expanded by day seven to include CCL2 and CXCL9 even as virus waned, accompanied by upregulation of leukocyte markers including CD163, CD226, CCR5, CCR6, CXCR1, CXCR2 and CXCR7 (Figure 3, B and C).\n9. The overall magnitude of the ferret upper respiratory response was lower than for a comparable influenza A virus infection, and influenza A virus produced the far larger antiviral signature including MX1, ISG20, OASL and Tetherin. SARS-CoV-2 produced a distinct signature enriched for cell death and leukocyte activation including IL1A and CXCL8. At day 14, with no viral reads, IL-6 and IL1RN remained elevated.\n10. In ferret trachea at day three, both viruses induced comparable monocyte and lymphocyte marker signatures, while SARS-CoV-2 uniquely induced genes aligning with haematopoietic progenitor cells. The authors state explicitly that whether this reflects induced haematopoiesis and whether it contributes to COVID-19 requires further work.\n11. Post-mortem lung from two COVID-19 patients compared with two healthy lungs showed about 2,000 differentially expressed genes, with a subset of interferon-stimulated genes induced, no type I or type III interferon detected by sequencing or semiquantitative PCR, and robust chemokine induction including CCL2, CCL8 and CCL11 (Figure 4A).\n12. Serum from 24 patients who tested positive and 24 controls showed no detectable interferon beta or lambda family interferons and significantly elevated IL-6, IL1RA, CCL2, CCL8, CXCL2, CXCL8, CXCL9 and CXCL16 (Figure 4, B and C). The authors state the sample size is not necessarily representative and that additional sampling is required.", "Mechanistic model": "The study does not establish a mechanism and the authors do not claim one. It defines a reproducible response phenotype, low type I and type III interferon with partial interferon-stimulated gene induction alongside strong chemokine and interleukin expression, and shows that this phenotype recurs across cell lines, primary epithelium, an animal model and human samples. The ruxolitinib experiment constrains the wiring by showing the chemokine arm does not require interferon signalling, and the interferon beta pretreatment shows the virus is sensitive to interferon when it is present, so the low interferon output is not a matter of resistance. What is not determined is why interferon induction is low. The paper proposes that an antagonist prevents engagement and is overcome at high multiplicity, offers the alternative that high multiplicity generates pathogen patterns that would not form physiologically, and notes a further possibility that a subset of cells refractory to the antagonist produces the limited interferon seen in vivo. No viral gene product is assigned this role here. The central interpretive claim, that reduced innate antiviral defence coupled with exuberant inflammatory cytokine production are the defining and driving features of COVID-19, is presented by the authors as a proposal drawn from correlated observations across systems, not as a demonstrated causal chain from response pattern to disease. The suggested parallel with cytokine release syndrome and the consequent suggestion that tocilizumab or anakinra might help are explicitly flagged by the authors as requiring formal testing, and the speculation about a restricted immune response in older people is labelled as such.", "Conceptual or technical advance": "Assembling one matched comparison across six respiratory viruses and four levels of biological system, produced within weeks of the pathogen's emergence, gave a reference description of the SARS-CoV-2 host response and a public dataset. The comparative framing is what makes the result interpretable, since a low interferon response only means something against viruses that produce a high one in the same cells. The demonstration that chemokine induction proceeds without interferon signalling separates two arms of the response that are usually considered together and identifies the inflammatory arm as the one that is intact. Practically, the work reoriented attention for treatment from boosting or mimicking interferon toward controlling inflammation, and it supplied specific circulating mediators to track. The observation that interferon induction depends strongly on multiplicity of infection is also a methodological caution for any cell culture study of this virus.", "Relationship to the broader research program": "The study applies to a newly emerged pathogen the comparative transcriptional profiling approach the laboratory had developed for influenza A virus and other RNA viruses, using the same reliance on sequencing viral and host reads from the same libraries and the same use of an NS1-deficient influenza A virus as the reference for an unantagonised response. Its reference list includes the laboratory's own review on the evolution of antiviral defence systems, its work on influenza A virus transmission bottlenecks in ferrets, its microRNA-based strategy for mitigating risk in gain-of-function influenza studies, and a companion preprint on host detection of negative-sense RNA viruses. It also cites Sharma and colleagues, 2003, for TBK1 as the kinase responsible for type I and type III interferon expression, which connects this work to the senior author's doctoral training period. Reading that connection as a continuous research arc would be category 3 synthesis.", "Related publications": "- Sharma and colleagues, 2003, methodological foundation. Cited here for the identification of TBK1 as the kinase driving type I and type III interferon expression, the node whose activation is assayed as a readout of detection.\n- Blanco-Melo and colleagues, 2020 preprint on ribonucleoprotein genomic structure and host detection of negative-sense RNA viruses, companion. Cited as the source of the recombinant parainfluenza virus 3 used here.\n- Varble and colleagues, 2014, predecessor. Cited for influenza A virus transmission bottlenecks defined by infection route and recipient host, supporting the inference about transmission potential from nasal virus.\n- Langlois and colleagues, 2013, methodological foundation. Cited as the source of the influenza A virus strains used.\n- tenOever, 2016, review or synthesis. The laboratory's review on the evolution of antiviral defence systems, cited for the framing of the cellular response.", "Limitations and boundaries": "The human tissue transcriptional analysis rests on two post-mortem COVID-19 lungs and two healthy lungs, all from males over 60, which the authors state plainly, and the serum study covers 24 cases and 24 controls, which they also describe as not necessarily representative. The comparison viruses were run at multiplicities and durations that differ between viruses, for example influenza A virus at multiplicity five for nine hours against SARS-CoV-2 at multiplicity 0.2 or two for 24 hours, so magnitude comparisons between viruses carry that caveat. SARS-CoV-1 and MERS-CoV data were taken from a previously published dataset rather than generated alongside. The A549 system depends on adenoviral ACE2 delivery, an artificial arrangement that also introduces a second virus vector. Interferon induction proved strongly dependent on multiplicity of infection, and the authors themselves note that high multiplicity conditions may not reflect physiological infection, which limits how firmly the low interferon phenotype can be stated. The ferret study used small groups, with two naive animals, six infected with SARS-CoV-2 and two per influenza comparison, and it samples the upper respiratory tract and trachea rather than the lower airway where severe disease occurs. No mechanism or viral antagonist is identified, no severity stratification of patients is available, and the inference that the observed response pattern drives COVID-19 pathology remains an interpretation of correlated observations. Therapeutic suggestions are raised by the authors as untested possibilities.", "Audience summaries": "### 25 words\n\nSARS-CoV-2 provokes unusually little interferon while driving strong chemokine and IL-6 output, a combination seen in cells, primary airway tissue, ferrets and patients, and linked to inflammatory disease.\n\n### 75 words\n\nComparing SARS-CoV-2 with five other respiratory viruses in the same systems showed a distinctive host response. Infected cells made little type I or type III interferon and only some interferon-stimulated genes, yet produced abundant chemokines and IL-6. The same pattern appeared in primary airway cells, in infected ferrets and in patient lung and serum. Blocking interferon signalling did not reduce the chemokine output, indicating the inflammatory arm operates independently of interferon.\n\n### 150 words\n\nMatched transcriptional profiling across respiratory cell lines, primary bronchial epithelium, ferrets and COVID-19 patient material positions the SARS-CoV-2 host response against SARS-CoV-1, MERS-CoV, influenza A virus, parainfluenza virus 3 and respiratory syncytial virus. In A549 cells made permissive by adenoviral ACE2, SARS-CoV-2 replicated to high levels at low multiplicity without activating TBK1 or inducing STAT1 and MX1, while high multiplicity did engage interferon, an observation the authors attribute either to saturation of an unidentified antagonist or to non-physiological pattern formation. The virus was sensitive to interferon beta, and ruxolitinib abolished interferon-stimulated gene induction while leaving chemokine induction largely intact. Primary bronchial cells infected with SARS-CoV-2 shared only eight induced genes with wild-type influenza A virus yet produced a broad chemokine program. Ferrets showed a cytokine response that outlasted viral clearance. Patient lung showed chemokines without detectable interferon, and patient serum showed raised IL-6, IL1RA and multiple chemokines." }, "discoveries": [ "claim-12" ], "relationships": [ { "from": "2003-sharma-triggering-the-interferon-antivira", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2003-sharma-triggering-the-interferon-antivira" }, { "from": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "to": "2003-sharma-triggering-the-interferon-antivira", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2020-blanco-melo-imbalanced-host-response-to-sars-c" }, { "from": "2020-bouhaddou-the-global-phosphorylation-landsca", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2020-bouhaddou-the-global-phosphorylation-landsca" }, { "from": "2020-yang-a-human-pluripotent-stem-cell-base", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2020-yang-a-human-pluripotent-stem-cell-base" }, { "from": "2021-eriksen-sars-cov-2-infects-human-adult-don", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-eriksen-sars-cov-2-infects-human-adult-don" }, { "from": "2021-guzman-solis-ancient-viral-genomes-reveal-intro", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "related", "evidence": "stated in the Related publications section of 2021-guzman-solis-ancient-viral-genomes-reveal-intro" }, { "from": "2021-hoagland-leveraging-the-antiviral-type-i-in", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-hoagland-leveraging-the-antiviral-type-i-in" }, { "from": "2021-horiuchi-immune-memory-from-sars-cov-2-infe", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-horiuchi-immune-memory-from-sars-cov-2-infe" }, { "from": "2021-nilsson-payant-reduced-nucleoprotein-availability", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "companion", "evidence": "stated in the Related publications section of 2021-nilsson-payant-reduced-nucleoprotein-availability" }, { "from": "2021-nilsson-payant-the-nf-b-transcriptional-footprint", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-nilsson-payant-the-nf-b-transcriptional-footprint" }, { "from": 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2022-yaron-host-protein-kinases-required-for-" }, { "from": "2022-zazhytska-non-cell-autonomous-disruption-of-", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2022-zazhytska-non-cell-autonomous-disruption-of-" }, { "from": "2023-carrau-delayed-engagement-of-host-defense", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2023-carrau-delayed-engagement-of-host-defense" } ], "canonical_url": "https://tenoeverlab.us/publications/2020-blanco-melo-imbalanced-host-response-to-sars-c/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sars-cov-2", "hpiv3", "rsv", "sars-cov", "mers-cov" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "pathway-enrichment-analysis", "immunoblotting", "multiplex-cytokine-assay", "small-molecule-inhibitor-profiling", "adenoviral-vector", "dimensionality-reduction" ] } }, { "id": "2020-bouhaddou-the-global-phosphorylation-landsca", "slug": "2020-bouhaddou-the-global-phosphorylation-landsca", "url": "/publications/2020-bouhaddou-the-global-phosphorylation-landsca/", "title": "The Global Phosphorylation Landscape of SARS-CoV-2 Infection", "authors": [ "Mehdi Bouhaddou", "Danish Memon", "Bjoern Meyer", "Kris M. White", "Veronica V. Rezelj", "Miguel Correa Marrero", "Benjamin J. Polacco", "James E. Melnyk", "Svenja Ulferts", "Robyn M. Kaake", "Jyoti Batra", "Alicia L. Richards", "Erica Stevenson", "David E. Gordon", "Ajda Rojc", "Kirsten Obernier", "Jacqueline M. Fabius", "Margaret Soucheray", "Lisa Miorin", "Elena Moreno", "Cassandra Koh", "Quang Dinh Tran", "Alexandra Hardy", "Rémy Robinot", "Thomas Vallet", "Benjamin E. Nilsson-Payant", "Claudia Hernandez-Armenta", "Alistair Dunham", "Sebastian Weigang", "Julian Knerr", "Maya Modak", "Diego Quintero", "Yuan Zhou", "Aurelien Dugourd", "Alberto Valdeolivas", "Trupti Patil", "Qiongyu Li", "Ruth Hüttenhain", "Merve Cakir", "Monita Muralidharan", "Minkyu Kim", "Gwendolyn Jang", "Beril Tutuncuoglu", "Joseph Hiatt", "Jeffrey Z. Guo", "Jiewei Xu", "Sophia Bouhaddou", "Christopher J.P. Mathy", "Anna Gaulton", "Emma J. Manners", "Eloy Félix", "Ying Shi", "Marisa Goff", "Jean K. Lim", "Timothy McBride", "Michael C. O’Neal", "Yiming Cai", "Jason C.J. Chang", "David J. Broadhurst", "Saker Klippsten", "Emmie De wit", "Andrew R. Leach", "Tanja Kortemme", "Brian Shoichet", "Melanie Ott", "Julio Saez-Rodriguez", "Benjamin R. tenOever", "R. Dyche Mullins", "Elizabeth R. Fischer", "Georg Kochs", "Robert Grosse", "Adolfo García-Sastre", "Marco Vignuzzi", "Jeffery R. Johnson", "Kevan M. Shokat", "Danielle L. Swaney", "Pedro Beltrao", "Nevan J. Krogan" ], "author_count": 78, "first_author": "Mehdi Bouhaddou", "senior_authors": [ "Nevan J. Krogan" ], "corresponding_authors": [ "Robert Grosse", "Adolfo García-Sastre", "Marco Vignuzzi", "Jeffery R. Johnson", "Kevan M. Shokat", "Danielle L. Swaney", "Pedro Beltrao", "Nevan J. Krogan" ], "tenoever_position": 67, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2020, "journal": "Cell", "volume": "182", "issue": "3", "pages": "685-712.e19", "doi": "10.1016/j.cell.2020.06.034", "doi_url": "https://doi.org/10.1016/j.cell.2020.06.034", "pmid": "32645325", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/32645325/", "pmcid": "PMC7321036", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321036/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321036/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "host-factors-and-druggable-signaling" ], "pathogens": [ "SARS-CoV-2" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "African green monkey", "human" ], "technologies": [ "quantitative mass spectrometry", "data-independent acquisition phosphoproteomics", "kinase activity inference", "transcription factor activity inference", "immunofluorescence microscopy", "scanning electron microscopy", "transmission electron microscopy", "flow cytometry DNA content analysis", "siRNA knockdown", "quantitative RT-PCR", "multiplexed ELISA", "plaque assay", "pharmacological dose response profiling" ], "biological_systems": [ "Vero E6 cells", "Caco-2 cells", "ACE2-expressing A549 cells", "Calu-3 cells", "primary human bronchial epithelial cells" ], "key_concepts": [ "phosphoproteomics", "kinase activity rewiring", "casein kinase II", "p38 MAPK signalling", "cell cycle arrest", "filopodial protrusions", "viral egress", "inflammatory cytokine production", "host-directed antiviral therapy", "kinase inhibitor repurposing" ], "keywords": [ "SARS-CoV-2", "COVID-19", "phosphoproteomics", "CK2", "p38 MAPK", "PIKFYVE", "CDK", "AXL", "silmitasertib", "apilimod" ], "one_sentence_contribution": "A time-resolved phosphoproteomic survey of SARS-CoV-2-infected cells showing that infection acts mainly through signalling rather than protein abundance, activating casein kinase II and the p38 cascade while shutting down mitotic kinases, and converting that kinase profile into inhibitors with antiviral activity.", "summary_25": "Time-resolved phosphoproteomics of infected cells showed SARS-CoV-2 acts mainly by rewiring signalling, activating casein kinase II and p38 while arresting the cell cycle, nominating several antiviral inhibitors.", "summary_75": "SARS-CoV-2-infected cells were sampled at six time points and analyzed for protein amount and phosphorylation. Almost all regulation occurred through phosphorylation. Casein kinase II and the p38 cascade were activated, mitotic kinases shut down, and cells arrested between S and G2. Infected human cells formed long branched filopodia carrying viral protein and budding particles. Inhibitors of casein kinase II, p38, AXL, PIKFYVE and cyclin-dependent kinases showed antiviral activity in culture.", "summary_150": "Kinases are readable from phosphorylation data and well supplied with inhibitors, which makes them a practical entry point for host-directed antivirals. Infected Vero E6 cells were sampled in triplicate at six time points, each sample split for abundance and phosphorylation measurement. Regulation was overwhelmingly post-translational, with almost no phosphorylation change accompanied by an abundance change. Activity was inferred for 97 kinases, with casein kinase II and p38 cascade members rising and cyclin-dependent and mitotic kinases falling, and flow cytometry confirmed arrest between S and G2. In human Caco-2 cells, infection induced long branched filopodia bearing viral M protein, with casein kinase II partially co-localizing with N protein, and electron microscopy showed particles apparently budding from these structures. p38 inhibition reduced both inflammatory cytokines and viral replication. Of 87 mapped compounds, 68 were tested, and inhibitors of casein kinase II, p38, AXL, PIKFYVE and cyclin-dependent kinases showed antiviral activity.", "citation": "Bouhaddou M, Memon D, Meyer B, White KM, Rezelj VV, Correa Marrero M, et al. The Global Phosphorylation Landscape of SARS-CoV-2 Infection. Cell, 2020, volume 182, issue 3, pages 685-712.e19. DOI 10.1016/j.cell.2020.06.034. PMID 32645325. PMCID PMC7321036.", "sections": { "Citation": "Bouhaddou M, Memon D, Meyer B, White KM, Rezelj VV, Correa Marrero M, et al. The Global Phosphorylation Landscape of SARS-CoV-2 Infection. Cell, 2020, volume 182, issue 3, pages 685-712.e19.\n\nDOI 10.1016/j.cell.2020.06.034. PMID 32645325. PMCID PMC7321036.", "One-sentence contribution": "A time-resolved phosphoproteomic survey of SARS-CoV-2-infected cells showing that infection acts mainly through signalling rather than protein abundance, activating casein kinase II and the p38 cascade while shutting down mitotic kinases, and converting that kinase profile into inhibitors with antiviral activity.", "Executive summary": "Early in the COVID-19 pandemic the options against SARS-CoV-2 were limited to remdesivir and supportive care, which made host-directed approaches attractive. This study asked how the virus rewires host signalling, on the reasoning that kinases are both readable from phosphorylation data and druggable. Vero E6 cells were infected and harvested in biological triplicate at six time points over 24 hours, and each sample was split for measurement of protein abundance and of phosphorylation by mass spectrometry, with sites mapped to human orthologs. Regulation was overwhelmingly at the level of phosphorylation rather than abundance, and almost no significantly changed phosphorylation site had a matching abundance change, which the authors read as evidence that signalling rather than transcription is the primary host response over this interval. Kinase activities were inferred for 97 of the 518 human kinases. Casein kinase II and multiple p38 pathway members rose, while cyclin-dependent and mitotic kinases fell, and flow cytometry confirmed arrest between S and G2. Imaging of infected human Caco-2 cells showed a marked increase in long branched filopodia bearing viral M protein, with casein kinase II partially co-localizing with N protein along them, and electron microscopy showed assembled particles on and apparently budding from these protrusions. Inhibition of p38 reduced inflammatory cytokines and viral replication. Mapping regulated kinases onto known inhibitors gave 87 candidates, of which 68 were tested at two institutions in two cell lines, with antiviral activity for inhibitors of casein kinase II, p38, AXL, PIKFYVE and cyclin-dependent kinases.", "Scientific context": "SARS-CoV-2 is an enveloped positive-sense RNA betacoronavirus closely related to SARS-CoV, entering cells through ACE2 with spike priming by TMPRSS2, translating ORF1a and ORF1ab into non-structural proteins that assemble the replication and transcription complex within remodelled endoplasmic reticulum, and assembling at the endoplasmic reticulum to Golgi intermediate compartment before release. At the time of writing, clinical management was largely supportive and remdesivir was the only agent with emergency authorisation. A virus to human protein interaction map for SARS-CoV-2 had recently been published by the same broad consortium, identifying 332 human proteins interacting with 27 viral proteins, which supplied a physical map but not a functional one. What remained unresolved was how infection alters the activity state of host signalling, which matters because the functional consequences of many phosphorylation events are annotated, because kinase activities can be inferred from substrate phosphorylation, and because kinases are a well-populated drug target class.", "Central question": "How does SARS-CoV-2 infection rewire host protein phosphorylation and kinase activity over the course of infection, and can the resulting activity profile be mapped onto existing kinase inhibitors to identify compounds with antiviral efficacy.", "Experimental strategy": "The design rests on treating phosphorylation as the readout with the right time resolution for an acute infection. Vero E6 cells were used for the discovery proteomics because of their permissiveness, and each sample was divided so that protein abundance and phosphorylation were measured from the same material, which allows the two layers to be compared directly rather than inferred against each other. Six time points spanning entry, replication and egress were used so that changes could be assigned to a phase of the life cycle, with biological triplicates and mock controls at both the start and end of the interval. Because the host is African green monkey, sequences were aligned to human and sites mapped to human orthologs, which makes the dataset interpretable against human annotation at the cost of introducing an orthology mapping step. Kinase activity was then inferred from the coordinated regulation of annotated substrates rather than measured directly, and the inferred profile was compared against a compendium of published phosphoproteomic conditions to place the infected state relative to known perturbations. Findings from that inference layer were followed with orthogonal assays in human cells, including immunoblotting for activating phosphorylations, imaging and electron microscopy, DNA content analysis, cytokine measurement by transcript and by protein, and siRNA knockdown. Pharmacological testing was deliberately spread across two institutions and two cell lines with overlapping and distinct inhibitor target profiles, which is what allows target attribution rather than compound-level observation alone.", "Key findings": "1. Phosphorylation, not protein abundance, is the dominant layer of the host response over 24 hours. High-quality quantification covered 4,624 human-orthologous phosphorylation sites and 3,036 proteins. The number of significantly regulated phosphorylation sites rose across the time course, while few proteins changed in abundance, and most that increased were viral (Fig. 1E to 1H). For nearly every significantly changed phosphorylation site there was no corresponding abundance change (Fig. 1J). The authors interpret this as evidence that signalling rather than transcriptional regulation is the primary host response in this window.\n\n2. Most host proteins that changed in abundance decreased, with Gene Ontology enrichment for platelet regulation among the downregulated set, including APOH, CD9, TSPAN14, AHSG, SERPINA1 and A2M (Fig. 1I). The suggestion that this may relate to coagulation and stroke in patients is explicitly framed by the authors as a possibility rather than a demonstrated link.\n\n3. Forty-nine phosphorylation sites across seven viral proteins were catalogued, 25 detected here and the remainder from a published dataset, with sites mapped for conservation, secondary structure and predicted interface status (Fig. 2A and 2B). Casein kinase II, cyclin-dependent kinase and protein kinase C families were the top predicted upstream kinases by sequence (Fig. 2C). The proposals that a C-terminal negative charge cluster in M protein is functional, that phosphorylation alters the surface charge of the N protein acidic wrist and thereby modulates RNA binding allosterically, and that the RS-rich region behaves as in SARS-CoV, are all presented by the authors as hypotheses.\n\n4. Forty of the 332 host proteins in the published virus to human interaction map were significantly differentially phosphorylated during infection (Fig. 3). Interpretations offered for individual cases, including decreased LARP1 phosphorylation favouring inhibition of ribosomal protein synthesis and increased NUP98 S888 phosphorylation possibly blocking host mRNA export, are author interpretation drawn from published functions of those sites rather than tested here.\n\n5. Regulated sites fell into five temporal clusters that the authors align with entry, replication and egress, with early clusters enriched for mRNA processing, cell cycle and apoptosis and later clusters enriched for RNA processing and DNA replication (Fig. 4A). Activity regulation was estimated for 97 kinases, strongest at 0 to 2 hours and at 24 hours. Predicted activation covered p38 pathway members including MAPK12, casein kinase II subunits, CAMK2G and PRKG1 and PRKG2, while predicted downregulation covered CDK1, CDK2, CDK5, AURKA, PRKACA, AKT1 and AKT2, MAPK1 and MAPK3, PIM1 and PAK1 (Fig. 4B).\n\n6. Comparison against a compendium of published phosphoproteomic conditions placed early and late infection near states induced by inhibition of mTOR, ERK, AKT and EGFR, and the middle of the time course near inhibition of PI3K, p70S6K and ROCK, with several time points resembling an S to G2 state and anticorrelating with mitosis (Fig. 4D). Complex-level analysis showed changes in spliceosome, proteasome and chromatin remodelling complexes (Fig. 4E).\n\n7. Cytoskeletal signalling is reorganised. PAK1 and PAK2 target sites in vimentin and stathmin were downregulated, while well-characterized casein kinase II target sites including CTNNA1 S641 and MYH9 S1943 rose (Fig. 5A and 5B). In SARS-CoV-2-infected human Caco-2 cells, M protein clusters localized along the shafts and tips of actin-rich filopodia, and infection increased filopodial number, length and branching relative to mock (Fig. 5C and 5D). Casein kinase II was present along these protrusions and partially co-localized with N protein (Fig. 5E and 5F). Scanning and transmission electron microscopy showed assembled particles along the filopodia with instances appearing to bud from them (Fig. 5G and 5H). Phosphoproteomics of Vero E6 cells overexpressing N protein alone showed significantly increased casein kinase II activity (Fig. S3E). The proposal that N protein allosterically controls casein kinase II activity to regulate the cytoskeleton is stated by the authors as a hypothesis.\n\n8. The p38 cascade is activated during infection. Inferred activation of MAP2K3, MAP2K6, MAPK12, MAPKAPK2 and MAPKAPK3 was confirmed by immunoblotting for phospho-p38 T180 and Y182, phospho-MK2 T334 and phospho-CREB and phospho-ATF1 in ACE2-expressing A549 human lung cells (Fig. 6B and 6C). Substrate sites including NELFE, HSPB1 and STAT1 rose late in the time course (Fig. 6D). Transcription factor activity inferred from published expression data in A549, Calu-3 and primary human bronchial epithelial cells showed p38-regulated factors among the most activated (Fig. 6E).\n\n9. The p38 inhibitor SB203580 reduced IL-6 and TNF-alpha transcripts dose dependently in infected ACE2-A549 cells, and multiplexed ELISA confirmed reductions in IL-6, CXCL8, CCL20 and CCL2 protein (Fig. 6F, Fig. S4A and S4B). The same treatment reduced SARS-CoV-2 subgenomic mRNA without major toxicity. The authors state explicitly that because replication is also inhibited, the contributions of p38 activity and of viral presence to cytokine production cannot be deconvolved in this experiment.\n\n10. Infection arrests the cell cycle. Phosphoproteomic profiles correlated with cells arrested at the S to G2 transition and anticorrelated with mitotic cells (Fig. 6G). CDK2 T14 and Y15 phosphorylation rose at 2 hours then declined, as did H2AX S140 (Fig. 6H). DNA content analysis at 24 hours showed a significant increase in cells in S phase and at G2 to M and a decrease in G0 to G1 (Fig. 6I). Whether the arrest is caused by p38 activity is raised as a possible mechanistic link from prior literature and is not tested.\n\n11. Mapping regulated kinase activities to inhibitors yielded 87 drugs and compounds, 10 approved by the US Food and Drug Administration, 53 in clinical testing and 24 preclinical, of which 68 were tested for antiviral efficacy and cytotoxicity at Mount Sinai and Institut Pasteur, in Vero E6 and ACE2-A549 cells (Fig. 7A). Remdesivir served as a positive control with a half maximal inhibitory concentration of 1.28 micromolar (Fig. 7B). Antiviral activity was found for the casein kinase II inhibitor silmitasertib at 2.34 micromolar, the AXL inhibitor gilteritinib at 0.807 micromolar, the MAPK11 and MAPK14 inhibitor ralimetinib at 0.873 micromolar, MAPK13-IN-1 at 4.63 micromolar, the MAPK14 inhibitor ARRY-797 at 0.913 micromolar in ACE2-A549 cells, the PIKFYVE inhibitor apilimod below 0.08 micromolar in Vero E6 and at 0.007 micromolar in ACE2-A549, and the CDK inhibitor dinaciclib at 0.127 and 0.032 micromolar in the two lines (Fig. 7C and 7E to 7K).\n\n12. Genetic perturbation supported the pharmacology for the p38 arm. Small interfering RNA knockdown of MAP2K3, MAPK13 and MAPK12 in ACE2-A549 cells significantly decreased viral replication with little or no effect on viability (Fig. 7I).", "Mechanistic model": "The study does not establish a single mechanism by which SARS-CoV-2 rewires host signalling, and the drug results identify host dependencies without resolving how each one is used by the virus. The authors state plainly that p38 inhibition suppresses cytokine production and impairs replication by a still unknown mechanism.\n\nWhat the data do constrain is the following. Over 24 hours the host response is enacted through phosphorylation rather than through changes in protein amount, which the paired abundance and phosphorylation measurement supports directly. Kinase activity changes are inferred, not measured, from coordinated substrate regulation, so statements about the activity of individual kinases carry the confidence of that inference except where confirmed by immunoblotting, as was done for the p38 arm. Two activity changes are supported by convergent evidence. Casein kinase II activity rises, which is supported by substrate phosphorylation, by the independent observation that N protein overexpression alone raises inferred casein kinase II activity, by physical interaction between N protein and casein kinase II subunits reported in the companion interaction map, by co-localization at filopodia, and by antiviral activity of silmitasertib. The p38 cascade is activated, supported by substrate phosphorylation, by immunoblotting for activating sites in human cells, by transcription factor activity inference from independent expression datasets, and by concordant pharmacological and siRNA results.\n\nThe proposed models beyond those points are the authors' own. That virus-containing filopodia serve egress or cell-to-cell spread is presented as a hypothesis, with the discussion noting the resemblance to Marburg virus more than to vaccinia and stating that further work is needed to determine whether the virus exploits Myosin X motor activity or actin assembly. That N protein allosterically controls casein kinase II is a hypothesis. That S to G2 arrest benefits replication by supplying nucleotides and DNA repair and replication proteins is imported from prior literature on other viruses. The co-localization and imaging data are correlative and do not establish that casein kinase II activity at filopodia is required for particle release.", "Conceptual or technical advance": "The work supplies a functional layer on top of the physical virus to host interaction map for SARS-CoV-2, and it demonstrates a workflow in which a time-resolved phosphoproteomic profile is converted into inferred kinase activities and then into a ranked set of testable compounds, with the compound set constructed so that overlapping and distinct target profiles allow attribution to targets rather than to molecules. Several specific host dependencies became testable as a result, including casein kinase II, the p38 cascade and its upstream activator AXL, PIKFYVE, and cyclin-dependent kinases. The observation that infection drives formation of long branched filopodia carrying viral protein and apparently budding particles opens a route of egress and spread for examination. The finding that p38 inhibition reduces both inflammatory cytokine output and viral replication distinguishes SARS-CoV-2 from earlier cases the authors cite for SARS-CoV, dengue virus and influenza A virus, where p38 inhibition affected the host response without directly impairing the virus, though the basis of that difference is not established here.", "Relationship to the broader research program": "The tenOever contribution to this paper is one component of a large multi-institution collaboration. The author contributions statement lists tenOever under work supervision, alongside fifteen others, and a member of his group appears among those who performed infection experiments. The transcription factor activity analysis draws on expression data from A549, Calu-3 and primary human bronchial epithelial cells reported in Blanco-Melo and colleagues 2020, work in which that laboratory was involved, which is the main point of contact between this study and the laboratory's own line of work on the transcriptional host response to SARS-CoV-2.\n\nCategory 3 synthesis, visible only across papers. The interferon and STAT1 axis appears here as a phosphoproteomic observation, with STAT1 among the p38 substrate sites rising late in infection, and the same protein is the object of direct mechanistic work in the 2007 Science report on IKKε. That is a shared subject rather than a claimed lineage, and neither paper cites the other. The SARS-CoV-2 hamster work reported by Horiuchi and colleagues in 2021 from the tenOever laboratory addresses immunity and transmission in an animal host, a different level of the same pathogen, and is not connected to this study by shared data or method.", "Related publications": "- Gordon et al. 2020, companion. The SARS-CoV-2 virus to human protein interaction map from the same consortium supplies the 332 interacting proteins used here and the physical interactions invoked for casein kinase II, Nsp7 with RHOA and Nsp2 with WASHC5.\n- Blanco-Melo et al. 2020, methodological foundation for the transcription factor activity analysis, supplying expression data from infected A549, Calu-3 and primary human bronchial epithelial cells.\n- Davidson et al. 2020, companion dataset from another group whose viral phosphorylation sites were combined with those detected here.\n- Ou et al. 2020, predecessor reporting antiviral capacity for apilimod, which this study extends by placing PIKFYVE within a phosphorylation-regulated context.\n- Horiuchi et al. 2021, Science Immunology, from the tenOever laboratory. Shares the pathogen but no data, method or claim with this study, so no substantive relationship is asserted.", "Limitations and boundaries": "The authors state their own principal limitation, that the discovery proteomics was performed in Vero E6, an African green monkey kidney line rather than a human respiratory cell, with the mitigation that pharmacological testing was repeated in human ACE2-A549 cells and that most drug effects replicated across lines. Several further boundaries follow from the design. Phosphorylation sites were mapped onto human orthologs, which adds an inference step between measurement and annotation. Kinase activities were estimated from substrate regulation for 97 of 518 human kinases, so the analysis is bounded by existing substrate annotation and cannot speak to poorly annotated kinases, and individual activity calls are predictions except where confirmed directly, as the paper itself illustrates for PRKACA, where substrate-based inference predicted decreased activity while an activation loop site increased. The viral phosphorylation site catalogue does not distinguish cleaved from uncleaved viral proteins. Structural interpretations of M protein and N protein phosphorylation are computational predictions without functional testing. The cytokine experiment cannot separate the effect of p38 activity from the effect of reduced viral replication, as stated in the text. The imaging and electron microscopy evidence for filopodial budding is correlative, and no perturbation establishes that these structures are required for egress or spread. Antiviral efficacy is measured in cell culture over 48 hours with cell viability controls, and no animal or clinical efficacy is shown, so the compounds are candidates rather than established therapies. Inhibitor selectivity is a general caveat the paper addresses by testing overlapping target profiles rather than by direct target engagement measurement. The single virus isolate and the single cell type used for discovery mean that variant-dependent and cell-type-dependent aspects of the signalling response are outside the study's scope.", "Audience summaries": "### 25 words\n\nTime-resolved phosphoproteomics of infected cells showed SARS-CoV-2 acts mainly by rewiring signalling, activating casein kinase II and p38 while arresting the cell cycle, nominating several antiviral inhibitors.\n\n### 75 words\n\nSARS-CoV-2-infected cells were sampled at six time points and analyzed for protein amount and phosphorylation. Almost all regulation occurred through phosphorylation. Casein kinase II and the p38 cascade were activated, mitotic kinases shut down, and cells arrested between S and G2. Infected human cells formed long branched filopodia carrying viral protein and budding particles. Inhibitors of casein kinase II, p38, AXL, PIKFYVE and cyclin-dependent kinases showed antiviral activity in culture.\n\n### 150 words\n\nKinases are readable from phosphorylation data and well supplied with inhibitors, which makes them a practical entry point for host-directed antivirals. Infected Vero E6 cells were sampled in triplicate at six time points, each sample split for abundance and phosphorylation measurement. Regulation was overwhelmingly post-translational, with almost no phosphorylation change accompanied by an abundance change. Activity was inferred for 97 kinases, with casein kinase II and p38 cascade members rising and cyclin-dependent and mitotic kinases falling, and flow cytometry confirmed arrest between S and G2. In human Caco-2 cells, infection induced long branched filopodia bearing viral M protein, with casein kinase II partially co-localizing with N protein, and electron microscopy showed particles apparently budding from these structures. p38 inhibition reduced both inflammatory cytokines and viral replication. Of 87 mapped compounds, 68 were tested, and inhibitors of casein kinase II, p38, AXL, PIKFYVE and cyclin-dependent kinases showed antiviral activity." }, "discoveries": [], "relationships": [ { "from": "2020-bouhaddou-the-global-phosphorylation-landsca", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2020-bouhaddou-the-global-phosphorylation-landsca" }, { "from": "2020-bouhaddou-the-global-phosphorylation-landsca", "to": "2021-horiuchi-immune-memory-from-sars-cov-2-infe", "relationship": "related", "evidence": "stated in the Related publications section of 2020-bouhaddou-the-global-phosphorylation-landsca" }, { "from": "2021-horiuchi-immune-memory-from-sars-cov-2-infe", "to": "2020-bouhaddou-the-global-phosphorylation-landsca", "relationship": "related", "evidence": "stated in the Related publications section of 2021-horiuchi-immune-memory-from-sars-cov-2-infe" } ], "canonical_url": "https://tenoeverlab.us/publications/2020-bouhaddou-the-global-phosphorylation-landsca/", "controlled_vocabulary": { "pathogens": [ "sars-cov-2" ], "technologies": [ "rt-qpcr", "plaque-assay", "immunofluorescence-microscopy", "sirna-knockdown", "flow-cytometry", "multiplex-cytokine-assay", "small-molecule-inhibitor-profiling", "mass-spectrometry-proteomics", "phosphoproteomics", "tf-activity-inference", "electron-microscopy", "kinase-activity-inference" ] } }, { "id": "2020-mccune-rapid-dissemination-and-monopoliza", "slug": "2020-mccune-rapid-dissemination-and-monopoliza", "url": "/publications/2020-mccune-rapid-dissemination-and-monopoliza/", "title": "Rapid Dissemination and Monopolization of Viral Populations in Mice Revealed Using a Panel of Barcoded Viruses", "authors": [ "Broc T. McCune", "Matthew R. Lanahan", "Benjamin R. tenOever", "Julie K. Pfeiffer" ], "author_count": 4, "first_author": "Broc T. McCune", "senior_authors": [ "Julie K. Pfeiffer" ], "corresponding_authors": [ "Julie K. Pfeiffer" ], "tenoever_position": 3, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2020, "journal": "Journal of Virology", "volume": "94", "issue": "2", "pages": "e01590-19", "doi": "10.1128/jvi.01590-19", "doi_url": "https://doi.org/10.1128/jvi.01590-19", "pmid": "31666382", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/31666382/", "pmcid": "PMC6955244", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955244/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955244/pdf/", "publication_type": "collaborative study", "declared_conflicts": null, "research_areas": [ "viral-populations-evolution" ], "themes": [ "transmission-bottlenecks" ], "pathogens": [ "coxsackievirus B3", "poliovirus" ], "viral_families": [ "Picornaviridae" ], "host_species": [ "mouse", "human" ], "technologies": [ "barcoded virus libraries", "deep sequencing of barcodes", "neutral red light-sensitive virus labeling", "plaque assay", "whole-genome consensus sequencing", "Shannon diversity analysis", "radiolabeled amino acid tracing" ], "biological_systems": [ "HeLa cells", "293T cells", "mouse gastrointestinal tract", "mesenteric lymph nodes", "pancreas", "liver", "stool" ], "key_concepts": [ "viral population dynamics", "population bottlenecks", "founder effects", "population monopolization", "enteric virus dissemination", "gastrointestinal barrier", "type I interferon receptor", "replication versus inoculum discrimination" ], "keywords": [ "coxsackievirus B3", "barcoded virus", "dissemination", "viral population diversity", "Ifnar", "poliovirus", "Shannon diversity", "enteric virus" ], "one_sentence_contribution": "A library of 135 barcoded coxsackievirus B3 clones shows that orally inoculated virus reaches systemic tissues within 20 minutes and replicates as a diverse population, after which fewer than three variants come to dominate every tissue in the animal without any detectable adaptive mutation.", "summary_25": "Tagged coxsackieviruses fed to mice reached the liver within twenty minutes and replicated as a diverse population, yet within two days two or three variants dominated everywhere.", "summary_75": "A library of 135 otherwise identical coxsackieviruses, each carrying a short sequence tag, was fed to mice. The full population spread through the gut within hours, and a dye-based label showed that many members genuinely replicated. Even so, by two days almost every tissue in each animal was dominated by three or fewer tags, with no mutation to explain it. Virus also reached pancreas and liver twenty minutes after feeding.", "summary_150": "How the intestinal barrier shapes enteric virus populations had been studied only at late time points, which cannot separate restricted entry from later loss. Here 135 coxsackievirus B3 clones carrying unique nine nucleotide tags were pooled and given orally to interferon receptor deficient mice. All 135 were recovered from the upper gastrointestinal tract at 7.5 and 19 hours, but by 48 and 72 hours generally three or fewer dominated every tissue sampled, with the dominant set shared within an animal and differing between animals. Neutral red labeling, which distinguishes inoculum from progeny virions by light sensitivity, showed that diverse members had replicated before diversity collapsed, and consensus sequencing found no adaptive mutation, though low-frequency variants would escape detection. Virus reached pancreas, liver and mesenteric lymph nodes within 20 minutes, not seen in wild-type mice or with poliovirus. The routes behind both rapid spread and monopolization remain undetermined.", "citation": "McCune BT, Lanahan MR, tenOever BR, Pfeiffer JK. Rapid Dissemination and Monopolization of Viral Populations in Mice Revealed Using a Panel of Barcoded Viruses. Journal of Virology. 2020. Volume 94, issue 2, article e01590-19. DOI 10.1128/jvi.01590-19. PMID 31666382. PMCID PMC6955244. Attribution note. This study was led by the Pfeiffer laboratory at the University of Texas Southwestern, where all mouse work was carried out and to whose corresponding author correspondence is addressed. Benjamin tenOever is the third of four authors. The paper carries no author contributions statement, so the s", "sections": { "Citation": "McCune BT, Lanahan MR, tenOever BR, Pfeiffer JK. Rapid Dissemination and Monopolization of Viral Populations in Mice Revealed Using a Panel of Barcoded Viruses. Journal of Virology. 2020. Volume 94, issue 2, article e01590-19.\n\nDOI 10.1128/jvi.01590-19. PMID 31666382. PMCID PMC6955244.\n\nAttribution note. This study was led by the Pfeiffer laboratory at the University of Texas Southwestern, where all mouse work was carried out and to whose corresponding author correspondence is addressed. Benjamin tenOever is the third of four authors. The paper carries no author contributions statement, so the specific tenOever contribution cannot be stated from the text.", "One-sentence contribution": "A library of 135 barcoded coxsackievirus B3 clones shows that orally inoculated virus reaches systemic tissues within 20 minutes and replicates as a diverse population, after which fewer than three variants come to dominate every tissue in the animal without any detectable adaptive mutation.", "Executive summary": "The gastrointestinal tract restricts what passes from the lumen into the body, and enteric viruses must cross it to spread systemically. How that barrier shapes viral population structure over time had not been resolved, because earlier studies of enteric virus bottlenecks sampled late, at disease onset, and so could not distinguish a restriction imposed on entry from one imposed after replication. This study builds 135 clones of coxsackievirus B3 carrying unique nine nucleotide barcodes in the 5' untranslated region, mixes them at equal titer, and inoculates interferon alpha beta receptor deficient mice orally, sampling gastrointestinal and extraintestinal tissues from 20 minutes to 72 hours. All 135 barcodes were recoverable from the upper gastrointestinal tract at 7.5 and 19 hours, while by 48 and 72 hours generally three or fewer barcodes made up most of the population in every tissue sampled, with the same small set shared across tissues within an animal and differing between animals. Neutral red labeling, which renders inoculum virions light sensitive while progeny are not, showed that diverse viruses had replicated before diversity was lost, so the collapse is not explained by only a few inoculum members replicating. Whole-genome consensus sequencing of later samples found no mutation that would account for the takeover. Virus was detectable in pancreas, liver and mesenteric lymph nodes 20 minutes after oral inoculation of receptor deficient mice, and this was not seen in wild-type mice or with poliovirus.", "Scientific context": "RNA virus populations are diverse because the polymerase lacks proofreading, and that diversity can aid replication and spread within a host. Diversity is lost through selection or through stochastic bottlenecks. Barcoded viruses, carrying short unique sequences that are otherwise neutral, had been used to quantify such changes for plant viruses, human immunodeficiency virus, hepatitis C virus, Zika virus, influenza virus and poliovirus, often revealing founder effects in which a small subpopulation seeds a new tissue.\n\nThe Pfeiffer laboratory had previously shown that the gastrointestinal tract limits dissemination of poliovirus and reduces population diversity in tissues after oral inoculation, and had attributed the loss to combined bottlenecks between mouth and gut and between gut and blood. Those experiments sampled at disease onset, generally 72 hours or later, so they lacked the temporal resolution needed to say when diversity is lost or whether the surviving members are the only ones that ever replicated. Coxsackievirus B3 spreads by the fecal-oral route and replicates in extraintestinal tissues before shedding. Immunocompetent mice are infectable but do not succumb, while mice lacking the interferon alpha beta receptor support high replication and disease, which had established them as the tractable model for oral enterovirus infection.", "Central question": "How does the population structure of an orally acquired enteric virus change across tissues and over time, and is the loss of diversity in systemic tissues caused by restricted entry, by selection of adapted variants, or by events occurring after replication.", "Experimental strategy": "The design separates questions that a single late time point confounds. Barcodes are placed at a single site in the 5' untranslated region between the internal ribosome entry site and the start codon, so that the 135 clones are otherwise isogenic and any change in their relative frequencies reflects population processes rather than encoded differences. Each clone is grown separately and the library assembled from equal plaque-forming unit amounts, so the starting distribution is known rather than assumed, and replicate sequencing of the stock establishes that the readout is even and reproducible. Time is then sampled from 20 minutes to 72 hours, and tissues are split into those directly exposed to lumenal virus and those reached only after systemic spread, so that spatial and temporal structure can be read together. Two diversity metrics are used, the raw count of barcodes and the Shannon index, the latter sensitive to evenness as well as richness.\n\nTwo additional strategies address the ambiguity of the frequency data. Neutral red labeling makes inoculum virions light sensitive, so that light exposure of a tissue homogenate followed by amplification in HeLa cells reports only the barcodes that replicated in the animal, which distinguishes a restriction on replication from a restriction imposed afterwards. Whole-genome consensus sequencing of the dominant populations tests whether adaptation explains the takeover. Finally, a 20 minute time point, poliovirus as a comparator enterovirus, wild-type versus receptor deficient mice, and orally administered radiolabeled amino acids together test whether rapid appearance in systemic tissues is a property of this virus, of enteroviruses in general, or of the barrier itself.", "Key findings": "1. Insertion of a nine nucleotide barcode reduced replication in HeLa cells only at the 5 hour point, and barcodes were retained through ten replication cycles, which the authors take as showing adequate fitness for in vivo use. Replicate sequencing of the pooled stock confirmed roughly equal representation and reproducibility (Figures 1C and 1D).\n2. At 7.5 and 19 hours after oral inoculation of interferon receptor deficient mice, all 135 barcodes were detected in the upper gastrointestinal tract, demonstrating both rapid dissemination of the whole population and that the sequencing pipeline is sensitive and unbiased in tissue (Figure 2).\n3. By 48 and 72 hours, generally three or fewer barcodes accounted for most of the population in each tissue, and nearly all tissues within an animal contained the same small set. With two exceptions among the mice, the dominant barcodes differed between animals, which the authors read as indicating that no barcode carried a selective advantage (Figure 2).\n4. Barcode counts and Shannon diversity were high across the gastrointestinal tract at 7.5 and 19 hours and fell by 48 and 72 hours, while extraintestinal tissues showed lower diversity at all time points (Figure 3).\n5. Whole-genome consensus sequencing of mesenteric lymph node and liver at 48 hours found no mutations in five of eight samples, and in the remainder two synonymous changes and one nonsynonymous change. The authors conclude that a major adaptive mutation did not drive monopolization, and state explicitly that consensus sequencing cannot detect low-frequency variants (Figure 4).\n6. Neutral red labeling reduced titer 250,000-fold on light exposure. Some replication had occurred in stool and colon by 7.5 hours, at a level tenfold above the light-insensitive background of the stock, while most virus in extraintestinal tissues was still light sensitive at that point. By 19 hours nearly all liver virus had replicated, and by 48 hours nearly all virus in all tissues had replicated (Figures 5B, 5C and 6).\n7. Sequencing only the replicated fraction showed that diversity among replicated viruses was high in stool at 7.5 hours and included all 135 barcodes in most mice by 19 hours, but was low in nearly all tissues at 48 hours, and low at every time point in extraintestinal tissues. The conclusion drawn is that the collapse in diversity at 48 hours is not explained by only a small fraction of the inoculum having replicated (Figures 7 and 8). Replicated diversity in colon at 7.5 hours was low while stool diversity was high, which the authors attribute to replication at gastrointestinal sites other than colon seeding the feces.\n8. Virus was detected in pancreas, liver and mesenteric lymph nodes 20 minutes after oral inoculation of interferon receptor deficient mice, with high barcode diversity at those sites, and this early extraintestinal dissemination was not observed in wild-type mice (Figures 9A and 9B).\n9. Poliovirus in receptor transgenic interferon receptor deficient mice did not reach extraintestinal tissues at the same frequency at 20 minutes, so rapid spread is not a shared enterovirus property (Figures 9C and 9D). Orally administered radiolabeled cysteine and methionine reached extraintestinal tissues within 20 minutes at levels equivalent in wild-type and receptor deficient mice (Figure 9E).", "Mechanistic model": "The study does not establish a mechanism for either of its two main observations, and the discussion frames both as open questions.\n\nFor monopolization, the data exclude two candidate explanations and leave two others open. Selection on a barcode is argued against because the dominant barcodes differ between animals. Selection on an adaptive mutation is argued against by consensus sequencing, with the acknowledged caveat that low-frequency variants would not be seen. Restriction of replication to a few inoculum members is excluded by the light-sensitivity experiments, since diverse viruses demonstrably replicated first. The authors offer two alternatives without deciding between them, that all the populations descend from one or two infectious events, or that populations in separate tissues disseminate, intermingle, and can be subsumed by an invading population. They list as open objectives how a subpopulation comes to dominate a tissue and a whole animal, what the anatomical source of the monopolizing population is, and whether clearance is what enables monopolization.\n\nFor rapid dissemination, no route is demonstrated. The authors raise passage across the intestinal epithelium or through M cells giving access to the lymphatics and then the portal vein and bloodstream, and note that other mechanisms may contribute. Because radiolabeled amino acids also appeared systemically within 20 minutes, they suggest the route may not be specific to viruses. The dependence on interferon receptor status is an observation rather than an explained effect. The authors note that interferon gamma has been reported to affect gastrointestinal permeability but that a role for interferon alpha and beta in rapid viral dissemination has not been described, and they identify the contribution of type I interferon signaling to barrier permeability as a subject for future work. Notably, the radiolabel reached systemic tissues equally in both mouse genotypes while virus did not, so barrier permeability alone does not account for the genotype difference.", "Conceptual or technical advance": "The combination of a barcoded library with a replication-reporting label separates two things that frequency data alone cannot distinguish, which members of a population arrived somewhere and which members replicated there. Applying that combination across a time course changes the interpretation of enteric virus bottlenecks. Earlier work sampling at disease onset supported a picture in which barriers restrict which viruses get through. Here the restriction is shown to occur after broad dissemination and after broad replication, which reframes the loss of diversity as a postreplication event, plausibly involving clearance, rather than as a gate at the intestinal barrier.\n\nThe 20 minute observation makes the timing of enteric virus dissemination a tractable question. Appearance in pancreas and liver that fast is too rapid for replication to be involved, is not shared by poliovirus, and depends on interferon receptor status, which identifies a specific and testable set of variables. The barcoded coxsackievirus B3 library itself is a reusable resource for population studies in this system.", "Relationship to the broader research program": "The tenOever laboratory has used barcoded virus libraries to measure population bottlenecks, and Varble and colleagues in 2014 reported that influenza A virus transmission bottlenecks are defined by infection route and recipient host, a study with tenOever as senior author that is cited here as one of the precedents for the approach. The laboratory has separately used libraries of marked or engineered viruses, including barcode libraries as drift controls, in its own in vivo screening work.\n\nCategory 3 synthesis. Read beside Varble and colleagues in 2014 on influenza transmission bottlenecks and the barcode control arm of Varble and colleagues in 2013, this paper places the tenOever contribution within a recurring methodological interest in reading population structure from sequence-tagged virus libraries passaged through animals, applied here to a different virus, a different route and a different laboratory's disease model. This connection is synthesis across papers and is not claimed in the article itself.", "Related publications": "- Kuss, Etheredge and Pfeiffer, 2008, Multiple host barriers restrict poliovirus trafficking in mice, cited as reference 7 from the senior author's laboratory. Predecessor, and the study whose interpretation this work revises.\n- Varble and colleagues, 2014, Influenza A virus transmission bottlenecks are defined by infection route and recipient host, cited as reference 4 with tenOever as senior author. Methodological foundation for the barcoded library approach.\n- Lancaster and Pfeiffer, 2010, cited as reference 19 and the source of the neutral red labeled barcoded virus strategy used here. Methodological foundation.\n- Xiao and colleagues, 2017, on poliovirus intrahost evolution overcoming tissue-specific innate responses, cited as reference 20 as the contrasting case in which tissue-adapted mutations are selected. Predecessor.\n- Varble and colleagues, 2013, An In Vivo RNAi Screening Approach to Identify Host Determinants of Virus Replication. Companion in method from the tenOever laboratory, using barcoded virus libraries as the neutral control for in vivo selection.", "Limitations and boundaries": "Nearly all in vivo work uses mice lacking the interferon alpha beta receptor, chosen because immunocompetent mice do not develop disease after oral coxsackievirus B3 infection. The authors state that examining population dynamics in wild-type mice and other immunodeficient strains is future work. Since the rapid dissemination phenotype was absent in wild-type mice, the possibility that the interferon status of the host also shapes the monopolization dynamics is not excluded by anything reported here.\n\nThe inoculum is a single very high dose, one times ten to the ninth plaque-forming units delivered by pipette into the mouth, so the population dynamics observed apply to that dose and route and not necessarily to natural exposure. Only one virus strain, coxsackievirus B3 H3, is characterized in depth, with poliovirus used only for the 20 minute comparison and in a different mouse background carrying the poliovirus receptor transgene, which weakens the comparison.\n\nThe evidence against adaptation is consensus sequencing of eight samples from two tissues at one time point, and the authors note that consensus sequencing cannot detect low-frequency mutations within a population, so selection on a minority variant is not excluded. Barcode assignment has an acknowledged technical floor, since 13 percent of the library lies within one nucleotide of another member and thirteen barcodes contain an ATG, with five of those out of frame, although all were well represented in the stock. Barcode insertion itself reduced replication at one point in the HeLa growth curve. The replication readout depends on a background correction, since roughly one in 4,000 plaque-forming units of the neutral red stock is already light insensitive, and the interpretation assumes labeled and unlabeled virions penetrate tissues equally. The colon and stool discrepancy at 7.5 hours is explained by inference about unsampled gastrointestinal sites rather than by measurement.\n\nFinally, neither central observation is mechanistically resolved. The source of the monopolizing population, the reason a subpopulation takes over an entire animal, the anatomical route of 20 minute dissemination, and the basis of its dependence on interferon receptor status are all stated by the authors as unresolved.", "Audience summaries": "### 25 words\n\nTagged coxsackieviruses fed to mice reached the liver within twenty minutes and replicated as a diverse population, yet within two days two or three variants dominated everywhere.\n\n### 75 words\n\nA library of 135 otherwise identical coxsackieviruses, each carrying a short sequence tag, was fed to mice. The full population spread through the gut within hours, and a dye-based label showed that many members genuinely replicated. Even so, by two days almost every tissue in each animal was dominated by three or fewer tags, with no mutation to explain it. Virus also reached pancreas and liver twenty minutes after feeding.\n\n### 150 words\n\nHow the intestinal barrier shapes enteric virus populations had been studied only at late time points, which cannot separate restricted entry from later loss. Here 135 coxsackievirus B3 clones carrying unique nine nucleotide tags were pooled and given orally to interferon receptor deficient mice. All 135 were recovered from the upper gastrointestinal tract at 7.5 and 19 hours, but by 48 and 72 hours generally three or fewer dominated every tissue sampled, with the dominant set shared within an animal and differing between animals. Neutral red labeling, which distinguishes inoculum from progeny virions by light sensitivity, showed that diverse members had replicated before diversity collapsed, and consensus sequencing found no adaptive mutation, though low-frequency variants would escape detection. Virus reached pancreas, liver and mesenteric lymph nodes within 20 minutes, not seen in wild-type mice or with poliovirus. The routes behind both rapid spread and monopolization remain undetermined." }, "discoveries": [ "claim-11" ], "relationships": [], "canonical_url": "https://tenoeverlab.us/publications/2020-mccune-rapid-dissemination-and-monopoliza/", "controlled_vocabulary": { "pathogens": [ "poliovirus", "coxsackievirus-b3" ], "technologies": [ "plaque-assay", "viral-population-deep-sequencing", "barcoded-virus-library", "translation-measurement", "population-diversity-statistics", "neutral-red-labeling" ] } }, { "id": "2020-yang-a-human-pluripotent-stem-cell-base", "slug": "2020-yang-a-human-pluripotent-stem-cell-base", "url": "/publications/2020-yang-a-human-pluripotent-stem-cell-base/", "title": "A Human Pluripotent Stem Cell-based Platform to Study SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids", "authors": [ "Liuliu Yang", "Yuling Han", "Benjamin E. Nilsson-Payant", "Vikas Gupta", "Pengfei Wang", "Xiaohua Duan", "Xuming Tang", "Jiajun Zhu", "Zeping Zhao", "Fabrice Jaffré", "Tuo Zhang", "Tae Wan Kim", "Oliver Harschnitz", "David Redmond", "Sean Houghton", "Chengyang Liu", "Ali Naji", "Gabriele Ciceri", "Sudha Guttikonda", "Yaron Bram", "Duc-Huy T. Nguyen", "Michele Cioffi", "Vasuretha Chandar", "Daisy A. Hoagland", "Yaoxing Huang", "Jenny Xiang", "Hui Wang", "David Lyden", "Alain Borczuk", "Huanhuan Joyce Chen", "Lorenz Studer", "Fong Cheng Pan", "David D. Ho", "Benjamin R. tenOever", "Todd Evans", "Robert E. Schwartz", "Shuibing Chen" ], "author_count": 37, "first_author": "Liuliu Yang", "senior_authors": [ "Shuibing Chen" ], "corresponding_authors": [ "Fong Cheng Pan", "David D. Ho", "Benjamin R. tenOever", "Todd Evans", "Robert E. Schwartz", "Shuibing Chen" ], "tenoever_position": 34, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2020, "journal": "Cell Stem Cell", "volume": "27", "issue": "1", "pages": "125-136.e7", "doi": "10.1016/j.stem.2020.06.015", "doi_url": "https://doi.org/10.1016/j.stem.2020.06.015", "pmid": "32579880", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/32579880/", "pmcid": "PMC7303620", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303620/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303620/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "tropism-and-permissive-tissues", "models-for-pandemic-virology" ], "pathogens": [ "SARS-CoV-2" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "human", "mouse" ], "technologies": [ "directed differentiation of human pluripotent stem cells", "organoid culture", "vesicular stomatitis virus pseudo-entry virus", "quantitative RT-PCR", "bulk RNA sequencing", "single-cell RNA sequencing", "confocal immunofluorescence", "kidney capsule xenotransplantation", "gene set enrichment analysis" ], "biological_systems": [ "human pluripotent stem cell derivatives", "pancreatic endocrine cells", "liver organoids", "adult primary human islets", "adult hepatocyte organoids", "adult cholangiocyte organoids", "cardiomyocytes", "dopaminergic neurons", "cortical neurons", "macrophages", "microglia", "endothelial cells", "SCID-beige mouse xenograft", "COVID-19 lung autopsy tissue" ], "key_concepts": [ "SARS-CoV-2 tropism", "ACE2 expression", "TMPRSS2", "cell-type permissiveness", "chemokine induction", "organoid disease modeling", "pancreatic beta cell infection", "hepatocyte and cholangiocyte infection", "pseudotyped entry virus", "COVID-19 extrapulmonary involvement" ], "keywords": [ "SARS-CoV-2", "COVID-19", "human pluripotent stem cells", "organoids", "ACE2", "tropism", "pancreatic beta cells", "liver organoids", "chemokines", "disease modeling" ], "one_sentence_contribution": "A panel of eight human pluripotent stem cell derivatives spanning all three germ layers, together with adult primary islets and liver organoids, identifies pancreatic alpha and beta cells, hepatocytes, cholangiocytes, cardiomyocytes and dopaminergic neurons as permissive to SARS-CoV-2 and shows that permissiveness does not track ACE2 expression alone.", "summary_25": "Stem cell derived human tissues and adult organoids show that SARS-CoV-2 infects pancreatic insulin and glucagon cells, liver cells, heart muscle and dopaminergic neurons.", "summary_75": "Laboratory study of SARS-CoV-2 largely relied on monkey and cancer cell lines. Differentiating human pluripotent stem cells into eight cell types and organoids allowed side-by-side comparison of which human tissues the virus can enter. Pancreatic alpha and beta cells, liver organoids, cardiomyocytes and dopaminergic neurons were permissive, while endothelial cells, macrophages, microglia and cortical neurons were not, even where the ACE2 receptor was present. Infected cells produced strong chemokine responses and lost tissue-specific metabolic gene expression.", "summary_150": "COVID-19 involves organs beyond the lung, and available infection models were monkey cells, mutation-bearing human cancer lines or ACE2 transgenic mice. Eight derivatives of human pluripotent stem cells spanning all three germ layers were stained for ACE2 and challenged with a Spike-pseudotyped vesicular stomatitis virus reporter, then with authentic SARS-CoV-2. Pancreatic alpha and beta cells, liver organoids, cardiomyocytes and dopaminergic neurons supported entry and replication, while endothelial cells, macrophages, microglia and cortical neurons did not, despite detectable ACE2 in several of them, indicating that additional host factors govern entry. Adult primary human islets and adult hepatocyte and cholangiocyte organoids confirmed the pancreatic and hepatic results, and endocrine cells grafted under the mouse kidney capsule took up pseudo-entry virus in vivo. Infected endocrine cells and liver organoids induced chemokines resembling those in COVID-19 lung autopsy tissue, while losing hormone signaling and cytochrome P450 metabolic gene expression.", "citation": "Yang L, Han Y, Nilsson-Payant BE, Gupta V, Wang P, Duan X, Tang X, Zhu J, Zhao Z, Jaffré F, Zhang T, Kim TW, Harschnitz O, Redmond D, Houghton S, Liu C, Naji A, Ciceri G, Guttikonda S, Bram Y, Nguyen DHT, Cioffi M, Chandar V, Hoagland DA, Huang Y, Xiang J, Wang H, Lyden D, Borczuk A, Chen HJ, Studer L, Pan FC, Ho DD, tenOever BR, Evans T, Schwartz RE, Chen S. A Human Pluripotent Stem Cell-based Platform to Study SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids. Cell Stem Cell. 2020. Volume 27, issue 1, pages 125-136.e7. DOI 10.1016/j.stem.2020.06.015. PMID 32579880. PM", "sections": { "Citation": "Yang L, Han Y, Nilsson-Payant BE, Gupta V, Wang P, Duan X, Tang X, Zhu J, Zhao Z, Jaffré F, Zhang T, Kim TW, Harschnitz O, Redmond D, Houghton S, Liu C, Naji A, Ciceri G, Guttikonda S, Bram Y, Nguyen DHT, Cioffi M, Chandar V, Hoagland DA, Huang Y, Xiang J, Wang H, Lyden D, Borczuk A, Chen HJ, Studer L, Pan FC, Ho DD, tenOever BR, Evans T, Schwartz RE, Chen S. A Human Pluripotent Stem Cell-based Platform to Study SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids. Cell Stem Cell. 2020. Volume 27, issue 1, pages 125-136.e7. DOI 10.1016/j.stem.2020.06.015. PMID 32579880. PMCID PMC7303620.", "One-sentence contribution": "A panel of eight human pluripotent stem cell derivatives spanning all three germ layers, together with adult primary islets and liver organoids, identifies pancreatic alpha and beta cells, hepatocytes, cholangiocytes, cardiomyocytes and dopaminergic neurons as permissive to SARS-CoV-2 and shows that permissiveness does not track ACE2 expression alone.", "Executive summary": "COVID-19 presents with respiratory failure but also with cardiac, metabolic, gastrointestinal and neurological manifestations, and clinical reports linked poor outcomes to diabetes. At the time of the study most laboratory work used African green monkey Vero cells, human cancer lines carrying tumor-associated mutations and in some cases defects in innate immune sensing, or mice engineered to express human ACE2, none of which represent the diversity of human cell types the virus might reach.\n\nThe authors built a platform by directed differentiation of human pluripotent stem cells into eight cell types and organoids representing endoderm, mesoderm and ectoderm, stained each for ACE2, and measured entry with a vesicular stomatitis virus particle pseudotyped with SARS-CoV-2 Spike. Cell types scoring positive were then tested with authentic SARS-CoV-2, and the key findings were checked in adult primary human islets, in adult hepatocyte and cholangiocyte organoids, and in a xenograft of stem cell derived pancreatic endocrine cells under the kidney capsule of immunodeficient mice.\n\nPancreatic alpha and beta cells, liver organoids, cardiomyocytes and dopaminergic neurons supported entry and authentic virus replication, while endothelial cells, macrophages, microglia and cortical neurons did not, despite ACE2 protein being detectable in several of the non-permissive types. Transcriptional profiling of infected endocrine cells and liver organoids showed strong chemokine induction alongside loss of tissue-specific metabolic programs, with a chemokine signature comparable to that in COVID-19 lung autopsy tissue.", "Scientific context": "Respiratory failure dominates severe COVID-19, but retrospective series had reported in-hospital cardiac injury in nearly 20 percent of patients, gastrointestinal manifestations in about 25 percent, and an association between poor outcomes and type 2 diabetes, with suggestions that infection can precipitate new-onset diabetes. ACE2 had been identified as the entry receptor and TMPRSS2 as a priming protease. The paper states the gap in terms of models. Vero cells are not human, the human lines in wide use are cancer derived, carry mutations such as in P53 that has been reported to regulate SARS coronavirus replication, include lines such as Huh7.5 with a known RIG-I defect that would obscure antiviral responses, and are proliferative and often unpolarized. Human pluripotent stem cell derivatives had already been used to study Zika virus neuropathology and to screen antivirals in work cited from several groups, which supplied the precedent for applying the same approach to SARS-CoV-2.", "Central question": "Which human cell types are permissive to SARS-CoV-2, how well does permissiveness correspond to ACE2 expression, and can human pluripotent stem cell derivatives and adult organoids serve as physiologically relevant models of infection and of the cellular response to it?", "Experimental strategy": "The design is a screen followed by orthogonal validation. Directed differentiation generates eight derivatives from one starting material, which keeps genetic background constant across cell types and avoids the confounds of comparing unrelated cancer lines. Entry is measured first with a replication-incompetent vesicular stomatitis virus particle bearing SARS-CoV-2 Spike and encoding luciferase, which reads out receptor engagement and membrane fusion alone and can be handled without high containment, with both a plate-level luciferase measurement and single-cell imaging so that permissiveness can be assigned to marker-defined cell types within a mixed culture.\n\nCell types positive in that screen were then challenged with authentic SARS-CoV-2 in the tenOever laboratory, with subgenomic N RNA as a replication readout that distinguishes replication from input genome, plus Spike protein staining. Because a criticism of stem cell derivatives is developmental immaturity, the pancreatic and hepatic results were repeated in adult primary human islets and in organoids derived from adult liver, and pancreatic endocrine cells were transplanted under the kidney capsule to test entry in vivo. Single-cell RNA sequencing of adult islets provides an independent, unbiased map of ACE2 and TMPRSS2 across islet cell types. Finally, transcriptional responses were compared against lung autopsy material from COVID-19 patients and healthy donors to ask whether the in vitro response resembles the human disease.", "Key findings": "1. ACE2 protein was detected in stem cell derived alpha and beta cells but not delta cells, in albumin positive hepatocytes, in endothelial cells, cardiomyocytes, microglia, macrophages and dopaminergic neurons, and only at low levels in cortical neurons, and was not detected in undifferentiated stem cells (Figure 1B).\n\n2. Pseudo-entry virus produced high luciferase in pancreatic endocrine cells, liver organoids, cardiomyocytes and dopaminergic neurons, and low or absent signal in endothelial cells, microglia, macrophages and cortical neurons, with the same pattern at 48 hours as at 24 hours (Figure 1C and Supplementary Figure 2E). Within mixed cultures, luciferase localized to alpha and beta cells but not delta cells, to albumin positive hepatocytes, to cardiomyocytes and to dopaminergic neurons (Figure 1D to 1L).\n\n3. Single-cell RNA sequencing of adult human islets resolved nine cell types and placed ACE2 and TMPRSS2 expression in acinar, ductal, beta, alpha, mesenchymal and endothelial cells (Figure 2A to 2D), with ACE2 protein in primary beta and alpha cells confirmed by imaging (Figure 2E).\n\n4. Authentic SARS-CoV-2 infected primary human islets, with Spike protein detected in both insulin positive and glucagon positive cells at 24 hours (Figure 2F).\n\n5. Stem cell derived pancreatic endocrine cells transplanted under the kidney capsule retained ACE2 expression after two months and took up pseudo-entry virus in vivo, with luciferase detected in insulin positive and glucagon positive cells of the xenograft (Figure 2G to 2J). This establishes entry in vivo. It does not establish authentic virus replication in vivo, which was not tested.\n\n6. Authentic SARS-CoV-2 replicated in stem cell derived pancreatic endocrine cells in a dose-dependent manner by subgenomic N RNA, with Spike protein in both endocrine subtypes and read coverage across the viral genome (Figure 3A to 3D).\n\n7. Infected endocrine cells separated from mock by principal component analysis, with enrichment of viral infection pathways and of the insulin resistance pathway, and downregulation of calcium signaling, glucagon signaling and metabolic pathways (Figure 3E and 3F). Caspase 3 positive fractions rose in both alpha and beta cells after infection, and the authors interpret the loss of identity-associated programs as arising mainly from increased apoptosis rather than from dedifferentiation. That attribution is an interpretation supported by the caspase staining and apoptosis gene signature and is not established against the alternative.\n\n8. Chemokines including CCL2, CXCL5 and CXCL6 were upregulated in COVID-19 lung autopsy tissue relative to healthy donor lung, and comparable chemokine and cytokine induction was seen in infected endocrine cells (Figure 3G and 3H). The resemblance is a correlation between two systems and is presented as such.\n\n9. Adult primary hepatocyte organoids and cholangiocyte organoids supported both pseudo-entry virus and authentic SARS-CoV-2, with high subgenomic N RNA, Spike protein in a significant fraction of cells, and genome-wide read coverage (Figure 4A to 4H and Supplementary Figure 4).\n\n10. Infected hepatocyte organoids induced CXCL1, CXCL3, CXCL5, CXCL6 and CCL20 and downregulated hepatocyte metabolic markers CYP7A1, CYP2A6, CYP1A2 and CYP2D6, with enrichment of cytokine-cytokine receptor interaction, IL-17, chemokine, TNF and NF-kappa B signaling (Figure 4I to 4K). Cholangiocyte organoids induced CXCL1, CXCL2, CXCL3 and CCL2 with similar pathway enrichment (Figure 4L to 4N).\n\n11. Authentic virus subgenomic RNA was high in cardiomyocytes and dopaminergic neurons and low or absent in cortical neurons, microglia and macrophages, matching the pseudo-entry virus results (Supplementary Figure 4I to 4M).\n\n12. Several ACE2-positive cell types, including endothelium, macrophages and cortical neurons, showed little or no permissiveness. The authors read this as evidence that factors beyond ACE2, such as TMPRSS2, govern entry. The non-correspondence is the observation. The specific role of TMPRSS2 in these cell types was not tested here.", "Mechanistic model": "The study does not establish a mechanism for cell-type permissiveness and does not claim to. It establishes a map. Entry-competent cell types were identified with a Spike-pseudotyped particle and confirmed with authentic virus, and the central negative observation is that ACE2 protein detection does not predict permissiveness, which the authors take as pointing to additional required host factors, naming TMPRSS2 as an example. No host factor was manipulated, so the requirement is inferred rather than demonstrated.\n\nFor the cellular response, what the data support is that infection of pancreatic endocrine cells and of hepatic organoids induces a chemokine-dominated program while tissue-identity metabolic programs fall, and that apoptosis increases in infected endocrine cultures. Whether infection of beta cells contributes to the clinical association between COVID-19 and diabetes is raised as a question by the authors and is not addressed by these experiments. Whether the cell types identified here are major sites of infection in patients is explicitly left open in the paper's own limitations statement.", "Conceptual or technical advance": "The work supplies a standing panel of genetically matched, non-transformed human cell types and organoids in which tropism questions can be asked side by side, and it demonstrates the value of that design by producing a result a cancer line panel could not, namely a systematic mismatch between receptor expression and permissiveness across lineages. It also makes pancreatic endocrine cells and hepatic and biliary organoids available as infection models, and adds a xenograft configuration in which human endocrine tissue can be challenged in an animal. The authors propose the platform for antiviral drug screening, which is a stated prospect.", "Relationship to the broader research program": "The tenOever laboratory contribution here is the authentic SARS-CoV-2 infection work and the transcriptional comparison to human lung autopsy material, performed by Nilsson-Payant, Hoagland and tenOever according to the author contributions statement. The COVID-19 lung autopsy dataset and the chemokine-dominated response signature come from the laboratory's own work published as Blanco-Melo and colleagues 2020, which is cited for that comparison. Category 3 synthesis, visible when this paper is set beside that one and the laboratory's other 2020 SARS-CoV-2 work, is that the laboratory supplied a consistent transcriptional benchmark, an induced chemokine program with a muted interferon response, against which many collaborating groups calibrated their own model systems during that period.", "Related publications": "- Blanco-Melo and colleagues 2020. Methodological foundation and companion. Source of the COVID-19 lung autopsy transcriptional data and of the response signature used for comparison here.\n- Han and colleagues 2020, on candidate COVID-19 therapeutics using stem cell derived lung organoids. Companion. Overlapping author group applying the same platform logic to the airway, cited here for consistency of the inflammatory signature.\n- Zhao and colleagues 2020, on SARS-CoV-2 infection of liver ductal organoids. Companion. Independent report from another group consistent with the biliary findings here.", "Limitations and boundaries": "The authors state their own boundaries. Whether any of these cell types are major targets in patients cannot be settled without analysis of primary patient material, and the work focuses on entry and to some extent replication, leaving viral release and secondary spread across lineages unexamined. The platform is simplified relative to intact organ systems and contains no immune component. Beyond that, most permissiveness calls at the screening stage rest on a pseudotyped particle that reports entry only, in a vesicular stomatitis virus context rather than a coronavirus one. Infections were analyzed at a single time point of 24 hours at low multiplicity, with n equal to three biological replicates for the quantitative comparisons. Stem cell derivatives are developmentally immature, which the authors mitigate for the pancreas and liver by using adult primary material but not for cardiomyocytes, neurons or the myeloid lineages. The in vivo xenograft experiment used pseudo-entry virus, not authentic SARS-CoV-2, and an immunodeficient host. The comparison to COVID-19 patients uses lung autopsy tissue as the reference for responses measured in pancreas and liver models, since pancreatic samples from patients were not obtainable. The assignment of downregulated identity programs to apoptosis rather than to loss of cell identity rests on caspase 3 staining and a gene signature. One authentic virus isolate, USA-WA1/2020, was used throughout.", "Audience summaries": "### 25 words\n\nStem cell derived human tissues and adult organoids show that SARS-CoV-2 infects pancreatic insulin and glucagon cells, liver cells, heart muscle and dopaminergic neurons.\n\n### 75 words\n\nLaboratory study of SARS-CoV-2 largely relied on monkey and cancer cell lines. Differentiating human pluripotent stem cells into eight cell types and organoids allowed side-by-side comparison of which human tissues the virus can enter. Pancreatic alpha and beta cells, liver organoids, cardiomyocytes and dopaminergic neurons were permissive, while endothelial cells, macrophages, microglia and cortical neurons were not, even where the ACE2 receptor was present. Infected cells produced strong chemokine responses and lost tissue-specific metabolic gene expression.\n\n### 150 words\n\nCOVID-19 involves organs beyond the lung, and available infection models were monkey cells, mutation-bearing human cancer lines or ACE2 transgenic mice. Eight derivatives of human pluripotent stem cells spanning all three germ layers were stained for ACE2 and challenged with a Spike-pseudotyped vesicular stomatitis virus reporter, then with authentic SARS-CoV-2. Pancreatic alpha and beta cells, liver organoids, cardiomyocytes and dopaminergic neurons supported entry and replication, while endothelial cells, macrophages, microglia and cortical neurons did not, despite detectable ACE2 in several of them, indicating that additional host factors govern entry. Adult primary human islets and adult hepatocyte and cholangiocyte organoids confirmed the pancreatic and hepatic results, and endocrine cells grafted under the mouse kidney capsule took up pseudo-entry virus in vivo. Infected endocrine cells and liver organoids induced chemokines resembling those in COVID-19 lung autopsy tissue, while losing hormone signaling and cytochrome P450 metabolic gene expression." }, "discoveries": [], "relationships": [ { "from": "2020-yang-a-human-pluripotent-stem-cell-base", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2020-yang-a-human-pluripotent-stem-cell-base" } ], "canonical_url": "https://tenoeverlab.us/publications/2020-yang-a-human-pluripotent-stem-cell-base/", "controlled_vocabulary": { "pathogens": [ "sars-cov-2" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "immunofluorescence-microscopy", "pathway-enrichment-analysis", "pseudotyped-entry-reporter", "single-cell-rna-seq", "hpsc-directed-differentiation", "xenotransplantation", "organoid-culture" ] } }, { "id": "2021-daniloski-identification-of-required-host-fa", "slug": "2021-daniloski-identification-of-required-host-fa", "url": "/publications/2021-daniloski-identification-of-required-host-fa/", "title": "Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells", "authors": [ "Zharko Daniloski", "Tristan X. Jordan", "Hans-Hermann Wessels", "Daisy A. Hoagland", "Silva Kasela", "Mateusz Legut", "Silas Maniatis", "Eleni P. Mimitou", "Lu Lu", "Evan Geller", "Oded Danziger", "Brad R. Rosenberg", "Hemali Phatnani", "Peter Smibert", "Tuuli Lappalainen", "Benjamin R. tenOever", "Neville E. Sanjana" ], "author_count": 17, "first_author": "Zharko Daniloski", "senior_authors": [ "Benjamin R. tenOever", "Neville E. Sanjana" ], "corresponding_authors": [ "Benjamin R. tenOever", "Neville E. Sanjana" ], "tenoever_position": 16, "tenoever_role": "senior", "contribution_character": "co-led", "year": 2021, "journal": "Cell", "volume": "184", "issue": "1", "pages": "92-105.e16", "doi": "10.1016/j.cell.2020.10.030", "doi_url": "https://doi.org/10.1016/j.cell.2020.10.030", "pmid": "33147445", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/33147445/", "pmcid": "PMC7584921", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584921/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584921/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response", "programmable-virology" ], "themes": [ "in-vivo-screening-through-fitness", "host-factors-and-druggable-signaling" ], "pathogens": [ "SARS-CoV-2" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "human" ], "technologies": [ "genome-scale CRISPR-Cas9 knockout screening", "GeCKOv2 library", "amplicon sequencing", "robust rank aggregation", "ECCITE-seq single-cell CRISPR screening", "RNA interference", "small-molecule inhibitor panels", "flow cytometry", "immunofluorescence microscopy", "bulk RNA sequencing", "cholesterol quantification", "plaque assay", "quantitative RT-PCR" ], "biological_systems": [ "A549-ACE2 cells", "Huh7.5-ACE2 cells", "Caco-2 cells", "Calu-3 cells", "Vero E6 cells" ], "key_concepts": [ "host dependency factors", "forward genetic screening", "endosomal trafficking", "vacuolar ATPase", "Retromer complex", "Commander complex", "ARP2 and ARP3 complex", "class 3 PI3K", "cholesterol biosynthesis", "ACE2 surface availability", "druggable target identification" ], "keywords": [ "SARS-CoV-2", "CRISPR screen", "host factors", "RAB7A", "PIK3C3", "ATP6AP1", "NPC1", "CCDC22", "cholesterol", "ACE2", "amlodipine", "COVID-19 therapeutics" ], "one_sentence_contribution": "A genome-scale CRISPR loss-of-function screen in ACE2-expressing human alveolar epithelial cells ranks every protein-coding gene by the effect of its loss on SARS-CoV-2 infection, converging on endosomal machinery, and links several top hits to increased cholesterol biosynthesis and, for RAB7A, to intracellular sequestration of ACE2.", "summary_25": "Switching off each human gene in turn showed which ones SARS-CoV-2 needs. Most were endosome trafficking genes, and losing several of them raised cholesterol and blocked infection.", "summary_75": "To find the human genes SARS-CoV-2 depends on, every protein-coding gene was disabled in turn across a pool of lung cells, which were then infected and sequenced to see which knockouts survived. Known entry factors appeared near the top, alongside whole complexes that move and acidify endosomes. Disabling six of these genes raised cellular cholesterol, and a drug that raises cholesterol also blocked infection. Losing RAB7A trapped the ACE2 receptor inside cells instead of at the surface.", "summary_150": "A GeCKOv2 genome-scale CRISPR knockout screen in ACE2-expressing A549 cells, selected by survival of SARS-CoV-2 infection at two multiplicities, ranked all 19,050 targeted genes. ACE2 and cathepsin L scored highly, and the remaining top hits formed coherent endosomal complexes including thirteen vacuolar ATPase subunits, Retromer, Commander, ARP2 and ARP3, and class 3 PI3K components. Thirty hits were confirmed with independent guides, small interfering RNA, a second cell line, and inhibitors, with PIK3C3 antagonists among the most potent. Pooled single-cell CRISPR profiling showed that loss of ATP6AP1, ATP6V1A, CCDC22, NPC1, PIK3C3 or RAB7A converges on upregulated cholesterol biosynthesis, with measured cholesterol rising 10 to 50 percent, and amlodipine reproduced both the cholesterol increase and the antiviral effect. RAB7A loss reduced surface ACE2 and accumulated it in EEA1-positive vesicles in A549, Caco-2 and Calu-3 cells. How cholesterol blocks infection was not determined, and the work is confined to transformed human cell lines.", "citation": "Daniloski Z, Jordan TX, Wessels HH, Hoagland DA, Kasela S, Legut M, Maniatis S, Mimitou EP, Lu L, Geller E, Danziger O, Rosenberg BR, Phatnani H, Smibert P, Lappalainen T, tenOever BR, Sanjana NE. Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells. Cell. 2021. Volume 184, issue 1, pages 92-105.e16. DOI 10.1016/j.cell.2020.10.030. PMID 33147445. PMCID PMC7584921.", "sections": { "Citation": "Daniloski Z, Jordan TX, Wessels HH, Hoagland DA, Kasela S, Legut M, Maniatis S, Mimitou EP, Lu L, Geller E, Danziger O, Rosenberg BR, Phatnani H, Smibert P, Lappalainen T, tenOever BR, Sanjana NE. Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells. Cell. 2021. Volume 184, issue 1, pages 92-105.e16.\n\nDOI 10.1016/j.cell.2020.10.030. PMID 33147445. PMCID PMC7584921.", "One-sentence contribution": "A genome-scale CRISPR loss-of-function screen in ACE2-expressing human alveolar epithelial cells ranks every protein-coding gene by the effect of its loss on SARS-CoV-2 infection, converging on endosomal machinery, and links several top hits to increased cholesterol biosynthesis and, for RAB7A, to intracellular sequestration of ACE2.", "Executive summary": "Early in the COVID-19 pandemic the set of human genes known to be required for SARS-CoV-2 infection was limited to a handful, chiefly ACE2 and cathepsin L, while proteomic interaction maps had proposed hundreds of virus-host contacts without establishing which mattered for infection. This study applies pooled genome-scale CRISPR knockout screening to the problem, using the GeCKOv2 library across 19,050 genes in A549 lung epithelial cells engineered to express ACE2, then selecting for survival of SARS-CoV-2 infection at two multiplicities. Guide enrichment analysis placed ACE2 and cathepsin L among the top hits, supporting the readout, and the remaining top-ranked genes fell into coherent complexes dominated by endosomal biology, including thirteen vacuolar ATPase subunits, four Retromer members, four Commander members, four ARP2 and ARP3 complex members and three class 3 PI3K pathway genes. Thirty top genes were retested individually with fresh guides, by small interfering RNA, in a second cell line, and against a panel of 26 small molecules, with PIK3C3 inhibitors among the most effective. Single-cell CRISPR profiling of the top hits found that six of them, all in the endosomal entry pathway, share upregulation of cholesterol biosynthesis when lost, and cellular cholesterol rose accordingly. Amlodipine, which raises intracellular cholesterol, reduced infection. RAB7A loss reduced ACE2 at the cell surface and accumulated it in EEA1-positive vesicles, an effect reproduced in Caco-2 and Calu-3 cells expressing endogenous ACE2.", "Scientific context": "At the time of writing, remdesivir was the only approved antiviral and roughly thirty vaccines were in trials. Compound screens had been run against SARS-CoV-2, and affinity purification and proximity labelling proteomics had mapped hundreds of high-confidence virus-host protein interactions, but interaction is not requirement, and the paper states the gap plainly. There were no genome-wide studies directly identifying human genes required for viral infection, and knowledge of essential host genes was limited to ACE2 and cathepsin L. The known entry biology framed the expected answer space. Spike binds ACE2, is proteolytically activated by host proteases including furin, TMPRSS2 and cathepsin L in either the secretory pathway or the target cell during entry, and fusion then releases viral RNA into the cytoplasm. Whether the genes that matter beyond these steps would be lung specific, or broadly expressed, was also open.", "Central question": "Which human genes are required for SARS-CoV-2 infection of human lung epithelial cells, and by what cellular mechanisms does loss of the top-ranked genes confer resistance.", "Experimental strategy": "The screen exploits the fact that SARS-CoV-2 kills A549-ACE2 cells, turning survival into a selection. Cells transduced at low multiplicity with an all-in-one Cas9 and guide vector receive on average one guide, so a surviving cell can be attributed to a single gene knockout, and guide abundance measured by amplicon sequencing before and after infection reports which knockouts confer resistance. Running the selection at two multiplicities, 0.01 and 0.3, tests whether hits depend on viral dose. Three independent enrichment statistics were applied to the same data to check that the gene ranking is not an artefact of one method.\n\nBecause pooled screens carry false positives, the validation strategy was deliberately multi-modal. Thirty genes from the top 200 were retested in arrayed format with three guides per gene that were absent from the screening library, which controls for guide-specific off-target effects. Small interfering RNA knockdown provides a perturbation that does not cut DNA. Repeating a subset in a human liver line tests cell type dependence. A panel of 26 inhibitors against nine druggable hits tests whether the dependency is chemically accessible, with remdesivir as a positive control and paired viability measurement to distinguish antiviral effect from toxicity, and combining the top PIK3C3 inhibitors with PIK3C3 knockout tests on-target specificity by asking whether the drug still works when its target is gone.\n\nTo ask why loss of these genes protects rather than only that it does, the authors coupled a minipool of the same guides to ECCITE-seq, which reads out guide identity, transcriptome and surface protein in single cells. Doing this in a pooled rather than arrayed format puts all perturbations through the same handling, so a shared transcriptional signature across different knockouts is unlikely to reflect batch structure. Finally, flow cytometry and immunofluorescence for ACE2 across the perturbed panel test whether any hit acts by controlling receptor availability, with Caco-2 and Calu-3 cells included so the conclusion is not confined to cells overexpressing ACE2.", "Key findings": "1. The screen recovers known entry factors. ACE2 ranked 8th in the low multiplicity screen and 12th in the high multiplicity screen, and cathepsin L was also among the top scoring genes (Table S1). Library representation was maintained through selection, and substantial guide dropout followed infection as expected (Figure S1A, Figures 1C and 1D).\n\n2. Approximately 1,000 genes reached significance by robust rank aggregation (Figure S1B), three enrichment methods largely agreed (Figure S1C), and 27 of the top 50 genes were shared between the two multiplicities (Figure 1F), which the authors read as indicating that several host dependencies function independently of viral dose.\n\n3. Top hits cluster into coherent complexes centred on endosomal biology, including 13 vacuolar ATPase proton pump subunits, four Retromer members, four Commander members, four ARP2 and ARP3 complex members, three class 3 PI3K pathway genes, ER to Golgi trafficking genes and two transcriptional modulators (Figures 2 and 3A). Gene set enrichment identified endosome processing, transport and acidification categories as significant (Figure 3B).\n\n4. Nearly all top hits are broadly expressed across 12 human tissues in GTEx, with ACE2 the exception in showing tissue-restricted expression enriched in testis, small intestine, kidney and heart (Figure 3C). The authors interpret the breadth as implying that these mechanisms may operate independent of cell or tissue type, which is an inference from expression rather than a tested claim.\n\n5. Twenty-two of the top 50 low multiplicity genes had been reported as direct interactors with SARS-CoV-2 proteins in published proteomics, a significant enrichment over random gene sets (Figure 3D). ATP6AP1 interacts with nsp6, ATP6V1A with the membrane protein and RAB7A with nsp7.\n\n6. Comparison with prior screens for Zika virus and pandemic H1N1 influenza showed greater similarity in enriched gene ontology categories between SARS-CoV-2 and Zika virus, with vacuolar ATPase subunits enriched in all three (Figure 3E, Figure S2E).\n\n7. Arrayed validation confirmed the hits. All 30 Cas9-perturbed lines showed reduced infected cell percentage, up to tenfold, at 36 hours after infection (Figures 4A and 4B), with a significant negative correlation between arrayed infection percentage and screen fold change (Figure 4C). Viral load was reduced across a full growth curve at 5, 10, 24 and 48 hours (Figure S3B). All eight genes tested in Huh7.5-ACE2 cells reduced infection (Figure S3C), and small interfering RNA knockdown reproduced the effect (Figure S3D).\n\n8. Seven of 26 inhibitors reduced viral load more than 100-fold, four of them targeting PIK3C3 (Figure 4E). Autophinib and ALLN exceeded 1000-fold. Combining PIK3C3 inhibitors with PIK3C3 knockout indicated that Compound-19, PIK-III and autophinib act on target while SAR405 gave greater inhibition in knockout cells, which the authors read as possible off-target activity. Panobinostat and pracinostat reduced viability by more than half, so their apparent antiviral readouts were flagged as unreliable (Figure 4F).\n\n9. Six endosomal entry pathway genes share a transcriptional response when lost. Single-cell profiling across 18,853 singly perturbed cells found that loss of ATP6AP1, ATP6V1A, CCDC22, NPC1, PIK3C3 or RAB7A upregulated lipid and cholesterol homeostasis pathways (Figures 5B, 5C and S5B), and measured cholesterol rose by 10 to 50 percent depending on the perturbation (Figure 5D). The authors note that only 11 of 30 target genes produced a detectable transcriptomic shift, and suggest the remainder produce subtler changes.\n\n10. Raising cholesterol pharmacologically reduces infection. Amlodipine increased cholesterol in A549-ACE2 cells and reduced infection by quantitative RT-PCR, plaque assay and viral read fraction, with modest impact on viability, and its transcriptional profile resembled the CRISPR perturbations with cholesterol biosynthesis as the top upregulated pathway (Figures S5C to S5I). The interpretation offered, that these perturbations counteract virus-mediated suppression of cholesterol synthesis, draws on the authors' separate work and is presented as a possibility rather than as demonstrated here.\n\n11. RAB7A loss relocates ACE2. Surface ACE2 measured by flow cytometry was significantly reduced in RAB7A knockout A549-ACE2 cells (Figures 6A and 6B), with Rab7a depletion confirmed by western blot. Immunofluorescence showed ACE2 accumulating in vesicle-like structures in about 35 percent of RAB7A knockout cells compared with predominantly membrane localisation in controls (Figures 6C and 6D), and these vesicles colocalised often with EEA1 and less often with LysoTracker (Figure 6E). The effect held in Caco-2 and Calu-3 cells expressing endogenous ACE2 (Figures 6F to 6I).", "Mechanistic model": "The study is a dependency map first and a mechanism study second, and it does not establish a single mechanism for the class of hits it identifies. The authors state in their own limitations section that the precise mechanism by which changes in cholesterol disrupt viral infection remains to be elucidated.\n\nWhat the data constrain is the following. A large fraction of the genes whose loss confers resistance operate in endosomal maturation, acidification, sorting and recycling, which is consistent with a virus that depends on endosomal entry and cathepsin L cleavage. Within that class, six genes converge on a shared downstream consequence, elevated cholesterol biosynthesis, and an independent pharmacological route to the same elevation also reduces infection, which makes cholesterol a plausible common effector rather than an incidental correlate. The authors propose that these perturbations counter a virus-driven suppression of cholesterol synthesis reported in their separate work, and they note precedents for lipid composition affecting virion maturation and infectivity in hepatitis C and influenza A. Neither the causal ordering between cholesterol change and infection block, nor the step of the viral life cycle affected, is established here.\n\nFor RAB7A the data support a more specific model. Loss of Rab7a reduces ACE2 at the plasma membrane and accumulates it in early endosomal compartments, which would limit viral attachment. The authors note that this cannot be the whole story, because Rab7a interacts with nsp7 and nsp7 is not present in the incoming virion, implying an additional post-entry role, and they point to RAB7A being the top performer in arrayed validation and showing both altered cholesterol and ACE2 sequestration as consistent with more than one contributing pathway. That multiplicity is offered as a possibility, not demonstrated.", "Conceptual or technical advance": "The screen converts the question of which host genes matter for SARS-CoV-2 from a list of candidates into a genome-scale quantitative ranking with an effect size for every protein-coding gene, which is the resource the paper positions as its main output. Coupling a minipool of validated guides to single-cell transcriptomic and surface protein readout makes it possible to ask not only which genes are required but whether mechanistically distinct knockouts converge on a shared cellular state, and that is how the cholesterol link was found rather than assumed. The authors also frame a methodological argument, that starting from forward genetics and moving to inhibitors yields therapeutic candidates whose mechanism of action is known from the outset, in contrast to compound-first screening where mechanism must be reconstructed afterwards.", "Relationship to the broader research program": "The virological work, including all BSL3 infections, sits with the tenOever laboratory while the screening platform and analysis sit with the Sanjana laboratory, and the two are joint corresponding authors with Sanjana as lead contact. The A549-ACE2 line used throughout, made by Danziger and Rosenberg, is the same engineered system used across the tenOever laboratory's SARS-CoV-2 work. The cholesterol connection draws on Hoagland and colleagues from the same collaboration network, which reported that SARS-CoV-2 downregulates cholesterol synthesis and that compounds raising it are antiviral.\n\nCategory 3 synthesis. Comparison of this screen with the laboratory's transcriptional and epigenetic work on SARS-CoV-2 would bear on whether host dependencies and host response pathways overlap, and the paper's own comparison with Zika virus and influenza screens raises the question of shared versus virus-specific dependency architecture. Both are cross-paper questions and neither is settled by this study alone.", "Related publications": "- Daniloski and colleagues, 2021, eLife, companion. The same first author and the same two corresponding laboratories, using the related A549-ACE2 and Caco-2 systems, on the Spike D614G substitution.\n- Hoagland and colleagues, 2020, companion. Cited here as the independent survey of more than 20,000 candidate treatments that identified induction of cholesterol biosynthesis as a mechanism of viral inhibition, and as the source of the claim that SARS-CoV-2 downregulates cholesterol synthesis.\n- Gordon and colleagues, 2020, predecessor. The SARS-CoV-2 protein interaction map against which top-ranked screen hits were cross-referenced.\n- Zhu and colleagues, 2020, companion. An independent genome-scale CRISPR screen in ACE2-overexpressing A549 cells with a different library, reported here as giving substantially overlapping top-ranked genes.\n- Wei and colleagues, 2020, and Heaton and colleagues, 2020, companion. Contemporaneous loss-of-function screens for SARS-CoV-2 host factors, with the African green monkey cell screen of Wei and colleagues overlapping only at ACE2 and cathepsin L.\n- Sanjana and colleagues, 2014, methodological foundation. Source of the GeCKOv2 library used for the screen.\n- Mimitou and colleagues, 2019, methodological foundation. Source of the ECCITE-seq method used for the single-cell CRISPR readout.", "Limitations and boundaries": "The authors provide their own limitations section and it is followed here. The screen was performed in A549 cells overexpressing ACE2, so transcriptional regulators of endogenous ACE2 would not be recovered, and A549 is a lung adenocarcinoma line rather than primary airway tissue. Tissue-specific host factors relevant to the other organs affected in COVID-19 are not addressed. The mechanism by which increased cholesterol blocks infection is not established. Integration with human genetic variants associated with COVID-19 risk is proposed as future work rather than performed.\n\nBeyond the authors' list, the selection is survival-based, so the screen reports resistance to virus-induced death rather than blockade of any defined step, and genes whose loss is itself lethal or strongly deleterious cannot score. Guide dropout after infection was substantial, which reduces effective library complexity in the selected population. Validation of protein loss by western blot was performed for a subset of genes only, and knockout lines are polyclonal, so residual protein is expected, a point the authors raise specifically in interpreting the PIK3C3 inhibitor specificity test. Inhibitors were tested at a single concentration of 10 micromolar with viability assessed at 36 hours, and two compounds were excluded on viability grounds. Only 11 of 30 perturbations produced detectable transcriptomic shifts in the single-cell data, so the cholesterol signature is established for a subset rather than for all validated hits. Comparison with the monkey cell screen of Wei and colleagues overlapped only at two genes, which the authors attribute to technical or biological differences without resolving which. All work uses a single early isolate, USA-WA1/2020, and there is no animal model or primary tissue component.", "Audience summaries": "### 25 words\n\nSwitching off each human gene in turn showed which ones SARS-CoV-2 needs. Most were endosome trafficking genes, and losing several of them raised cholesterol and blocked infection.\n\n### 75 words\n\nTo find the human genes SARS-CoV-2 depends on, every protein-coding gene was disabled in turn across a pool of lung cells, which were then infected and sequenced to see which knockouts survived. Known entry factors appeared near the top, alongside whole complexes that move and acidify endosomes. Disabling six of these genes raised cellular cholesterol, and a drug that raises cholesterol also blocked infection. Losing RAB7A trapped the ACE2 receptor inside cells instead of at the surface.\n\n### 150 words\n\nA GeCKOv2 genome-scale CRISPR knockout screen in ACE2-expressing A549 cells, selected by survival of SARS-CoV-2 infection at two multiplicities, ranked all 19,050 targeted genes. ACE2 and cathepsin L scored highly, and the remaining top hits formed coherent endosomal complexes including thirteen vacuolar ATPase subunits, Retromer, Commander, ARP2 and ARP3, and class 3 PI3K components. Thirty hits were confirmed with independent guides, small interfering RNA, a second cell line, and inhibitors, with PIK3C3 antagonists among the most potent. Pooled single-cell CRISPR profiling showed that loss of ATP6AP1, ATP6V1A, CCDC22, NPC1, PIK3C3 or RAB7A converges on upregulated cholesterol biosynthesis, with measured cholesterol rising 10 to 50 percent, and amlodipine reproduced both the cholesterol increase and the antiviral effect. RAB7A loss reduced surface ACE2 and accumulated it in EEA1-positive vesicles in A549, Caco-2 and Calu-3 cells. How cholesterol blocks infection was not determined, and the work is confined to transformed human cell lines." }, "discoveries": [ "claim-10" ], "relationships": [ { "from": "2021-daniloski-identification-of-required-host-fa", "to": "2021-daniloski-the-spike-d614g-mutation-increases", "relationship": "companion", "evidence": "stated in the Related publications section of 2021-daniloski-identification-of-required-host-fa" }, { "from": "2021-daniloski-identification-of-required-host-fa", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "companion", "evidence": "stated in the Related publications section of 2021-daniloski-identification-of-required-host-fa" }, { "from": "2021-daniloski-the-spike-d614g-mutation-increases", "to": "2021-daniloski-identification-of-required-host-fa", "relationship": "companion", "evidence": "stated in the Related publications section of 2021-daniloski-the-spike-d614g-mutation-increases" } ], "canonical_url": "https://tenoeverlab.us/publications/2021-daniloski-identification-of-required-host-fa/", "controlled_vocabulary": { "pathogens": [ "sars-cov-2" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "immunofluorescence-microscopy", "sirna-knockdown", "flow-cytometry", "small-molecule-inhibitor-profiling", "amplicon-sequencing", "genome-wide-crispr-screen", "metabolite-quantification", "single-cell-crispr-screen" ] } }, { "id": "2021-daniloski-the-spike-d614g-mutation-increases", "slug": "2021-daniloski-the-spike-d614g-mutation-increases", "url": "/publications/2021-daniloski-the-spike-d614g-mutation-increases/", "title": "The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types", "authors": [ "Zharko Daniloski", "Tristan X Jordan", "Juliana K Ilmain", "Xinyi Guo", "Gira Bhabha", "Benjamin R tenOever", "Neville E Sanjana" ], "author_count": 7, "first_author": "Zharko Daniloski", "senior_authors": [ "Benjamin R tenOever", "Neville E Sanjana" ], "corresponding_authors": [ "Benjamin R tenOever", "Neville E Sanjana" ], "tenoever_position": 6, "tenoever_role": "senior", "contribution_character": "co-led", "year": 2021, "journal": "eLife", "volume": "10", "issue": null, "pages": "e65365", "doi": "10.7554/elife.65365", "doi_url": "https://doi.org/10.7554/elife.65365", "pmid": "33570490", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/33570490/", "pmcid": "PMC7891930", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891930/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891930/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "tropism-and-permissive-tissues" ], "pathogens": [ "SARS-CoV-2" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "human" ], "technologies": [ "site-directed mutagenesis", "lentiviral pseudotyping", "flow cytometry", "bio-layer interferometry", "western blotting", "trans-complementation infection assay", "imaging cytometry", "quantitative PCR", "MHC epitope prediction" ], "biological_systems": [ "A549-ACE2 cells", "Huh7.5-ACE2 cells", "Caco-2 cells", "Calu-3 cells", "HEK293T cells", "HEK293FT cells" ], "key_concepts": [ "Spike D614G", "viral entry efficiency", "pseudotyped particle systems", "ACE2 binding kinetics", "proteolytic processing of Spike", "linkage disequilibrium with ORF1b P314L", "isogenic variant comparison", "vaccine antigen sequence choice" ], "keywords": [ "SARS-CoV-2", "Spike", "D614G", "pseudotyped lentivirus", "ACE2", "bio-layer interferometry", "Spike cleavage", "trans-complementation", "viral transduction", "COVID-19 variant" ], "one_sentence_contribution": "Isolating the SARS-CoV-2 Spike D614G substitution from the ORF1b P314L variant it travels with, through pseudotyped lentiviral particles and a trans-complementation assay with replication-competent virus, shows that the single Spike change raises entry efficiency across human lung, liver and colon cell lines without altering S1 affinity for ACE2.", "summary_25": "A single amino acid change in the SARS-CoV-2 Spike protein, tested in isolation, made the virus enter human lung, liver and colon cells more efficiently.", "summary_75": "Early in the pandemic a Spike variant called D614G took over globally, but it always travelled with a second mutation, so its own effect was unclear. Building the change into Spike on its own showed that particles carrying it entered four types of human cell more efficiently, and the same held for live virus supplied with the variant. Receptor binding by the outer Spike fragment was unchanged, but the variant Spike was cut less readily by host proteases.", "summary_150": "The SARS-CoV-2 Spike D614G substitution sits in linkage disequilibrium with an ORF1b P314L variant, so its phenotype cannot be read from sequence databases. Introducing it alone into a codon-optimised Spike, EGFP lentiviral particles pseudotyped with G614 transduced Caco-2 and Calu-3 cells 1.3 to 2.4-fold better and A549-ACE2 and Huh7.5-ACE2 cells 1.5 to 7.7-fold better than D614 particles, across four doses, with particle RNA content differing by only about 7 percent in the opposite direction. Bio-layer interferometry with purified S1 subunit showed comparable ACE2 binding for both variants, while western blotting showed roughly 2.5-fold less cleavage of G614 Spike in transfected cells and roughly 1.4-fold less on virions, with no difference in Spike incorporation. A trans-complementation assay, supplying ACE2 and one Spike variant to cells before infection with replication-competent virus, reproduced the advantage at 12, 18 and 24 hours. The cleavage explanation is proposed, not established.", "citation": "Daniloski Z, Jordan TX, Ilmain JK, Guo X, Bhabha G, tenOever BR, Sanjana NE. The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types. eLife. 2021. Volume 10, article e65365. DOI 10.7554/elife.65365. PMID 33570490. PMCID PMC7891930.", "sections": { "Citation": "Daniloski Z, Jordan TX, Ilmain JK, Guo X, Bhabha G, tenOever BR, Sanjana NE. The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types. eLife. 2021. Volume 10, article e65365.\n\nDOI 10.7554/elife.65365. PMID 33570490. PMCID PMC7891930.", "One-sentence contribution": "Isolating the SARS-CoV-2 Spike D614G substitution from the ORF1b P314L variant it travels with, through pseudotyped lentiviral particles and a trans-complementation assay with replication-competent virus, shows that the single Spike change raises entry efficiency across human lung, liver and colon cell lines without altering S1 affinity for ACE2.", "Executive summary": "By mid-2020 a SARS-CoV-2 Spike variant carrying glycine rather than aspartate at position 614 had become dominant worldwide, but because that change is in linkage disequilibrium with an ORF1b P314L variant within the same clade, population genetics could not attribute any phenotype to Spike alone, and groups disagreed about whether the rise reflected selection or a founder effect. This study separates the two by building the substitution into a human-codon-optimised Spike coding sequence and testing it in isolation. EGFP lentiviral particles pseudotyped with either D614 or G614 Spike were used to transduce four human cell lines, two with endogenous ACE2 and two engineered to overexpress it, across four viral doses. G614 particles transduced more cells at every dose in every line, by 1.3 to 2.4-fold in the endogenous ACE2 lines and 1.5 to 7.7-fold in the overexpressing lines, with only a small opposing difference in particle titre. Bio-layer interferometry found comparable binding of D614 and G614 Spike S1 subunit to immobilised human ACE2, so affinity of that subunit does not account for the difference. Western blotting showed the G614 Spike to be more resistant to proteolytic cleavage both in transfected cells and on virions, without a difference in Spike incorporation. A trans-complementation assay, co-transfecting ACE2 and one Spike variant into HEK293T cells before infection with replication-competent virus, reproduced the increase.", "Scientific context": "The first sequenced SARS-CoV-2 isolate and most January and February 2020 sequences carried aspartate at Spike position 614. From February onward the glycine variant rose, and in a survey of 22,103 GISAID genomes in early June 2020 the authors found roughly 72 percent carrying G614. Whether this reflected a functional advantage was contested, with some groups proposing increased transmissibility under positive selection and others arguing that the available evidence did not support selection. The interpretive obstacle the paper identifies is specific. In the A2a clade the Spike change is in linkage disequilibrium with an ORF1b P314L variant, so any association measured in sequence databases cannot be assigned to Spike. Two clinical datasets, from Sheffield and from the University of Washington, had reported roughly threefold higher viral RNA by diagnostic PCR in patients carrying G614. Separately, most SARS-CoV-2 vaccines then in development were built on the original D614 Spike sequence, which made the functional status of the substitution relevant beyond virology.", "Central question": "Does the Spike D614G substitution by itself, separated from the linked ORF1b P314L variant, alter the efficiency with which SARS-CoV-2 enters human cells, and if so through what step.", "Experimental strategy": "The design is built around the linkage problem. Because natural isolates cannot supply an isogenic comparison, the substitution is introduced by site-directed mutagenesis into a cloned Spike coding sequence, so the only difference between the two arms is the single codon.\n\nPseudotyped lentiviral particles then isolate entry from every other stage of the viral life cycle. Because the particles carry an EGFP reporter and cannot replicate, the flow cytometry readout at three days counts successful entry and integration events rather than rounds of spread, so a difference cannot be attributed to downstream replication or to the linked polymerase variant. Four cell lines spanning lung, liver and colon, two relying on endogenous ACE2 and two overexpressing it, test whether any effect depends on receptor abundance or tissue of origin. Four viral volumes per line guard against a dose-specific artefact, a no-pseudotype arm establishes the background, and quantitative PCR of particle RNA checks that the two preparations were comparably titred.\n\nMechanism is then approached at the two steps the Spike protein performs. Bio-layer interferometry with purified S1 subunit and immobilised human ACE2 asks whether receptor affinity differs. Western blotting with a C-terminal C9 tag, which allows full-length Spike and the S2 and S2 prime fragments to be resolved on the same blot, asks whether proteolytic processing differs, and doing this both in transfected cells and on purified virions distinguishes cleavage during production from the state of the delivered particle. Normalising total Spike to the p24 capsid protein separately tests whether the variants differ in how much Spike each particle carries, which is an alternative explanation for a transduction difference.\n\nFinally, because pseudotypes may not report the behaviour of Spike in its native context, a trans-complementation assay tests the substitution against replication-competent virus without requiring a reverse genetics system. Co-transfecting ACE2 together with one Spike variant into cells that are otherwise poorly infectable means that only transfected cells are readily infected, so the supplied Spike variant shapes the outcome. Low multiplicities and early time points were used because the authors reasoned that higher doses and longer infections would mask the contribution of the transfected Spike.", "Key findings": "1. The G614 variant rose to roughly 72 percent of 22,103 surveyed genomes by early June 2020 (Figure 1a). Across 56 countries, G614 prevalence showed a small but significant positive correlation with case-fatality rate (r = 0.29, p = 0.04) (Figure 1b), which the authors present as a smaller effect than a previously reported correlation computed on a roughly tenfold smaller dataset. This is an ecological correlation and is not offered as evidence of causation.\n\n2. Reanalysis of published Sheffield and University of Washington patient PCR data showed a roughly 5 threshold cycle offset between the two sites, attributable to methodological differences, but a consistent difference between variants within each site of 1.6 and 1.8 threshold cycles (Figure 1c and 1d). The authors read the consistency as suggesting a biological difference.\n\n3. G614-pseudotyped particles transduce human cells more efficiently than D614 particles, at every dose in all four lines (Figure 2c). Fold increases were 1.4 to 1.9 in Calu-3, 1.3 to 2.4 in Caco-2, 1.8 to 4.6 in A549-ACE2 and 1.5 to 7.7 in Huh7.5-ACE2 (Figure 2d). Particles lacking an attachment protein gave negligible transduction.\n\n4. The difference is not a titre artefact. Viral RNA measured with two independent primer sets differed by about 7 percent in favour of D614, which if anything means the transduction advantage of G614 is slightly underestimated (Figure 2 supplement 2).\n\n5. ACE2 binding by the S1 subunit is comparable between variants. Bio-layer interferometry fit best to a 2 to 1 heterogeneous binding model, giving KD1 of 8.45 nanomolar and KD2 of 127 nanomolar for D614 and KD1 of 18.0 nanomolar and KD2 of 92.7 nanomolar for G614 (Figure 3b, 3c and Table 1). The authors read this as indicating the transduction phenotype is independent of S1 affinity for ACE2.\n\n6. G614 Spike is more resistant to proteolytic cleavage. In transfected HEK293FT cells the ratio of cleaved to full-length Spike was roughly 2.5-fold lower for G614 (Figure 3d to 3f), and on purified pseudotyped particles it was roughly 1.4-fold lower (Figure 3g to 3i). No significant difference was found in Spike incorporation into particles when total Spike was normalised to p24 (Figure 3 supplement 1).\n\n7. The substitution alters predicted MHC binding for at least one epitope, with predicted affinity for HLA-A 02 01 shifting from 58 to 221 nanomolar (Figure 3 supplement 2). This is computational prediction, not measured immunogenicity.\n\n8. The effect holds for replication-competent virus. In the trans-complementation assay, cells supplied with G614 Spike showed significantly more infection than those supplied with D614 Spike at 12, 18 and 24 hours after infection, at multiplicities of both 0.01 and 0.1 (Figure 4b to 4d). Untransfected cells showed minimal infection, under 1 percent in most cases.", "Mechanistic model": "The study does not establish a definitive mechanism, and the authors describe the cleavage result as suggesting a possible mechanism rather than demonstrating one.\n\nWhat the data constrain is the following. The entry advantage of G614 is a property of Spike alone, since it is reproduced when the substitution is the only difference between two otherwise identical constructs and when the ORF1b variant is absent entirely. It does not arise from higher affinity of the S1 subunit for ACE2 under the conditions measured, and it does not arise from more Spike per particle. It is accompanied by less proteolytic cleavage of Spike, both during production in cells and on the assembled particle.\n\nThe model the authors propose is that reduced cleavage leaves a greater fraction of functional Spike, uncleaved and retaining the receptor-binding domain, on each newly assembled virion, which would raise the probability of productive attachment. They are careful about the boundaries of this proposal. They note that other groups working with full-length Spike trimers reported that G614 shifts the trimer toward an ACE2-binding competent conformation, which they suggest means trimers behave differently from the S1 monomers measured here, so the absence of an affinity difference in their assay does not exclude a conformational contribution. They also note that two studies using isogenic replication-competent viruses found no difference in Spike cleavage or incorporation, and that reported differences across the field may partly reflect the choice of pseudoviral system, producer cell handling and transfection reagents, which can affect protease activity. They state that future work is required to establish whether G614 Spike is processed differently on live virus, and cite a report that G614 may additionally be processed by elastase-2.", "Conceptual or technical advance": "The trans-complementation assay is the methodological contribution. It allows a Spike variant to be tested against replication-competent SARS-CoV-2 in an isogenic fashion without a reverse genetics system, by supplying both the receptor and the Spike variant to cells that are otherwise poorly permissive, which puts such experiments within reach of laboratories that cannot build recombinant coronaviruses. The concordance the authors report between the pseudotyped lentiviral result and the replication-competent result also supports the broader use of pseudotypes for studying Spike variants, which they note explicitly as a practical implication. Conceptually, the work separates a phenotype from a linked genetic marker, converting a contested population-genetic observation into a measurable property of one amino acid.", "Relationship to the broader research program": "This study shares its first author, its two corresponding laboratories and much of its cell line panel, including A549-ACE2, Huh7.5-ACE2, Caco-2 and Calu-3, with the genome-scale CRISPR screen published by the same collaboration in Cell in 2021. The tenOever laboratory contributed the work with replication-competent virus while the Sanjana laboratory contributed the pseudotype and genetic engineering components, and the two are joint corresponding authors.\n\nCategory 3 synthesis. Read alongside the CRISPR screen, which found that RAB7A loss reduces surface ACE2 and that entry depends on endosomal machinery, this paper addresses the complementary side of the same entry step from the viral rather than the host direction. Whether receptor availability and Spike cleavage state interact is a question the two papers raise together and neither answers.", "Related publications": "- Daniloski and colleagues, 2021, Cell, companion. Same first author and same two corresponding laboratories, sharing the engineered A549-ACE2 and Huh7.5-ACE2 lines and the Caco-2 and Calu-3 panel, addressing host requirements for entry where this paper addresses a viral determinant of it.\n- Korber and colleagues, 2020, predecessor. Source of the Sheffield patient PCR data reanalysed here and of the argument about G614 spread.\n- Wagner and colleagues, 2020, predecessor. Source of the University of Washington patient PCR data reanalysed here.\n- Yurkovetskiy and colleagues, 2020, companion. Reported that G614 shifts full-length Spike trimers toward an ACE2-binding competent conformation, a result the authors discuss as complementary to and possibly reconciling with their monomer binding data.\n- Plante and colleagues, 2020, and Hou and colleagues, 2020, companion. Isogenic replication-competent SARS-CoV-2 comparisons that found no difference in Spike cleavage or incorporation, discussed here as discrepant with the pseudotype result.\n- Shang and colleagues, 2020, methodological foundation. Source of the human-codon-optimised Spike coding sequence used for mutagenesis.", "Limitations and boundaries": "All experiments are in transformed human cell lines, two of which overexpress ACE2 at non-physiological levels, and the largest fold effects were seen in those overexpressing lines, so the magnitude of the effect is system dependent. There is no primary airway tissue, organoid or animal component, and nothing in the study addresses transmissibility between hosts or clinical severity, which the authors note remains uncertain and uncorrelated with variant status in the two clinical studies they cite. The binding measurement used purified S1 subunit monomers rather than full-length trimers, and the authors themselves identify this as the likely reason their result differs from a study using trimers, so the absence of an affinity difference is bounded to that construct. The cleavage measurement is made on transfected cells and on lentiviral particles, not on authentic SARS-CoV-2 virions, and the authors state that whether the same holds on live virus requires further work, noting that two isogenic virus studies found no such difference. The trans-complementation assay depends on transient overexpression of both ACE2 and Spike, so the ratio of supplied Spike to virus-encoded Spike is not controlled, and readouts are restricted to 12 to 24 hours at low multiplicity because later times were expected to mask the effect. The correlation between G614 prevalence and case-fatality rate is ecological, drawn across countries, and subject to every confounder that applies to such comparisons. The MHC binding result is prediction from an algorithm with no experimental immunology. Only one isolate, USA-WA1/2020, was used for the live virus work, and the study predates the variants of concern that followed.", "Audience summaries": "### 25 words\n\nA single amino acid change in the SARS-CoV-2 Spike protein, tested in isolation, made the virus enter human lung, liver and colon cells more efficiently.\n\n### 75 words\n\nEarly in the pandemic a Spike variant called D614G took over globally, but it always travelled with a second mutation, so its own effect was unclear. Building the change into Spike on its own showed that particles carrying it entered four types of human cell more efficiently, and the same held for live virus supplied with the variant. Receptor binding by the outer Spike fragment was unchanged, but the variant Spike was cut less readily by host proteases.\n\n### 150 words\n\nThe SARS-CoV-2 Spike D614G substitution sits in linkage disequilibrium with an ORF1b P314L variant, so its phenotype cannot be read from sequence databases. Introducing it alone into a codon-optimised Spike, EGFP lentiviral particles pseudotyped with G614 transduced Caco-2 and Calu-3 cells 1.3 to 2.4-fold better and A549-ACE2 and Huh7.5-ACE2 cells 1.5 to 7.7-fold better than D614 particles, across four doses, with particle RNA content differing by only about 7 percent in the opposite direction. Bio-layer interferometry with purified S1 subunit showed comparable ACE2 binding for both variants, while western blotting showed roughly 2.5-fold less cleavage of G614 Spike in transfected cells and roughly 1.4-fold less on virions, with no difference in Spike incorporation. A trans-complementation assay, supplying ACE2 and one Spike variant to cells before infection with replication-competent virus, reproduced the advantage at 12, 18 and 24 hours. The cleavage explanation is proposed, not established." }, "discoveries": [], "relationships": [ { "from": "2021-daniloski-identification-of-required-host-fa", "to": "2021-daniloski-the-spike-d614g-mutation-increases", "relationship": "companion", "evidence": "stated in the Related publications section of 2021-daniloski-identification-of-required-host-fa" }, { "from": "2021-daniloski-the-spike-d614g-mutation-increases", "to": "2021-daniloski-identification-of-required-host-fa", "relationship": "companion", "evidence": "stated in the Related publications section of 2021-daniloski-the-spike-d614g-mutation-increases" } ], "canonical_url": "https://tenoeverlab.us/publications/2021-daniloski-the-spike-d614g-mutation-increases/", "controlled_vocabulary": { "pathogens": [ "sars-cov-2" ], "technologies": [ "rt-qpcr", "flow-cytometry", "immunoblotting", "site-directed-mutagenesis", "pseudotyped-entry-reporter", "biolayer-interferometry", "epitope-prediction", "trans-complementation-assay" ] } }, { "id": "2021-eriksen-sars-cov-2-infects-human-adult-don", "slug": "2021-eriksen-sars-cov-2-infects-human-adult-don", "url": "/publications/2021-eriksen-sars-cov-2-infects-human-adult-don/", "title": "SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium", "authors": [ "Anne Z. Eriksen", "Rasmus Møller", "Bar Makovoz", "Skyler A. Uhl", "Benjamin R. tenOever", "Timothy A. Blenkinsop" ], "author_count": 6, "first_author": "Anne Z. Eriksen", "senior_authors": [ "Benjamin R. tenOever", "Timothy A. Blenkinsop" ], "corresponding_authors": [ "Benjamin R. tenOever", "Timothy A. Blenkinsop" ], "tenoever_position": 5, "tenoever_role": "senior", "contribution_character": "co-led", "year": 2021, "journal": "Cell Stem Cell", "volume": "28", "issue": "7", "pages": "1205-1220.e7", "doi": "10.1016/j.stem.2021.04.028", "doi_url": "https://doi.org/10.1016/j.stem.2021.04.028", "pmid": "34022129", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/34022129/", "pmcid": "PMC8126605", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126605/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126605/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "tropism-and-permissive-tissues" ], "pathogens": [ "SARS-CoV-2" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "human" ], "technologies": [ "immunofluorescence", "bulk RNA sequencing", "single-cell RNA sequencing", "quantitative RT-PCR", "plaque assay", "protease inhibition with TPCK", "gene set enrichment with Enrichr" ], "biological_systems": [ "post-mortem human ocular surface tissue", "primary adult human cornea limbus sclera iris retinal pigment epithelium and choroid cultures", "human embryonic stem cell derived SEAM whole-eye cultures" ], "key_concepts": [ "ocular route of SARS-CoV-2 entry", "limbal stem cell niche", "ACE2 and TMPRSS2 expression", "TMPRSS4 as an alternative protease", "NF-kB-driven chemokine response", "attenuated type I and III interferon signaling", "bystander versus infected cell responses", "SEAM whole-eye organoid model" ], "keywords": [ "SARS-CoV-2", "eye", "limbus", "cornea", "ACE2", "TMPRSS2", "organoid", "single-cell RNA sequencing", "interferon", "NF-kB" ], "one_sentence_contribution": "Human ocular surface tissue carries SARS-CoV-2 entry machinery and supports productive replication, with the limbus most permissive in both adult donor cells and stem cell derived whole-eye cultures, where infection drives NF-kB chemokine induction and blunted interferon signaling.", "summary_25": "Human ocular surface cells, especially at the limbus, carry SARS-CoV-2 entry factors and support productive infection, both in donor tissue and in stem cell derived eye cultures.", "summary_75": "Viral RNA had been found in tears, but it was unclear whether eye cells are actually infected. Antigen was present in ocular surface tissue from three deceased COVID-19 patients, and cells cultured from donor eyes supported infection, most strongly in the limbus, the ring of tissue holding the corneal stem cell niche. Stem cell derived eye cultures reproduced this and released infectious virus. Infected cells mounted a strong inflammatory response with weak interferon signaling.", "summary_150": "Combining post-mortem tissue from SARS-CoV-2 positive donors, primary cultures from six regions of healthy cadaver globes, and human embryonic stem cell derived whole-eye cultures, this study shows that human ocular surface cells are directly infectable and productively support SARS-CoV-2. Spike antigen was present in donor ocular surface epithelium co-localizing with ACE2. Among cultured tissues the limbus carried the highest ACE2 and TMPRSS2 expression and the most viral reads, and a broad serine protease inhibitor nearly abolished infection. Transcriptionally, infection drove an NF-kB chemokine program while suppressing genes underlying normal limbal barrier and secretory function. Stem cell derived cultures concentrated entry factors in conjunctival and limbal clusters, released a hundredfold more infectious virus than the input, and yielded a heavily infected limbal cluster in which interferon-stimulated genes were lower than in bystander cells, which the authors interpret as intracellular antagonism superimposed on paracrine interferon signaling. Whether the eye is a genuine entry portal remains untested.", "citation": "Eriksen AZ, Møller R, Makovoz B, Uhl SA, tenOever BR, Blenkinsop TA. SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium. Cell Stem Cell. 2021. 28(7), 1205-1220.e7. DOI 10.1016/j.stem.2021.04.028. PMID 34022129. PMCID PMC8126605. Correspondence is addressed to both Benjamin R. tenOever and Timothy A. Blenkinsop, who are also listed as joint on conceptualization, supervision, project administration and funding in the author contributions statement.", "sections": { "Citation": "Eriksen AZ, Møller R, Makovoz B, Uhl SA, tenOever BR, Blenkinsop TA. SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium. Cell Stem Cell. 2021. 28(7), 1205-1220.e7.\n\nDOI 10.1016/j.stem.2021.04.028. PMID 34022129. PMCID PMC8126605.\n\nCorrespondence is addressed to both Benjamin R. tenOever and Timothy A. Blenkinsop, who are also listed as joint on conceptualization, supervision, project administration and funding in the author contributions statement.", "One-sentence contribution": "Human ocular surface tissue carries SARS-CoV-2 entry machinery and supports productive replication, with the limbus most permissive in both adult donor cells and stem cell derived whole-eye cultures, where infection drives NF-kB chemokine induction and blunted interferon signaling.", "Executive summary": "Viral RNA had been detected in tears and conjunctiva during COVID-19, but whether ocular surface cells are themselves infected, and which ones, was unresolved. This study combines three human systems. Anterior segments from three post-mortem donors who had tested positive for SARS-CoV-2 were stained for viral antigen, and spike protein was found in the ocular surface epithelium of all three, co-localizing extensively with ACE2 and detectable in both limbal keratin 15 positive and negative cells. Cells cultured from six regions of healthy cadaver globes were then challenged directly. Spike protein appeared in cornea, limbus, sclera and retinal pigment epithelium but not in iris or choroid, while subgenomic nucleocapsid transcript was detected across all six, and ACE2 and TMPRSS2 messenger RNA were highest in the limbus. Whole-transcriptome sequencing of genetically matched cornea, limbus and sclera showed the limbus most heavily infected and dominated by an NF-kB chemokine signature, with limbal identity genes suppressed. A serine protease inhibitor nearly abolished infection. To supply a renewable model, human embryonic stem cell derived SEAM cultures were profiled at single-cell resolution, entry factors were concentrated in conjunctival and limbal clusters, and infection produced a hundredfold increase in infectious virus and a heavily infected limbal cluster. Interferon-stimulated genes were induced across the culture but were lower in the cells carrying the most viral reads.", "Scientific context": "Aerosol transmission was understood to be the dominant route for SARS-CoV-2, and masks leave the eyes exposed. Viral RNA had been reported in tears and ocular secretions of a small percentage of patients, coronaviral RNA had previously been found in the eye for SARS-CoV-1 and MERS-CoV, and conjunctival inoculation produced respiratory infection in rhesus monkeys and in golden hamsters. On the host side, immunohistochemistry, bulk RNA sequencing and chromatin accessibility work by other groups had shown that cornea, limbus and conjunctiva express ACE2 and TMPRSS2, though TMPRSS2 is low in ocular tissue and other serine proteases are more abundant, and published screens had widened the list of candidate entry-associated genes. What remained unknown was whether virus in tears reflects direct infection of ocular surface cells or drainage from the respiratory tract, whether some ocular tissues are more permissive than others, and what host program infection triggers there. Systemic seeding was considered unlikely by the authors on the grounds that no transfusion-associated transmission had been reported.", "Central question": "Can SARS-CoV-2 directly infect human ocular surface cells and replicate productively there, which ocular cell types are most permissive and why, and what transcriptional response does infection provoke in those cells?", "Experimental strategy": "Three complementary human systems each answer a different part of the question and cover one another's weaknesses. Post-mortem tissue from infected donors establishes that viral antigen is present in the eye in real disease, but cannot say where infection started. Cells cultured from dissected regions of healthy cadaver globes allow direct experimental challenge with a defined multiplicity and a side-by-side comparison of six ocular tissues, with cell identity verified by marker staining first, and with genetically matched cornea, limbus and sclera used for the transcriptional comparison so that donor background does not confound it. Because human eyes are available only through donation, a human embryonic stem cell derived self-formed ectodermal autonomous multizone culture provides a renewable model that contains several ocular lineages at once, and single-cell sequencing of that culture allows infected cells, uninfected cells in the same culture, and cells of a separate uninfected culture to be compared, which is what separates cell-autonomous responses from paracrine ones. Productive replication, as opposed to entry alone, is tested by plaque assay on supernatant. Entry route is probed pharmacologically with TPCK, an inhibitor of serine proteases including the TMPRSS family.", "Key findings": "1. Ocular surface tissue from all three SARS-CoV-2 positive post-mortem donors stained positive for spike protein, predominantly in epithelium and absent from corneal stroma, in cells both positive and negative for the limbal marker keratin 15, with strong co-localization with ACE2 and some spike positive cells lacking ACE2 (Fig. 1, Fig. S1 and S2). Nucleocapsid protein was found in p63 positive cells at the limbus and co-stained with ACE2 and TMPRSS2 (Fig. S3). The authors state explicitly that these samples cannot establish whether the eye was infected first or seeded from another organ.\n2. Cells cultured from six regions of healthy donor globes, with identity confirmed by marker staining (Fig. 2A), showed spike protein after challenge in cornea, limbus, sclera and retinal pigment epithelium but not in iris or choroid (Fig. 2B), while subgenomic nucleocapsid transcript was detected in all six (Fig. 2E). The discrepancy between protein and subgenomic RNA detection is not resolved in the text.\n3. ACE2 and TMPRSS2 messenger RNA were significantly higher in limbus than in the other tissues, with ACE2 in retinal pigment epithelium the exception (Fig. 2C and 2D). Iris and choroid had lower average expression, though that difference was not statistically significant.\n4. In genetically matched donor material, sequencing reads mapping to the viral genome were present in cornea, limbus and sclera, and were significantly higher in limbus (Fig. 3A and 3B).\n5. The host response in all three tissues was dominated by NF-kB-associated genes, including CXCL1, CXCL2, CXCL3 and CXCL6, TNFAIP2, TNFAIP3 and TNFAIP6, IL6 and RELB (Fig. 3F). Chemokine induction was greater in limbus and sclera than in cornea, and SOD2, RIPOR3, TNFAIP3 and C3 rose in all three (Fig. 3F and 3G). Limbus was the only tissue with a large set of genes decreasing on infection (Fig. 3D).\n6. Pre-treatment with the serine protease inhibitor TPCK almost completely abolished infection of limbal cells (Fig. 3H), which the authors take as confirmation of entry and of serine protease dependence.\n7. Genes higher in limbus than cornea included ACE2, TMPRSS2, TMPRSS4, TMPRSS11E, SPINT1 and SPINT2, and donor limbal cells expressed TMPRSS4 about elevenfold above corneal cells. The authors propose TMPRSS4 as a candidate explanation for limbal permissivity and say directly that further studies are required to assess it.\n8. Genes suppressed in infected limbal cells mapped to normal limbal functions including O-glycan biosynthesis, mucin glycosylation, tight junction interactions and glycosphingolipid biosynthesis, and overlapped with gene sets downregulated by SARS-CoV-2 in ferret nasal wash, intestinal organoids, Calu-3 cells and lung (Table S2C).\n9. Single-cell profiling of the SEAM culture resolved eleven clusters covering surface ectoderm, ocular surface ectoderm, retinal pigment epithelium, neural lineages, dorsal optic cup and periocular mesenchyme, plus some non-ocular populations (Fig. 4 and Fig. S7). ACE2 was expressed above baseline in 11.7 percent of cells with 73.7 percent of those in ocular surface ectoderm clusters, TMPRSS2 in 266 cells with 78.2 percent in the same clusters, and 113 cells co-expressed both, concentrated in the conjunctiva and limbus population (Fig. 5).\n10. SEAM cultures supported productive infection, with infectious virus in the supernatant increasing a hundredfold over input and plateauing after 24 hours for at least three days (Fig. 6B).\n11. Integrated single-cell analysis of infected and uninfected SEAM cultures revealed a second limbal cluster present only in the infected culture, in which viral E, M, N and S transcripts were high in nearly all cells (Fig. 6E, 6F and 6H). The authors attribute the separation of this cluster to the abundance of viral and response transcripts.\n12. NF-kB and TNF-associated genes were elevated in the heavily infected limbal cluster above both uninfected clusters and bystander cells, which the authors interpret as a virus-driven rather than cytokine-driven signature.\n13. Interferon-stimulated genes rose across the infected culture including in the uninfected limbal cluster, but were lower in the cluster carrying the most viral reads, though still above the uninfected culture (Fig. 6H). The authors read the broad induction as paracrine interferon signaling and the relative deficit in infected cells as viral antagonism, and they note that the pathway as a whole apart from IRF1 and ZFP36 was less induced, suggesting antagonism at a major node.\n14. Of genes expressed in the SEAM limbus, 87 percent were also expressed in adult limbus, and 80 percent of corneal cluster genes were expressed in adult cornea. Genes downregulated in both systems converged on injury response, immune cell recruitment, cell-cell communication, limbal and conjunctival function, and barrier function (Fig. 7).", "Mechanistic model": "The study does not establish a mechanism for why the limbus is preferentially infected, and it says so. What it constrains is the following. Entry into ocular surface cells requires serine protease activity, since a broad TMPRSS-family inhibitor blocks infection, and permissivity correlates with expression of ACE2 together with several serine proteases, with TMPRSS4 standing out as elevated in limbus relative to cornea in donor cells and enriched in limbal clusters in the stem cell derived model. That correlation is offered as a candidate explanation and is not tested by loss of function. The host response has two arms whose relationship is interpreted rather than demonstrated. NF-kB-dependent chemokine induction is highest in cells carrying the most viral RNA, which the authors read as driven by something in the virus rather than by soluble mediators, while interferon-stimulated gene induction is broad across the culture but comparatively lower in those same cells, which they read as intracellular antagonism of interferon induction layered on top of paracrine signaling from neighboring cells or from debris. Two alternatives are acknowledged in the discussion, namely that interferon induction may simply not be efficiently inhibited in infected cells, and that NF-kB signaling can itself suppress interferon output. The suppression of limbal identity genes is described as possibly a consequence of the virus commandeering host transcriptional machinery, or possibly part of an injury and epithelial-to-mesenchymal response, and neither is distinguished experimentally.", "Conceptual or technical advance": "The work places the ocular surface on the map of tissues that can be directly and productively infected by SARS-CoV-2 in human material, and it localizes the vulnerability to a specific anatomical compartment rather than to the eye as a whole. Doing this required a model that could be infected repeatedly, since human eyes come only from donation, and the adaptation of the SEAM whole-eye differentiation protocol for virology, combined with single-cell profiling before and after infection, provides one. The single-cell design also makes a distinction that bulk work cannot, separating what a cell does because it contains virus from what it does because its neighbors do, and that distinction is what allows the interferon and NF-kB arms to be described separately. The finding that limbal identity genes are suppressed connects infection to potential loss of ocular surface function rather than only to viral output.", "Relationship to the broader research program": "The transcriptional framing here belongs to a line running through the tenOever laboratory's COVID-19 work, in which SARS-CoV-2 elicits a strong NF-kB-driven inflammatory program alongside a muted type I and type III interferon response, established for lung and airway systems in Blanco-Melo and colleagues and cited repeatedly in this paper for direct comparison. The same laboratory's hamster work on ocular inoculation and on nucleocapsid transcript release from dying cells is likewise cited, and the interferon antagonism framework draws on the senior author's review of antiviral defense evolution. Applying that framework to an entirely different tissue, provided and characterized by the Blenkinsop laboratory, is what the collaboration adds. Whether the ocular surface constitutes a meaningful transmission route or a viral reservoir is raised as an open question by the authors and is not answered here. Comparisons across corpus entries on tissue-specific host responses to SARS-CoV-2 would be category 3 synthesis and belong to central assembly.", "Related publications": "- Blanco-Melo et al. 2020, imbalanced host response to SARS-CoV-2 drives development of COVID-19. Predecessor from the tenOever laboratory, the source of the NF-kB high and interferon low signature that this paper compares against throughout.\n- Hoagland et al. 2021, work from the same laboratory on SARS-CoV-2 in golden hamsters including ocular inoculation and interferon dynamics. Predecessor, cited for ocular route infection in animals and for nucleocapsid transcript release from dying cells.\n- tenOever 2016, the evolution of antiviral defense systems. Review or synthesis from the same laboratory, cited for interferon as first line of defense and viral antagonism.\n- Hayashi et al. 2016, self-formed ectodermal autonomous multizone cultures. Methodological foundation from another group, the differentiation protocol adopted here.\n- Collin et al. 2021 and Mencucci et al. 2021, expression of SARS-CoV-2 entry factors in ocular surface tissue. Predecessors from other groups, providing the entry factor expression data this study builds on and compares with.", "Limitations and boundaries": "The authors list their own limitations plainly. Only three to seven genetically distinct donors were available per ocular cell type, and the three infected post-mortem donors cannot show whether the eye was the portal of entry or was seeded from an established airway infection, so the transmission question the paper motivates remains open. The SEAM cultures are stem cell derived and immature, indicated by low KRT3 and KRT12, and they contain cells at different developmental stages, which may itself contribute to the higher infectivity observed in the limbal compartment given that the limbus is a stem cell niche. Single-cell sequencing of infected cadaver donor cells was not performed, which the authors name as the experiment that would test whether a specific niche with stem cell properties is preferentially infected. Infections are at a single multiplicity of one and largely a single 24 hour time point, with growth followed for three days only in the SEAM model. The TMPRSS4 hypothesis rests on expression correlation, and the protease requirement is established with a broad inhibitor that does not identify which protease matters. The infected SEAM culture and the uninfected comparison culture were differentiated for different lengths of time, 54 days and 31 days respectively, which is a difference between the compared conditions beyond infection itself. Nothing here addresses clinical ocular disease, infectivity of tears, or whether ocular infection contributes to spread within or between people.", "Audience summaries": "### 25 words\n\nHuman ocular surface cells, especially at the limbus, carry SARS-CoV-2 entry factors and support productive infection, both in donor tissue and in stem cell derived eye cultures.\n\n### 75 words\n\nViral RNA had been found in tears, but it was unclear whether eye cells are actually infected. Antigen was present in ocular surface tissue from three deceased COVID-19 patients, and cells cultured from donor eyes supported infection, most strongly in the limbus, the ring of tissue holding the corneal stem cell niche. Stem cell derived eye cultures reproduced this and released infectious virus. Infected cells mounted a strong inflammatory response with weak interferon signaling.\n\n### 150 words\n\nCombining post-mortem tissue from SARS-CoV-2 positive donors, primary cultures from six regions of healthy cadaver globes, and human embryonic stem cell derived whole-eye cultures, this study shows that human ocular surface cells are directly infectable and productively support SARS-CoV-2. Spike antigen was present in donor ocular surface epithelium co-localizing with ACE2. Among cultured tissues the limbus carried the highest ACE2 and TMPRSS2 expression and the most viral reads, and a broad serine protease inhibitor nearly abolished infection. Transcriptionally, infection drove an NF-kB chemokine program while suppressing genes underlying normal limbal barrier and secretory function. Stem cell derived cultures concentrated entry factors in conjunctival and limbal clusters, released a hundredfold more infectious virus than the input, and yielded a heavily infected limbal cluster in which interferon-stimulated genes were lower than in bystander cells, which the authors interpret as intracellular antagonism superimposed on paracrine interferon signaling. Whether the eye is a genuine entry portal remains untested." }, "discoveries": [], "relationships": [ { "from": "2021-eriksen-sars-cov-2-infects-human-adult-don", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-eriksen-sars-cov-2-infects-human-adult-don" }, { "from": "2021-eriksen-sars-cov-2-infects-human-adult-don", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-eriksen-sars-cov-2-infects-human-adult-don" } ], "canonical_url": "https://tenoeverlab.us/publications/2021-eriksen-sars-cov-2-infects-human-adult-don/", "controlled_vocabulary": { "pathogens": [ "sars-cov-2" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "immunofluorescence-microscopy", "pathway-enrichment-analysis", "small-molecule-inhibitor-profiling", "single-cell-rna-seq" ] } }, { "id": "2021-guzman-solis-ancient-viral-genomes-reveal-intro", "slug": "2021-guzman-solis-ancient-viral-genomes-reveal-intro", "url": "/publications/2021-guzman-solis-ancient-viral-genomes-reveal-intro/", "title": "Ancient viral genomes reveal introduction of human pathogenic viruses into Mexico during the transatlantic slave trade", "authors": [ "Axel A Guzmán-Solís", "Viridiana Villa-Islas", "Miriam J Bravo-López", "Marcela Sandoval-Velasco", "Julie K Wesp", "Jorge A Gómez-Valdés", "María de la Luz Moreno-Cabrera", "Alejandro Meraz", "Gabriela Solís-Pichardo", "Peter Schaaf", "Benjamin R TenOever", "Daniel Blanco-Melo", "María C Ávila Arcos" ], "author_count": 13, "first_author": "Axel A Guzmán-Solís", "senior_authors": [ "María C Ávila Arcos", "Daniel Blanco-Melo" ], "corresponding_authors": [ "Daniel Blanco-Melo", "María C Ávila Arcos" ], "tenoever_position": 11, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2021, "journal": "eLife", "volume": "10", "issue": null, "pages": "e68612", "doi": "10.7554/elife.68612", "doi_url": "https://doi.org/10.7554/elife.68612", "pmid": "34350829", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/34350829/", "pmcid": "PMC8423449", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8423449/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8423449/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "viral-populations-evolution" ], "themes": [ "historical-virus-movement" ], "pathogens": [ "human parvovirus B19", "hepatitis B virus" ], "viral_families": [ "Parvoviridae", "Hepadnaviridae" ], "host_species": [ "human" ], "technologies": [ "ancient DNA extraction", "targeted in-solution hybridisation capture", "shotgun metagenomic sequencing", "deamination damage analysis", "maximum likelihood phylogenetics", "dated coalescent analysis", "strontium isotope analysis", "radiocarbon dating", "principal component analysis of ancient genomes", "ADMIXTURE ancestry analysis" ], "biological_systems": [ "archaeological human dental remains", "tooth enamel", "parietal bone", "phalanx" ], "key_concepts": [ "paleovirology", "ancient DNA authentication", "viral genotype geography", "transatlantic slave trade", "Colonial epidemics", "Cocoliztli", "host genetic ancestry", "molecular tip calibration", "cross-population transmission" ], "keywords": [ "ancient DNA", "parvovirus B19", "hepatitis B virus", "paleovirology", "Colonial Mexico", "New Spain", "slave trade", "phylogenetics", "strontium isotopes", "genotype 3" ], "one_sentence_contribution": "Targeted ancient DNA capture applied to skeletal remains from a Colonial hospital and chapel in Mexico City recovers three human parvovirus B19 genomes and one hepatitis B virus genome whose closest relatives are African strains, in hosts whose genetic ancestry and strontium isotope signatures point to African birth, giving direct molecular evidence that these viruses reached New Spain during the transatlantic slave trade.", "summary_25": "Viral DNA recovered from teeth in Colonial Mexico City burials yields African-lineage parvovirus B19 and hepatitis B virus genomes, direct evidence of introduction during the transatlantic slave trade.", "summary_75": "Which pathogens reached the Americas after European colonisation has been argued mostly from historical records. Using a capture assay for ancient viral DNA on teeth from a Colonial hospital and chapel in Mexico City, the authors reconstructed three parvovirus B19 genomes and one hepatitis B virus genome. All belong to lineages associated with Africa, and the individuals carrying them show African ancestry and West African birth signatures. One Indigenous individual also carried the virus.", "summary_150": "Targeted hybridisation capture applied to ancient DNA from 26 dental samples at two Colonial sites in Mexico City yielded three human parvovirus B19 genomes at 92 to 99 percent coverage of the coding region and one hepatitis B virus genome at 90 percent coverage, all authenticated by capture-negative controls, best-hit filtering and terminal deamination damage. The hepatitis B virus genome carries the genotype A core insertion and clusters with sub-genotype A4, never before reported in the Americas. The parvovirus B19 genomes fall in genotype 3, predominant in West Africa, whereas only genotype 1 had been described molecularly in Mexico. Three hosts fall within African genetic variation and carry L mitochondrial haplogroups, and enamel strontium ratios indicate West African birth against bone values matching the Mexico City valley, with radiocarbon dates in the early Colonial period. A fourth individual, of Indigenous ancestry from a separate site, also carried genotype 3.", "citation": "Guzmán-Solís AA, Villa-Islas V, Bravo-López MJ, Sandoval-Velasco M, Wesp JK, Gómez-Valdés JA, Moreno-Cabrera ML, Meraz A, Solís-Pichardo G, Schaaf P, TenOever BR, Blanco-Melo D, Ávila Arcos MC. Ancient viral genomes reveal introduction of human pathogenic viruses into Mexico during the transatlantic slave trade. *eLife* 2021, volume 10, article e68612. DOI 10.7554/elife.68612. PMID 34350829. PMCID PMC8423449.", "sections": { "Citation": "Guzmán-Solís AA, Villa-Islas V, Bravo-López MJ, Sandoval-Velasco M, Wesp JK, Gómez-Valdés JA, Moreno-Cabrera ML, Meraz A, Solís-Pichardo G, Schaaf P, TenOever BR, Blanco-Melo D, Ávila Arcos MC. Ancient viral genomes reveal introduction of human pathogenic viruses into Mexico during the transatlantic slave trade. *eLife* 2021, volume 10, article e68612.\n\nDOI 10.7554/elife.68612. PMID 34350829. PMCID PMC8423449.", "One-sentence contribution": "Targeted ancient DNA capture applied to skeletal remains from a Colonial hospital and chapel in Mexico City recovers three human parvovirus B19 genomes and one hepatitis B virus genome whose closest relatives are African strains, in hosts whose genetic ancestry and strontium isotope signatures point to African birth, giving direct molecular evidence that these viruses reached New Spain during the transatlantic slave trade.", "Executive summary": "The collapse of Indigenous populations after European colonisation of the Americas is attributed in part to introduced pathogens, but the identity of the agents responsible for the Colonial epidemics of New Spain has rested on historical accounts and on inference from modern viral strains rather than on material from the period. This study asks what can be recovered directly. Dental remains from 26 individuals were sampled at two Colonial sites in Mexico City, a hospital where mass burials indicate urgent simultaneous disposal of bodies and a chapel ten kilometres away. Shotgun sequencing with metagenomic classification identified samples carrying viral signal, and a designed panel of RNA probes targeting clinically important human viruses was used to enrich those libraries, with a virus-negative library carried through as a control. Enrichment yielded three human parvovirus B19 genomes and one hepatitis B virus genome, authenticated by best-hit filtering against the full nucleotide database and by the terminal deamination damage characteristic of ancient DNA. The hepatitis B virus genome belongs to sub-genotype A4, not previously reported in the Americas, and the parvovirus B19 genomes belong to genotype 3, which predominates in West Africa, whereas only genotype 1 had been described molecularly in Mexico. Ancestry analysis of the same sequencing data placed three hosts within African genetic variation with L mitochondrial haplogroups, strontium ratios indicated West African birth with later life in the Mexico City valley, and radiocarbon dates fell in the early Colonial period. A fourth individual, from the chapel site and of Indigenous ancestry, also carried genotype 3.", "Scientific context": "European colonisation of the Americas was followed by a severe collapse of Indigenous populations, attributed in part to introduced pathogens. Smallpox, measles and mumps have long been proposed as agents of the Colonial epidemics, and the deadliest outbreaks in New Spain, referred to as Cocoliztli, have no established cause. Prior work on those epidemics has rested on historical accounts, including autopsy descriptions from the Hospital Real de San José de los Naturales, on bioarchaeology and on phylogenetic inference from modern viral strains, not on direct recovery of viral material from the period. Ancient viral genomics had by then produced results for several viral families from Eurasian and some American contexts, including ancient hepatitis B virus and parvovirus B19 genomes, and tooth roots had been shown to be a usable source of pathogen DNA for blood-borne agents. Bioarchaeological and paleogenomic work on the hospital collection had already identified individuals of sub-Saharan African ancestry. The gap this study addresses is the absence of direct molecular evidence for specific viruses circulating in Colonial Mexico.", "Central question": "Which viral pathogens can be recovered directly from human remains buried in Colonial epidemic contexts in Mexico City, and do their genome sequences, read alongside the ancestry and geographic origin of the individuals who carried them, indicate introduction from Africa during the transatlantic slave trade?", "Experimental strategy": "The design joins three independent lines of evidence about the same individuals, which is what allows a claim about geographic introduction rather than merely about viral presence. Sampling targets dental remains, chosen because vascularised tooth roots can retain DNA from blood-borne agents, from two archaeologically distinct Colonial sites, a hospital associated with mass burials suggestive of urgent simultaneous disposal and a chapel ten kilometres away. Shotgun sequencing with metagenomic classification first identifies which samples carry any plausible viral signal, and a biotinylated RNA probe set then enriches for clinically important human viruses chosen on three criteria, DNA viruses already recovered from archaeological remains, representatives of families that integrate into the human genome, and RNA viruses with a DNA intermediate. A virus-negative library is carried through capture as the control that measures enrichment specificity. Authentication proceeds by several separate tests rather than one, including retention only of reads whose best database hit is the virus in question, characteristic deamination damage at read termini, and coverage structure across the genome. Human ancestry is then read from the same shotgun data by mitochondrial haplogroup, principal component analysis against a reference panel and admixture analysis, while strontium isotope ratios in enamel against bone distinguish place of birth from place of later life, and radiocarbon dating places the individuals in time. Viral origin is finally tested by phylogenetic placement against modern genotype and sub-genotype diversity of known geographic distribution.", "Key findings": "1. Metagenomic screening of 26 dental samples identified 17 with at least one normalised viral hit, with signals resembling Hepadnaviridae, Herpesviridae, Parvoviridae and Poxviridae, and twelve samples were carried forward to capture (Figure 1c).\n2. After capture, one library showed roughly a hundredfold increase in hepatitis B virus hits and three showed roughly fifty to two hundredfold increases in parvovirus B19 hits, while the negative control library showed negligible enrichment (Figure 1c).\n3. All recovered reads displayed cytosine to thymine accumulation toward the five prime terminus with a near-symmetrical guanine to adenine pattern at the three prime end, the damage signature expected of ancient DNA (Figure 2a).\n4. Three parvovirus B19 genomes were reconstructed at 92.37 to 99.1 percent coverage of the single-stranded coding region with average depths of 2.98 to 15.36, and one hepatitis B virus genome at 89.9 percent coverage and 30.8 average depth (Figure 2, b and c).\n5. Coverage was markedly higher over the double-stranded inverted terminal repeats of parvovirus B19 and lower over the single-stranded region of hepatitis B virus. The authors interpret this uneven structure two ways, as reflecting better postmortem survival of double-stranded DNA and as an argument that the reads come from replication intermediates rather than from virus integrated into the human genome, since integration would give even coverage. Both are interpretations of the coverage pattern.\n6. The hepatitis B virus genome carries the six-nucleotide core gene insertion characteristic of genotype A and clusters phylogenetically with sub-genotype A4, which had previously been recovered only from African individuals in Belgium and never in the Americas (Figure 3a).\n7. All three parvovirus B19 genomes fall in genotype 3. Two are similar to sub-genotype 3b sequences from immigrants in Germany and one is closer to a divergent sub-genotype 3a strain isolated from a child with severe anaemia in France (Figure 3b). Genotype 3 is predominant in West Africa, and only genotype 1 had previously been described molecularly in Mexico.\n8. Testing for temporal structure found little for the full hepatitis B virus phylogeny, a stronger signal within genotype A analysed alone, and a usable signal for parvovirus B19 across all three genotypes, corroborated by date randomisation. A dated coalescent analysis gave a parvovirus B19 substitution rate of 1.03 by 10 to the minus five substitutions per site per year under a strict clock, with divergence times from the most recent common ancestor of genotypes 1, 2 and 3 of 7.19, 2.11 and 3.64 thousand years.\n9. The three individuals from the hospital fall within African genetic variation by principal component analysis and carry L mitochondrial haplogroups (Figure 4a).\n10. Strontium ratios in tooth enamel from two hospital individuals, 0.71098 and 0.71109, resemble West African soils and rocks and values reported for first-generation Africans in the Americas, while their parietal and phalanx values, 0.70672 and 0.70755, resemble the Trans Mexican Volcanic Belt where Mexico City lies. The isotope data therefore support birth in West Africa and later life in New Spain.\n11. Radiocarbon dating placed two individuals at 1442 to 1608 and 1472 to 1625 CE, the early Colonial period when arrivals of enslaved people were highest.\n12. The individual from the chapel site, who carried a genotype 3 parvovirus B19 genome, clusters with present-day Mexicans and Peruvians and shows a Native American component by admixture analysis (Figure 4b). The authors describe this as the first genotype 3 genome obtained from a non-African individual and read it as evidence that the virus spread across ancestries after introduction.", "Mechanistic model": "This is a historical and phylogeographic study rather than a mechanistic one, and it establishes no disease mechanism. What the evidence supports is a chain of inference about origin. Viral genomes of African-associated lineages were recovered from individuals whose nuclear ancestry, mitochondrial haplogroup and enamel strontium signature indicate African birth, at a date when forced transport from Africa to New Spain was at its height, which the authors read as direct molecular evidence of introduction during the transatlantic slave trade. The recovery of one of those lineages from an individual of Indigenous ancestry at a separate site is read as onward transmission between populations. Several further propositions in the discussion are offered explicitly as possibilities rather than findings. The authors state that they cannot establish where the African-born individuals acquired either infection, whether in Africa, in the Americas or during the crossing, nor whether infection caused their deaths, nor the age at acquisition, nor whether the hepatitis B virus infection was acute or chronic or acquired vertically. The suggestion that parvovirus B19 infection might explain the skeletal anaemia markers in one individual is framed as possible and is accompanied by the acknowledgement that genetic anaemias cannot be excluded because the relevant loci were not covered. The suggestion that some cases recorded as measles in sixteenth-century Mexico might have been parvovirus B19 is presented as a hypothesis requiring further study and is accompanied by an explicit statement that the study does not reject the role of measles. The overlap between the radiocarbon dates and the hepatitis-like symptoms recorded in Cocoliztli autopsies is noted, with the authors stating that additional analyses are needed before any link is established.", "Conceptual or technical advance": "The study demonstrates that a designed capture panel spanning clinically important human DNA viruses, applied to dental remains from documented Colonial burial contexts, can recover near-complete viral genomes and that those genomes can be authenticated and placed phylogenetically with enough resolution to assign sub-genotype. Combining that recovery with host nuclear and mitochondrial ancestry, strontium isotopes and radiocarbon dating on the same individuals is what converts a viral sequence into evidence about human movement, and it provides a template for investigating introduced pathogens in other Colonial contexts. The ancient parvovirus B19 genotype 3 genomes also serve as calibration points that had not previously existed for that genotype, allowing dated coalescent inference. Substantively, the work moves the discussion of Colonial epidemic aetiology from historical inference and modern-strain phylogeography onto direct molecular evidence, while stopping short of assigning causation.", "Relationship to the broader research program": "The paper is led by the Ávila Arcos group at the Universidad Nacional Autónoma de México, with Daniel Blanco-Melo, formerly of the tenOever laboratory and here affiliated with Mount Sinai and the Fred Hutchinson Cancer Research Center, as co-corresponding author and a principal conceptual contributor. The stated contribution of Benjamin tenOever is resources and manuscript review, and he is neither a corresponding author nor a senior author, so this record is classified as collaborative rather than lab-led. The connection to the wider corpus runs through the alumnus rather than through a research theme, and the paper's questions, methods and material are those of ancient DNA and paleovirology rather than of the laboratory's work on virus-host interaction in living systems. Asserting a thematic continuity would be category 3 synthesis and is not supported by this paper alone.", "Related publications": "- Blanco-Melo and colleagues, 2020, and other tenOever laboratory work, no relationship asserted. Nothing in this paper's reference list or content links it to the tenOever laboratory's own published research, and the connection is one of personnel rather than of scientific lineage.\n\nThe list is deliberately empty of corpus links. The references this paper builds on are from the ancient DNA, paleovirology, bioarchaeology and virus phylogenetics literature produced by other groups.", "Limitations and boundaries": "Four viral genomes from four individuals, drawn from 26 sampled, is a small basis for statements about what circulated in Colonial Mexico City, and the authors say that larger sample sizes from varied contexts are needed. Coverage is thin for two of the parvovirus B19 genomes, with average depths under four over the coding region. The capture panel covers only DNA viruses and RNA viruses with a DNA intermediate, and for some families only a few genes were tiled because of probe kit size limits, so the leading candidate agents of the Colonial epidemics, which are RNA viruses, could not be sought at all, a constraint the authors state and attribute to rapid RNA degradation. Sampling was biased by design, since hospital individuals were selected on morphological and dental indicators previously interpreted as African ancestry, which shapes what ancestry and viral lineage combinations could be found. Strontium and radiocarbon data exist for only two individuals each, and not for all four virus-positive individuals. Temporal structure was weak for hepatitis B virus across genotypes, so no dated analysis was performed for that virus, and the parvovirus B19 divergence estimates rest on one clock model choice among two reported. Genotype geography is inferred from modern sampling that is itself uneven, and sub-genotype A4 in particular has a contested origin resting on sequences from African immigrants in Europe. No causal link to any epidemic, to Cocoliztli or to the individuals' deaths is established, and the authors repeatedly say so. The authors also note that both viruses have been found in ancient DNA datasets with no association to disease or epidemics.", "Audience summaries": "### 25 words\n\nViral DNA recovered from teeth in Colonial Mexico City burials yields African-lineage parvovirus B19 and hepatitis B virus genomes, direct evidence of introduction during the transatlantic slave trade.\n\n### 75 words\n\nWhich pathogens reached the Americas after European colonisation has been argued mostly from historical records. Using a capture assay for ancient viral DNA on teeth from a Colonial hospital and chapel in Mexico City, the authors reconstructed three parvovirus B19 genomes and one hepatitis B virus genome. All belong to lineages associated with Africa, and the individuals carrying them show African ancestry and West African birth signatures. One Indigenous individual also carried the virus.\n\n### 150 words\n\nTargeted hybridisation capture applied to ancient DNA from 26 dental samples at two Colonial sites in Mexico City yielded three human parvovirus B19 genomes at 92 to 99 percent coverage of the coding region and one hepatitis B virus genome at 90 percent coverage, all authenticated by capture-negative controls, best-hit filtering and terminal deamination damage. The hepatitis B virus genome carries the genotype A core insertion and clusters with sub-genotype A4, never before reported in the Americas. The parvovirus B19 genomes fall in genotype 3, predominant in West Africa, whereas only genotype 1 had been described molecularly in Mexico. Three hosts fall within African genetic variation and carry L mitochondrial haplogroups, and enamel strontium ratios indicate West African birth against bone values matching the Mexico City valley, with radiocarbon dates in the early Colonial period. A fourth individual, of Indigenous ancestry from a separate site, also carried genotype 3." }, "discoveries": [], "relationships": [ { "from": "2021-guzman-solis-ancient-viral-genomes-reveal-intro", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "related", "evidence": "stated in the Related publications section of 2021-guzman-solis-ancient-viral-genomes-reveal-intro" } ], "canonical_url": "https://tenoeverlab.us/publications/2021-guzman-solis-ancient-viral-genomes-reveal-intro/", "controlled_vocabulary": { "pathogens": [ "hbv", "parvovirus-b19" ], "technologies": [ "phylogenetics", "targeted-capture", "ancient-dna", "ancient-dna-authentication", "host-ancestry-analysis", "archaeometric-dating", "metagenomic-sequencing" ] } }, { "id": "2021-hoagland-leveraging-the-antiviral-type-i-in", "slug": "2021-hoagland-leveraging-the-antiviral-type-i-in", "url": "/publications/2021-hoagland-leveraging-the-antiviral-type-i-in/", "title": "Leveraging the antiviral type I interferon system as a first line of defense against SARS-CoV-2 pathogenicity", "authors": [ "Daisy A. Hoagland", "Rasmus Møller", "Skyler A. Uhl", "Kohei Oishi", "Justin Frere", "Ilona Golynker", "Shu Horiuchi", "Maryline Panis", "Daniel Blanco-Melo", "David Sachs", "Knarik Arkun", "Jean K. Lim", "Benjamin R. tenOever" ], "author_count": 13, "first_author": "Daisy A. Hoagland", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 13, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2021, "journal": "Immunity", "volume": "54", "issue": "3", "pages": "557-570.e5", "doi": "10.1016/j.immuni.2021.01.017", "doi_url": "https://doi.org/10.1016/j.immuni.2021.01.017", "pmid": "33577760", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/33577760/", "pmcid": "PMC7846242", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846242/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846242/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling", "pandemic-host-response" ], "themes": [ "calibration-of-interferon-in-vivo", "interferon-as-intervention", "models-for-pandemic-virology" ], "pathogens": [ "SARS-CoV-2", "SARS-CoV-2 USA-WA1/2020", "influenza A virus", "influenza A/California/04/2009" ], "viral_families": [ "Coronaviridae", "Orthomyxoviridae" ], "host_species": [ "golden hamster", "hamster cells", "human cells" ], "technologies": [ "mRNA sequencing", "real-time quantitative RT-PCR", "plaque assay", "immunohistochemistry", "histopathology", "de novo transcriptome assembly", "gene ontology enrichment analysis", "ELISA", "intranasal interferon administration" ], "biological_systems": [ "golden hamster", "hamster lung", "hamster trachea", "olfactory bulb", "brain", "small intestine", "BHK-21 cells" ], "key_concepts": [ "type I interferon", "interferon-stimulated genes", "chemokine induction", "imbalanced host response", "systemic inflammation", "subgenomic RNA", "pathogen-associated molecular patterns", "prophylaxis", "transmission blocking", "COVID-19 pathogenesis", "small animal model" ], "keywords": [ "SARS-CoV-2", "COVID-19", "golden hamster model", "type I interferon", "intranasal interferon", "transcriptional atlas", "systemic inflammation", "double-stranded RNA mimetic", "subgenomic nucleocapsid RNA", "broad-spectrum antiviral" ], "one_sentence_contribution": "Longitudinal transcriptional and histological profiling of SARS-CoV-2 infected golden hamsters maps a wave of inflammation that reaches tissues with little or no productive replication, and shows that intranasal type I interferon given before or after challenge lowers viral load and disease burden.", "summary_25": "Hamsters infected with SARS-CoV-2 develop inflammation in organs the virus barely reaches, and interferon sprayed into the nose before or after exposure lowers virus and disease.", "summary_75": "SARS-CoV-2 provokes strong chemokine signaling but weak interferon signaling, a combination linked to severe COVID-19. Profiling infected golden hamsters across tissues and time showed inflammation moving from the upper to the lower airway and appearing in brain, olfactory bulb and intestine despite almost no virus there. Delivering type I interferon directly into the nose, before or one day after infection, reduced viral load, tissue damage and onward transmission.", "summary_150": "Golden hamsters infected with an unmodified clinical SARS-CoV-2 isolate were profiled longitudinally by mRNA sequencing, quantitative RT-PCR, plaque assay and histopathology across trachea, lung, olfactory bulb, brain and small intestine. Compared against influenza A virus at matched viral load, SARS-CoV-2 drove a stronger inflammatory and neutrophil associated signature. A newly assembled and functionally validated hamster interferon beta gene allowed interferon induction to be measured, and it remained low while chemokines rose. Interferon stimulated gene and chemokine peaks occurred in the trachea several days before the lung, and antiviral programs appeared in distal organs where infectious virus was scarce, which the authors attribute tentatively to disseminated viral RNA acting as a pattern recognition ligand. Intranasal universal interferon alpha A/D given before challenge, or beginning one day after, lowered infectious virus and inflammatory transcripts, shifted the lung infiltrate toward macrophages, and blocked transmission in three of five contact exposed animals.", "citation": "Hoagland DA, Møller R, Uhl SA, Oishi K, Frere J, Golynker I, Horiuchi S, Panis M, Blanco-Melo D, Sachs D, Arkun K, Lim JK, tenOever BR. Leveraging the antiviral type I interferon system as a first line of defense against SARS-CoV-2 pathogenicity. Immunity. 2021. Volume 54, issue 3, pages 557-570.e5. DOI 10.1016/j.immuni.2021.01.017. PMID 33577760. PMCID PMC7846242.", "sections": { "Citation": "Hoagland DA, Møller R, Uhl SA, Oishi K, Frere J, Golynker I, Horiuchi S, Panis M, Blanco-Melo D, Sachs D, Arkun K, Lim JK, tenOever BR. Leveraging the antiviral type I interferon system as a first line of defense against SARS-CoV-2 pathogenicity. Immunity. 2021. Volume 54, issue 3, pages 557-570.e5.\n\nDOI 10.1016/j.immuni.2021.01.017. PMID 33577760. PMCID PMC7846242.", "One-sentence contribution": "Longitudinal transcriptional and histological profiling of SARS-CoV-2 infected golden hamsters maps a wave of inflammation that reaches tissues with little or no productive replication, and shows that intranasal type I interferon given before or after challenge lowers viral load and disease burden.", "Executive summary": "Severe COVID-19 is associated with a host response in which type I interferon induction is muted while chemokine expression is high, and this combination has been proposed to drive the neutrophil and monocyte infiltration that characterizes the disease. Testing that idea and any intervention derived from it requires an animal model in which an unmodified clinical isolate causes progressive lower respiratory tract disease. The authors used golden hamsters, which are naturally permissive, and profiled infection across time and across tissues by mRNA sequencing, quantitative RT-PCR, plaque assay and histopathology. Comparison against pandemic H1N1 influenza A virus at matched viral loads showed that SARS-CoV-2 drives a stronger inflammatory and neutrophil associated signature. Infection could be established by intranasal, ocular, contact and, less efficiently, fomite exposure, and as few as ten plaque forming units sufficed to seed the lower respiratory tract. A longitudinal series showed interferon stimulated gene and chemokine induction that peaked in the trachea several days before the lungs, and an antiviral transcriptional response in olfactory bulb, brain and small intestine despite viral reads orders of magnitude below respiratory levels. Intranasal universal interferon alpha A/D, given prophylactically or starting one day after infection, reduced infectious virus, reduced proinflammatory transcripts, shifted the lung infiltrate away from neutrophils, and prevented transmission in three of five contact exposed animals. An intranasal double-stranded RNA mimetic gave comparable antiviral activity.", "Scientific context": "By late 2020 several groups had reported that SARS-CoV-2 elicits an unbalanced host response in which interferon induction is low relative to chemokine induction, and this had been described in cell culture, in a ferret model, in autopsy material and in patient cohorts. Severe COVID-19 had also been linked to autoantibodies against interferons and to inborn errors of innate immunity affecting TLR3 and IRF7, which together argued that an inadequate interferon response contributes to disease severity. At the same time systemic interferon administration had shown poor concordance in trials and can produce unwanted effects, and the SOLIDARITY trial did not show a mortality benefit. What remained undefined, as the paper states, was the transcriptional host response across the full course of infection and across tissues, including early infection and the period after viral clearance, because clinical sampling cannot readily provide it. Golden hamsters had been introduced as a permissive small animal model, but the hamster genome annotation was incomplete for immune genes, including Ifnb1, which limited transcriptomic interpretation.", "Central question": "What is the systemic and longitudinal host response to SARS-CoV-2 across respiratory and distal tissues, and can locally delivered type I interferon, rather than systemic interferon, be used to limit viral replication, tissue damage and transmission?", "Experimental strategy": "The design pairs a descriptive arm with an interventional arm in the same model. Golden hamsters were chosen because an unmodified clinical isolate replicates and causes progressive lower respiratory tract pathology without host genetic modification, which avoids the confounds of transgenic or adenovirus transduced ACE2 mouse systems. A matched influenza A virus comparison at equivalent viral read depth was used to separate what is specific to SARS-CoV-2 biology from what accompanies any respiratory virus at that load. Route and dose were varied deliberately, since the amount and site of the inoculum shape the response and the authors wanted to know which route is reliable before committing to it for the rest of the study. A longitudinal bulk mRNA sequencing series across trachea, lung, olfactory bulb, brain and small intestine at two inoculum doses provides the temporal and spatial map, with curated interferon stimulated gene and chemokine lists and gene ontology enrichment as the readouts. Because Ifnb1 is not annotated in the golden hamster genome, the authors performed a de novo assembly, identified a candidate transcript, cloned it, and showed it induces interferon stimulated genes in hamster but not human cells, which makes interferon beta measurable in this model. Sensitive subgenomic RNA assays combined with plaque assay were used to distinguish replicating virus from viral RNA in distal tissues. For the intervention, a universal interferon alpha A/D preparation was selected after comparing candidate interferons for potency on hamster cells, then delivered intranasally in prophylactic, therapeutic and contact transmission designs.", "Key findings": "1. At comparable viral loads five days after infection, SARS-CoV-2 and influenza A virus produced distinct lung transcriptional signatures, with SARS-CoV-2 driving higher inflammatory transcripts including Ccl5, Gzmb and Il1rn and higher neutrophil associated transcripts Fcgr3, Ccrl2 and Cf2 (Figures 1A to 1C).\n\n2. A functional golden hamster Ifnb1 transcript was identified by de novo assembly and validated by cloning and supernatant transfer, which induced interferon stimulated genes in hamster but not human cells (Figures S1A to S1E). Using this annotation, no significant Ifnb1 induction was detected in either SARS-CoV-2 or influenza A virus infected animals (Figure S1F), which the authors interpret as high chemokine expression in the setting of a muted type I interferon response.\n\n3. Infection was established by intranasal inoculation, ocular inoculation and direct contact, with fomite exposure the least efficient route, and all routes including the ocular route produced a respiratory host response with elevated Isg15 and Cxcl11 (Figures 2B, 2C and S2A).\n\n4. Ten plaque forming units were sufficient to establish lower respiratory tract infection. Raising the inoculum as high as one hundred thousand plaque forming units did not increase replication and tended to lower subgenomic nucleocapsid and nsp14 levels, while eliciting a comparable host response (Figures 2D, 2E, S2B and S2C). Intranasal infection was reproducible across twelve animals (Figures 2F to 2H).\n\n5. Infection produced progressive lower respiratory tract pathology scored by a certified pathologist, with nucleocapsid protein maximal at day four and still detectable at day fourteen, epithelial degeneration and alveolar necrosis, type II pneumocyte hyperplasia peaking at days four and eight, cumulative inflammation maximal at day eight, and atypical adenomatous hyperplasia at day fourteen after most inflammation had resolved (Figures 3A to 3E). Apoptosis in bronchial epithelium, nested neutrophil accumulations and severe vascular edema were observed at day three (Figures 3F to 3H).\n\n6. The interferon stimulated gene response tracked viral load in the trachea but did not contain replication, and high lung viral load followed on day four (Figures 4A and 4B). A hundredfold higher inoculum removed the delay in interferon stimulated gene induction in both tissues without restricting the virus. Irrespective of dose, induction of many classical interferon stimulated genes appeared limited.\n\n7. Inflammation moved from the upper to the lower respiratory tract. With a one hundred plaque forming unit challenge, Ccl4 and Ccl5 peaked at day two in trachea but not until day six in lung, and the tracheal peak preceded the lung peak by roughly four days (Figures 5A, 5B and S5A). Tnf and Il1b were high in the upper tract and largely absent in the lower, while Il36a, Cxcl6 and Ccl22 were higher in lung. Detectable Ifnb and Ifnl reads appeared only in trachea one day after a high dose infection and in no other tissue at any time (Figures 5C and 5D). Cytokine expression fell by day fourteen, coinciding with peak spike specific serum IgG (Figure 5E).\n\n8. Distal tissues showed a strong antiviral transcriptional response despite viral reads orders of magnitude below the respiratory tract. Interferon signaling enrichment was evident in brain, olfactory bulb and small intestine, brain differential expression peaked at day two coinciding with the upper respiratory tract viral peak, and the most sustained interferon response was in the olfactory bulb (Figures 6A to 6F and S6).\n\n9. Subgenomic nucleocapsid RNA was amplifiable in olfactory bulb, brain and small intestine on days one, two and four, and as far out as day eight in olfactory bulb at roughly one two hundredth of day four lung levels, but did not consistently correlate with infectious particles by plaque assay (Figures 6G and 6H). The authors interpret this as suggesting that dissemination of virus derived pathogen associated molecular patterns, rather than productive infection, may underlie the distal inflammation, and they state that further study is needed to substantiate it.\n\n10. Prophylactic intranasal interferon alpha A/D induced a broad interferon stimulated gene program in hamster lung (Figure 7A, Table S4) and, when started twenty four hours before challenge, reduced infectious virus and subgenomic nucleocapsid RNA and lowered Cxcl11 and Il6 (Figures 7B to 7E), with reduced nucleocapsid and increased MxA protein by immunohistochemistry and less inflammation by histology (Figures 7F to 7H).\n\n11. Therapeutic intranasal interferon alpha A/D started twenty four hours after infection reduced infectious virions and raised Il10 at day three, and at day six reduced subgenomic membrane and nucleocapsid RNA and Il6, with an infiltrate weighted toward reactive macrophages rather than neutrophils and increased bronchial epithelial mitosis (Figures 7I to 7M).\n\n12. In a contact transmission design, prophylactic intranasal interferon alpha A/D reduced titers in infected animals by more than two logs and prevented transmission in three of five exposed animals, corroborated by nucleocapsid and MxA immunohistochemistry and by lung subgenomic nucleocapsid levels (Figures S7E to S7H). Intranasal administration of a double-stranded RNA mimetic gave antiviral activity comparable to interferon alpha A/D (Figure S7I).", "Mechanistic model": "The study does not establish a definitive mechanism for either the systemic inflammation or the protective effect of local interferon. What the data support is a sequence rather than a molecular chain. Virus replicates first and most heavily in the upper respiratory tract, where chemokine and interferon stimulated gene induction begins, then spreads to the lower respiratory tract where the same programs appear several days later, and inflammation appears in olfactory bulb, brain and small intestine at times that track the upper respiratory tract viral peak rather than local replication. For the distal inflammation the authors propose, explicitly as speculation, that abundant subgenomic viral RNA, possibly protected as a nucleocapsid bound ribonucleoprotein, is secreted or spills over from the primary replication site and acts as a disseminated pathogen associated molecular pattern. The supporting observation is the discordance between subgenomic nucleocapsid detection and infectious particle recovery, which is suggestive rather than demonstrative, since neither transfer of such material nor its recognition by a specific sensor was tested. For the intervention, the data show that raising the interferon stimulated gene set in the airway before or shortly after challenge lowers viral load, lowers proinflammatory transcripts, and changes the composition of the infiltrate. Whether the reduced neutrophil presence follows from the lower viral load, from a direct effect of interferon on recruitment, or from both is not resolved. The authors also note it remains unclear whether the heightened distal antiviral state itself restricts viral tropism in this model.", "Conceptual or technical advance": "The study provides a longitudinal, multi tissue transcriptional atlas of SARS-CoV-2 infection in a naturally permissive small animal, annotated well enough to read interferon biology, since the previously unannotated hamster Ifnb1 was identified and functionally validated as part of the work. That makes the golden hamster usable for interferon focused questions rather than only for viral load and pathology endpoints. Conceptually, the separation between where virus replicates and where inflammation appears reframes distal COVID-19 manifestations as potentially driven by disseminated viral material rather than requiring distal productive infection. Practically, the work moves interferon from a systemic therapeutic, where tolerability has limited it, to a local airway prophylactic and early therapeutic, and shows that the same benefit can be obtained by delivering a pattern recognition receptor agonist that induces endogenous interferon, which the authors note is cheaper and easier to manufacture and store.", "Relationship to the broader research program": "The authors state their general strategy as developing broad-spectrum antiviral countermeasures that exploit the host response rather than pathogen specific interventions for each new outbreak, and they frame airway delivery of type I interferon as an instance of that strategy. The work connects directly to the laboratory's earlier description of an imbalanced host response to SARS-CoV-2 across cell, ferret and patient systems, which it extends into a systemic and longitudinal frame. The proposal that viral RNA can act as a disseminated pathogen associated molecular pattern is linked by the authors to their own earlier work on unrelated RNA viruses. Category 3 synthesis, requiring several corpus papers side by side, is that the laboratory repeatedly builds animal models in which the host transcriptional response, rather than viral titer alone, is the primary readout, and then intervenes on that response. That statement is not asserted from this paper alone.", "Related publications": "- Blanco-Melo et al. 2020, imbalanced host response to SARS-CoV-2 drives development of COVID-19, predecessor. Cited throughout as the source of the low interferon and high chemokine framing and of the ferret model comparison, and shares authors with this study.\n- Langlois et al. 2013, microRNA based strategy to mitigate the risk of gain of function influenza studies, methodological foundation. Appears in the reference list as prior laboratory work on engineered antiviral approaches.\n- tenOever et al. 2002 and 2004, cited by the authors as prior support for the idea that viral RNA species can be disseminated and detected apart from infectious particles, conceptual extension.\n- Si et al. 2020, human organ chip enabled pipeline to rapidly repurpose therapeutics during viral pandemics, companion. Shares several authors and belongs to the same effort to identify host directed countermeasures.", "Limitations and boundaries": "The authors provide their own limitations section. The study uses young golden hamsters that clear the virus and survive, so it does not represent older animals or lethal disease, and both age and severity may alter the host response and tropism. Extrapolation to human disease is explicitly flagged. The value of intranasal interferon late in the disease course was not tested, and the authors call for work in a lethal model and in animals genetically lacking innate immune sensors such as TLR3 and TLR7. Beyond the stated limitations, the transcriptomics are bulk rather than single cell, so cell type attributions such as T cell or macrophage origin of particular transcripts are inferences from marker genes rather than direct measurements. Distal tissue conclusions rest on small group sizes, with as few as two animals in one small intestine condition, and on pooled RNA for some subgenomic RNA measurements. The evidence that disseminated viral RNA drives distal inflammation is circumstantial. Hamster genome annotation is incomplete, gene names are given by human ortholog, and the Ifnb1 transcript used here was newly assigned by the authors. The inoculum dose strongly shapes the response, which the authors note should be considered before extrapolating any of these data to human disease. Finally, the interferon intervention was tested as intranasal administration in a rodent airway and does not establish efficacy or dosing in humans.", "Audience summaries": "### 25 words\n\nHamsters infected with SARS-CoV-2 develop inflammation in organs the virus barely reaches, and interferon sprayed into the nose before or after exposure lowers virus and disease.\n\n### 75 words\n\nSARS-CoV-2 provokes strong chemokine signaling but weak interferon signaling, a combination linked to severe COVID-19. Profiling infected golden hamsters across tissues and time showed inflammation moving from the upper to the lower airway and appearing in brain, olfactory bulb and intestine despite almost no virus there. Delivering type I interferon directly into the nose, before or one day after infection, reduced viral load, tissue damage and onward transmission.\n\n### 150 words\n\nGolden hamsters infected with an unmodified clinical SARS-CoV-2 isolate were profiled longitudinally by mRNA sequencing, quantitative RT-PCR, plaque assay and histopathology across trachea, lung, olfactory bulb, brain and small intestine. Compared against influenza A virus at matched viral load, SARS-CoV-2 drove a stronger inflammatory and neutrophil associated signature. A newly assembled and functionally validated hamster interferon beta gene allowed interferon induction to be measured, and it remained low while chemokines rose. Interferon stimulated gene and chemokine peaks occurred in the trachea several days before the lung, and antiviral programs appeared in distal organs where infectious virus was scarce, which the authors attribute tentatively to disseminated viral RNA acting as a pattern recognition ligand. Intranasal universal interferon alpha A/D given before challenge, or beginning one day after, lowered infectious virus and inflammatory transcripts, shifted the lung infiltrate toward macrophages, and blocked transmission in three of five contact exposed animals." }, "discoveries": [ "claim-13" ], "relationships": [ { "from": "2021-daniloski-identification-of-required-host-fa", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "companion", "evidence": "stated in the Related publications section of 2021-daniloski-identification-of-required-host-fa" }, { "from": "2021-eriksen-sars-cov-2-infects-human-adult-don", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-eriksen-sars-cov-2-infects-human-adult-don" }, { "from": "2021-hoagland-leveraging-the-antiviral-type-i-in", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-hoagland-leveraging-the-antiviral-type-i-in" }, { "from": "2022-frere-sars-cov-2-infection-in-hamsters-a", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2022-frere-sars-cov-2-infection-in-hamsters-a" }, { "from": "2022-oishi-a-diminished-immune-response-under", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2022-oishi-a-diminished-immune-response-under" }, { "from": "2022-zazhytska-non-cell-autonomous-disruption-of-", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2022-zazhytska-non-cell-autonomous-disruption-of-" }, { "from": "2022-zazhytska-non-cell-autonomous-disruption-of-", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2022-zazhytska-non-cell-autonomous-disruption-of-" }, { "from": "2023-carrau-delayed-engagement-of-host-defense", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2023-carrau-delayed-engagement-of-host-defense" }, { "from": "2023-zhang-mouse-genome-rewriting-and-tailori", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2023-zhang-mouse-genome-rewriting-and-tailori" } ], "canonical_url": "https://tenoeverlab.us/publications/2021-hoagland-leveraging-the-antiviral-type-i-in/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sars-cov-2" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "pathway-enrichment-analysis", "in-vivo-infection-route", "histopathology", "elisa", "immunohistochemistry", "de-novo-transcriptome-assembly" ] } }, { "id": "2021-horiuchi-immune-memory-from-sars-cov-2-infe", "slug": "2021-horiuchi-immune-memory-from-sars-cov-2-infe", "url": "/publications/2021-horiuchi-immune-memory-from-sars-cov-2-infe/", "title": "Immune memory from SARS-CoV-2 infection in hamsters provides variant-independent protection but still allows virus transmission", "authors": [ "Shu Horiuchi", "Kohei Oishi", "Lucia Carrau", "Justin Frere", "Rasmus Møller", "Maryline Panis", "Benjamin R. tenOever" ], "author_count": 7, "first_author": "Shu Horiuchi", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 7, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2021, "journal": "Science Immunology", "volume": "6", "issue": "66", "pages": "eabm3131", "doi": "10.1126/sciimmunol.abm3131", "doi_url": "https://doi.org/10.1126/sciimmunol.abm3131", "pmid": "34699266", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/34699266/", "pmcid": null, "pmc_url": null, "pmc_pdf_url": null, "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "immunity-age-and-reinfection" ], "pathogens": [ "SARS-CoV-2", "SARS-CoV-2 USA-WA1/2020", "SARS-CoV-2 B.1.351 beta variant", "influenza A/California/04/2009" ], "viral_families": [ "Coronaviridae", "Orthomyxoviridae" ], "host_species": [ "golden hamster" ], "technologies": [ "flow cytometry with cross-reactive antibodies", "mRNA sequencing", "gene set enrichment analysis", "quantitative RT-PCR", "plaque assay", "plaque reduction neutralization test", "anti-RBD ELISA", "peptide restimulation assay", "biotinylated antigen B cell probe", "adoptive cell transfer", "CellTrace Violet labeling", "cohousing transmission model" ], "biological_systems": [ "golden hamster", "hamster lung", "mediastinal lymph node", "peripheral blood mononuclear cells", "spleen", "Vero E6 cells", "MDCK cells" ], "key_concepts": [ "immune memory", "adaptive immune response", "antigen-specific T cells", "antigen-specific B cells", "delayed innate response", "variant of concern", "neutralizing antibody", "adoptive transfer", "transmission despite immunity", "golden hamster model development" ], "keywords": [ "SARS-CoV-2", "golden hamster", "immune memory", "beta variant", "transmission", "T cells", "B cells", "neutralizing antibodies", "influenza A virus", "reinfection" ], "one_sentence_contribution": "Longitudinal tracking of antigen-specific lymphocytes in golden hamsters shows that memory from the founder strain of SARS-CoV-2 clears a beta variant rechallenge and that transferred memory T cells alone lower viral load, yet protected animals still infected every cohoused naive partner.", "summary_25": "Hamsters recovered from SARS-CoV-2 cleared a beta variant rechallenge and transferred memory T cells alone lowered viral load, yet protected animals still infected cohoused naive partners.", "summary_75": "Reagents for immune profiling in golden hamsters were assembled from cross-reactive antibodies, then used to follow SARS-CoV-2 against influenza as a benchmark. The innate response to SARS-CoV-2 was delayed but the adaptive response was stronger, and antigen-specific T and B cells persisted past 40 days. Recovered animals rechallenged four months later showed no detectable virus yet still infected cohoused naive animals. Memory raised against the founder strain cleared a beta variant their serum neutralized poorly in vitro.", "summary_150": "Human COVID-19 immunology lacked controlled rechallenge and transmission experiments, and the golden hamster lacked reagents for immune phenotyping. Cross-reactive antibodies, peptide restimulation and biotinylated spike probes resolved antigen-specific T and B cells in this species. Compared with influenza at matched peak lung titer, SARS-CoV-2 delayed NF-kB and chemokine induction by about two days and cleared more slowly, but elicited more CD8 and T follicular helper cells and fewer regulatory T cells, with antigen-specific populations persisting beyond 40 days. Adoptive transfer of B cell-depleted lymphocytes from recovered animals lowered nasal titers in naive recipients and was followed by rapid spike-specific B cell appearance. Animals rechallenged with the homologous strain at four months yielded no detectable virus yet infected all eight cohoused naive partners. Rechallenge with the B.1.351 beta variant gave equal early titers but complete clearance by day four, despite poor in vitro neutralization of that variant.", "citation": "Horiuchi S, Oishi K, Carrau L, Frere J, Møller R, Panis M, tenOever BR. Immune memory from SARS-CoV-2 infection in hamsters provides variant-independent protection but still allows virus transmission. Science Immunology, 2021, volume 6, issue 66, article eabm3131. DOI 10.1126/sciimmunol.abm3131. PMID 34699266.", "sections": { "Citation": "Horiuchi S, Oishi K, Carrau L, Frere J, Møller R, Panis M, tenOever BR. Immune memory from SARS-CoV-2 infection in hamsters provides variant-independent protection but still allows virus transmission. Science Immunology, 2021, volume 6, issue 66, article eabm3131.\n\nDOI 10.1126/sciimmunol.abm3131. PMID 34699266.", "One-sentence contribution": "Longitudinal tracking of antigen-specific lymphocytes in golden hamsters shows that memory from the founder strain of SARS-CoV-2 clears a beta variant rechallenge and that transferred memory T cells alone lower viral load, yet protected animals still infected every cohoused naive partner.", "Executive summary": "Clinical immunology of COVID-19 was constrained by the absence of controlled rechallenge and transmission experiments, and the golden hamster had been shown to reproduce much of COVID-19 biology without the reagent base needed for detailed immune phenotyping. This study built that reagent base, using cross-reactive and commercial anti-hamster antibodies to resolve T cell and B cell populations by flow cytometry, and then used it to follow the response to SARS-CoV-2 longitudinally against influenza A virus as a benchmark. Both viruses reached comparable peak lung titers, but influenza was cleared by 7 days while SARS-CoV-2 persisted, and transcriptional profiling showed that NF-kB-associated and chemokine gene induction was delayed by about two days after SARS-CoV-2 infection. The delayed innate response was followed by an adaptive response that exceeded the one to influenza, with more CD8 T cells and T follicular helper cells and fewer regulatory T cells. Antigen-specific CD4 T cells and spike-specific B cells were detectable at 7 and 14 days and persisted beyond 40 days in lung, spleen and blood. Recovered animals rechallenged four months later with the same strain showed no detectable infectious virus, yet every cohoused naive animal became infected. Adoptive transfer of B cell-depleted lymphocytes from recovered animals reduced nasal wash titers in naive recipients and was followed by rapid appearance of spike-specific B cells. Rechallenge with the B.1.351 beta variant gave equal early titers but complete clearance by 4 days in previously exposed animals, with induction of antibodies neutralizing both the founder strain and the variant.", "Scientific context": "The host response to virus infection begins with recognition of pathogen-associated molecular patterns, activating the interferon regulatory factor and NF-kB pathways, inducing type I interferon and the interferon-stimulated genes, and recruiting the adaptive response through chemokines such as CXCL10 and CCL5. Influenza A virus masks aberrant RNA through NS1, giving a minimal transcriptional response in the infected cell. SARS-CoV-2 was known to diminish type I interferon while nonetheless driving high NF-kB activation for reasons the authors state remain unclear, a combination associated with poor local control and high proinflammatory output. At the time of the study, vaccination against spike was established as effective at preventing COVID-19, and virus-specific T and B cells had been detected in human blood for up to eight months after vaccination. What remained unknown was whether that memory blocks transmission or reinfection, including reinfection by variants under positive selection, because human data lacked the controls and designs available in a small animal model. The golden hamster had been shown to largely phenocopy COVID-19 biology, but commercial immunological reagents for the species were scarce.", "Central question": "Does immune memory established by SARS-CoV-2 infection protect the host against rechallenge, including by a variant of concern with a divergent spike protein, and does that protection prevent onward transmission.", "Experimental strategy": "The design has two halves, one methodological and one comparative. Because the golden hamster lacks a reagent base, the first task was to assemble a flow cytometry panel from antibodies raised against human and mouse targets that cross-react with hamster orthologs, combined with the few commercial anti-hamster reagents, with titrations chosen to maximize separation between populations. Antigen specificity was then read in two ways, by restimulating lymphocytes with pooled spike, nucleocapsid and matrix peptides and scoring Ki67 and IRF4 co-expression in CD4 T cells, and by labeling B cells with biotinylated spike detected through two differently colored streptavidins, with specificity controlled using splenocytes from influenza-infected animals.\n\nThe comparative half uses influenza A/California/04/2009 as a benchmark rather than as an object of study, on the reasoning that a well-characterized respiratory RNA virus with similar peak titer provides the calibration needed to say what is distinctive about the SARS-CoV-2 response in this host. Three perturbations then separate the components of memory. Rechallenge with the homologous strain at more than four months tests durability, with cohousing of naive animals one day after rechallenge added to test transmission, and the one-day delay before moving animals to a new cage is what distinguishes transmission from direct carryover of inoculum. Adoptive transfer of lymphocytes depleted of B cells using anti-MHC class II, anti-IgG and anti-IgM tests whether T cells contribute independently, and it also circumvents the confound of residual antibody in the recovered animals themselves. Rechallenge with the B.1.351 beta variant tests whether memory raised against the founder spike covers a divergent one, in a setting where T cells and other components act together, which a serum neutralization assay in vitro cannot capture.", "Key findings": "1. Peak lung titers were comparable between viruses, around 10^8 plaque-forming units at 3 days after infection, from inocula of 10^5 plaque-forming units for influenza and 10^3 for SARS-CoV-2. Influenza was undetectable by 7 days, whereas SARS-CoV-2 titers continued with little change until 7 days (Fig. 1A and 1B), and SARS-CoV-2-infected animals showed delayed growth relative to mock and influenza.\n\n2. The innate response to SARS-CoV-2 is delayed. mRNA sequencing of lung at 3 and 5 days showed influenza driving NF-kB-associated and chemokine gene expression at 3 days that returned toward baseline by 5 days, while SARS-CoV-2 required two additional days to reach a comparable response despite similar viral load (Fig. 1C to 1E). Quantitative RT-PCR for Isg15, Irf7, Cxcl10 and Ccl5 gave the same pattern (Fig. 1F). The authors read this as SARS-CoV-2 antagonizing the host response more potently than influenza in this model.\n\n3. The adaptive response to SARS-CoV-2 exceeds the response to influenza. Total lymphoid cell yield from lung rose at 7 days after SARS-CoV-2, as did total CD3 T cell frequency, returning to baseline by 14 days, with the increase attributable to CD8 cells (Fig. 2B to 2D). Both viruses induced CXCR3-positive T helper 1 cells, but SARS-CoV-2 gave significantly lower CXCR3-negative FoxP3-positive regulatory T cells than influenza, a more robust and sustained CXCR5 and Bcl6 double-positive T follicular helper population, and stronger CXCR3-positive CD8 T cells (Fig. 2E to 2G). B cell frequencies did not differ significantly between the two infections.\n\n4. Antigen-specific cells persist as memory. Ki67 and IRF4 double-positive CD4 T cells responding to spike, nucleocapsid and matrix peptides, and spike-specific B cells, were present at 7 and 14 days, with lung frequencies highest early and blood frequencies rising later (Fig. 3B and 3C). Both populations were still present beyond 40 days, with antigen-specific CD4 T cells in lung and spleen but below detection in blood, and spike-specific B cells at high frequency in lung, spleen and blood (Fig. 3D and 3E).\n\n5. Anti-RBD IgG and IgG2 and neutralizing titers peaked within two weeks and then declined to a plateau that remained above mock (Fig. 3F and 3G), with reported 50 percent plaque reduction neutralization values of 30.63 at 5 days, 1550.00 at 7 days, 829.30 at 14 days and 988.30 at 35 days.\n\n6. Homologous rechallenge at more than 120 days gave near-complete protection, with no infectious virus recoverable from nasal wash at 2 and 5 days or from lung at 5 days (Fig. 4B). Despite this, all eight cohoused naive animals became infected, with robust titers in both nasal wash and lung (Fig. 4C). The authors interpret the discrepancy as transmission of virus present below the limit of detection, citing prior work that very low amounts suffice for transmission in this model. This is interpretation, since no infectious particle was detected in the transmitting animals.\n\n7. A caution the authors raise about their own rechallenge result. Antibody amounts in rechallenged animals were similar to those at 72 days, and spike-specific B cell frequency did not correlate with anti-RBD titer after homologous reinfection, which they read as indicating that the antibody and B cell measurements do not reflect a memory recall and that protection in this experiment may instead have come from residual antibody from the primary infection.\n\n8. Memory T cells contribute independently. Adoptive transfer was first validated by CellTrace Violet labeling, showing donor lymphoid cells persisting in recipient organs for up to 4 days. Transfer of unfractionated cells from recovered animals produced a significant boost in anti-RBD antibody at 3 days in recipients, indicating functional transferred B cells, with the effect lost by 8 days as the primary response took over. Transfer of B cell-depleted lymphocytes from lung, blood and spleen of recovered animals into naive recipients, one day before challenge, significantly lowered nasal wash titers at 1, 3 and 5 days relative to matched control transfers (Fig. 5B), with lung titers trending downward (Fig. 5C), and spike-specific B cells were readily detectable in all recipients of T cells from recovered donors (Fig. 5D). The authors note baseline differences in viral load between cohorts receiving different cell lineages and state that comparisons were therefore made only within treatment cohorts.\n\n9. Memory raised against the founder strain clears a beta variant challenge. Serum from animals infected with the founder strain neutralized the beta variant far less well in vitro, with 50 percent plaque reduction values of 1030.00 and 896.20 against the founder strain at 7 and 14 days against 12.01 and 5.53 against the variant (Fig. 6A). In vivo, previously exposed and naive animals rechallenged with the beta variant at 120 days showed comparable nasal wash titers at 1 and 2 days, but previously exposed animals had cleared infectious virus by 4 days while the control group remained positive to 5 days, a pattern mirrored in lung (Fig. 6C). Spike-specific B cells and anti-RBD antibodies were robustly induced, their frequencies and titers correlated, and neutralizing titers rose against both the founder strain and the variant (Fig. 6D to 6G).", "Mechanistic model": "The study establishes several causal relationships and stops short of a full mechanism, and the paper is explicit about which is which.\n\nWhat the data support directly. Memory T cells are sufficient, in the near absence of B cells, to lower SARS-CoV-2 titers in the upper respiratory tract of a naive recipient, which the adoptive transfer of B cell-depleted lymphocytes shows. Their presence is followed by rapid appearance of spike-specific B cells and virus-specific antibody in recipients that received no memory B cells, which the authors read as memory T cells driving early induction of virus-specific antibody. Memory established against the founder spike is sufficient to clear a variant whose spike escapes neutralization by the same animals' serum in vitro, and the difference between the in vitro and in vivo outcomes is the basis for the paper's argument that serum neutralization assays understate protection because they omit T cells and the rest of the response.\n\nWhat is not established. The study does not identify which effector functions of the transferred T cells reduce viral load, does not distinguish CD4 from CD8 contributions, and by the authors' own account of their reagent limitations could not resolve antigen-specific CD8 T cells at all. The mechanism by which SARS-CoV-2 delays the innate response in this host is not addressed here, only observed. The explanation for transmission from animals in which no infectious particle was detected, that the transmitted dose lay below the assay's limit of detection, is a proposal consistent with prior observations in this model rather than a measured quantity. The relationship between the homologous rechallenge protection and memory is left deliberately unresolved by the authors, who raise residual antibody as an alternative account for that particular experiment. The clearance of the beta variant is therefore the stronger of the two rechallenge results, since it occurred in animals whose serum neutralized the challenge virus poorly.", "Conceptual or technical advance": "Two things became possible because of this work. The first is technical. A flow cytometry approach for the golden hamster was assembled from cross-reactive antibodies, with the paper reporting CXCR3, CXCR5 and Bcl6 as newly usable in this species, together with peptide restimulation and biotinylated antigen probes for detecting antigen-specific T and B cells and a validated adoptive transfer procedure that exploits the inbred status of the animals. This turns a model previously valued for reproducing disease into one in which the cellular components of the immune response can be dissected.\n\nThe second is conceptual. By running rechallenge and transmission in the same animals, the study separates protection of the host from interruption of spread, and finds them dissociated. By running a variant rechallenge in vivo alongside serum neutralization in vitro on the same animals, it shows the two measures giving different answers, which bears directly on how reduced neutralization titers against variants should be interpreted. The authors extend both findings to the human situation in their discussion, noting that recovered or vaccinated individuals may transmit without knowing it and that immune memory nonetheless appears sufficient against the variant tested. Those extensions are the authors' reading of a hamster experiment and are not demonstrated in humans here.", "Relationship to the broader research program": "This is a tenOever laboratory study with the corresponding author and the senior position held by tenOever, and it extends that laboratory's established use of the golden hamster for SARS-CoV-2, cited here as prior work. It also continues a recurring pattern in the corpus, the use of influenza A virus as a calibrated comparator against which the behavior of another virus is measured, and the treatment of the interferon and NF-kB transcriptional response as the primary readout of what a virus does to its host, with Isg15, Irf7, Cxcl10 and Ccl5 used as the index genes.\n\nCategory 3 synthesis, visible only across papers. The framing that morbidity follows from a virus antagonizing host defense, stated in the introduction here through the contrast between NS1-mediated masking by influenza and the delayed response to SARS-CoV-2, is the same view of host and virus that organizes the 2013 Nature Reviews Microbiology article, where the chordate protein-based defense is presented as the system viruses must evade. The interferon-stimulated gene readouts used here sit downstream of the machinery dissected in the 2007 Science report on IKKε. Neither connection is claimed by the paper and both require the other papers to see.", "Related publications": "- Blanco-Melo et al. 2020 and the earlier hamster work cited in this paper from the same laboratory, predecessor, establishing the transcriptional response to SARS-CoV-2 and the golden hamster model that this study builds on.\n- Bouhaddou et al. 2020, Cell, shares the pathogen and includes tenOever among its authors, but no data, method or claim connects it to this study, so no relationship is asserted beyond a shared subject.\n- tenOever 2019, Cold Spring Harbor Perspectives in Medicine, and tenOever 2013, Nature Reviews Microbiology, from the same author. Conceptually adjacent through the shared framing of virus and host antagonism and the use of influenza A virus as a reference system, but neither is cited here and neither supplies method or data.", "Limitations and boundaries": "The authors set out their own reagent-driven limits. The absence of commercial hamster antibodies meant that B cell-specific and CD8 T cell-specific populations could not be identified directly, that CD3 staining required fixation and so live cells of that population could not be sorted, that chemokine markers were not detectable after stimulation and culture, and that Ki67 and IRF4 background was too high in the absence of CD8 staining to resolve antigen-specific CD8 T cells or CD4 subsets. The T cell adoptive transfers therefore test a B cell-depleted lymphocyte population rather than a defined T cell subset, and the paper notes baseline viral load differences between cohorts receiving different cell lineages, restricting comparisons to within-cohort. Beyond these, the work is confined to the golden hamster, a species chosen for phenocopying COVID-19 but not equivalent to humans in immune repertoire or reagent-verified cell definitions. One founder strain and one variant of concern were tested, so the conclusion about variant-independent protection is bounded by B.1.351 and does not extend to variants that arose later. Challenge was by a single intranasal dose of 10^3 plaque-forming units under anesthesia, which is not equivalent to natural exposure. Rechallenge intervals were four months, so durability beyond that is untested. The transmission result rests on cohousing at a one to one ratio in eight pairs with no infectious virus detected in the donors, so the transmitted dose was never measured. The homologous rechallenge protection cannot be attributed to memory rather than residual antibody, as the authors themselves state. Immunity was generated by infection rather than by vaccination, so conclusions do not transfer directly to vaccinated hosts, a point the discussion addresses by analogy rather than by experiment.", "Audience summaries": "### 25 words\n\nHamsters recovered from SARS-CoV-2 cleared a beta variant rechallenge and transferred memory T cells alone lowered viral load, yet protected animals still infected cohoused naive partners.\n\n### 75 words\n\nReagents for immune profiling in golden hamsters were assembled from cross-reactive antibodies, then used to follow SARS-CoV-2 against influenza as a benchmark. The innate response to SARS-CoV-2 was delayed but the adaptive response was stronger, and antigen-specific T and B cells persisted past 40 days. Recovered animals rechallenged four months later showed no detectable virus yet still infected cohoused naive animals. Memory raised against the founder strain cleared a beta variant their serum neutralized poorly in vitro.\n\n### 150 words\n\nHuman COVID-19 immunology lacked controlled rechallenge and transmission experiments, and the golden hamster lacked reagents for immune phenotyping. Cross-reactive antibodies, peptide restimulation and biotinylated spike probes resolved antigen-specific T and B cells in this species. Compared with influenza at matched peak lung titer, SARS-CoV-2 delayed NF-kB and chemokine induction by about two days and cleared more slowly, but elicited more CD8 and T follicular helper cells and fewer regulatory T cells, with antigen-specific populations persisting beyond 40 days. Adoptive transfer of B cell-depleted lymphocytes from recovered animals lowered nasal titers in naive recipients and was followed by rapid spike-specific B cell appearance. Animals rechallenged with the homologous strain at four months yielded no detectable virus yet infected all eight cohoused naive partners. Rechallenge with the B.1.351 beta variant gave equal early titers but complete clearance by day four, despite poor in vitro neutralization of that variant." }, "discoveries": [], "relationships": [ { "from": "2020-bouhaddou-the-global-phosphorylation-landsca", "to": "2021-horiuchi-immune-memory-from-sars-cov-2-infe", "relationship": "related", "evidence": "stated in the Related publications section of 2020-bouhaddou-the-global-phosphorylation-landsca" }, { "from": "2021-horiuchi-immune-memory-from-sars-cov-2-infe", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-horiuchi-immune-memory-from-sars-cov-2-infe" }, { "from": "2021-horiuchi-immune-memory-from-sars-cov-2-infe", "to": "2020-bouhaddou-the-global-phosphorylation-landsca", "relationship": "related", "evidence": "stated in the Related publications section of 2021-horiuchi-immune-memory-from-sars-cov-2-infe" }, { "from": "2022-oishi-a-diminished-immune-response-under", "to": "2021-horiuchi-immune-memory-from-sars-cov-2-infe", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2022-oishi-a-diminished-immune-response-under" } ], "canonical_url": "https://tenoeverlab.us/publications/2021-horiuchi-immune-memory-from-sars-cov-2-infe/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sars-cov-2" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "flow-cytometry", "pathway-enrichment-analysis", "elisa", "animal-transmission-model", "antigen-specific-b-cell-detection", "cell-division-tracking", "neutralization-assay", "adoptive-transfer", "t-cell-restimulation" ] } }, { "id": "2021-nilsson-payant-reduced-nucleoprotein-availability", "slug": "2021-nilsson-payant-reduced-nucleoprotein-availability", "url": "/publications/2021-nilsson-payant-reduced-nucleoprotein-availability/", "title": "Reduced Nucleoprotein Availability Impairs Negative-Sense RNA Virus Replication and Promotes Host Recognition", "authors": [ "Benjamin E. Nilsson-Payant", "Daniel Blanco-Melo", "Skyler Uhl", "Beatriz Escudero-Pérez", "Silke Olschewski", "Patricia Thibault", "Maryline Panis", "Maria Rosenthal", "César Muñoz-Fontela", "Benhur Lee", "Benjamin R. tenOever" ], "author_count": 11, "first_author": "Benjamin E. Nilsson-Payant", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 11, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2021, "journal": "Journal of Virology", "volume": "95", "issue": "9", "pages": "e02274-20", "doi": "10.1128/jvi.02274-20", "doi_url": "https://doi.org/10.1128/jvi.02274-20", "pmid": "33568513", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/33568513/", "pmcid": "PMC8104106", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104106/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104106/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "influenza-genome-regulation", "innate-immune-signaling" ], "themes": [ "sensing-aberrant-rna", "polymerase-nucleoprotein-host-factors" ], "pathogens": [ "influenza A virus", "Sendai virus", "human parainfluenza virus 3", "measles virus", "human respiratory syncytial virus", "vesicular stomatitis virus", "Ebola virus", "Lassa virus", "SARS-CoV-2" ], "viral_families": [ "Orthomyxoviridae", "Paramyxoviridae", "Pneumoviridae", "Rhabdoviridae", "Filoviridae", "Arenaviridae", "Coronaviridae" ], "host_species": [ "human" ], "technologies": [ "microRNA target site insertion into viral genomes", "reverse genetics", "small interfering RNA knockdown", "bulk mRNA sequencing", "ribosomal RNA-depleted total RNA sequencing", "noncanonical junction read analysis", "Northern blot", "interferon-stimulated response element luciferase reporter", "flow cytometry", "immunoblotting", "reconstituted influenza replication complex" ], "biological_systems": [ "A549 cells", "A549-ACE2 cells", "A549-Dual reporter cells", "RIG-I-deficient A549 cells", "MDA5-deficient A549 cells", "MAVS-deficient A549 cells", "HEK-293T cells", "HEK-293T NoDice cells", "Vero E6 cells", "BHK-21 cells", "MDCK cells", "HeLa cells" ], "key_concepts": [ "nucleoprotein scaffold", "viral ribonucleoprotein complex", "defective viral genomes", "mini-viral RNA", "copy-back defective genomes", "polymerase processivity", "RIG-I and MAVS signalling", "pathogen-associated molecular patterns", "interferon induction", "antiviral drug target selection", "bystander priming" ], "keywords": [ "nucleoprotein", "negative-sense RNA virus", "defective viral genomes", "RIG-I", "interferon", "influenza A virus", "Sendai virus", "nucleozin", "baloxavir marboxil", "SARS-CoV-2 nucleocapsid" ], "one_sentence_contribution": "Restricting nucleoprotein availability in negative-sense RNA virus infections both blocks full-length genome replication and increases production of aberrant replication products that are sensed through RIG-I and MAVS, so a strong interferon response accompanies rather than follows successful infection.", "summary_25": "Starving a negative-sense RNA virus of its nucleoprotein stops it replicating but makes it far more visible, because the polymerase then churns out short immune-triggering fragments.", "summary_75": "Negative-sense RNA viruses wrap their genomes in nucleoprotein, which the polymerase also needs to copy full-length templates. Silencing nucleoprotein in influenza and Sendai virus infections blocked replication yet produced a much stronger interferon response, because the polymerase generated short defective genomes that RIG-I detects. The same pairing held for seven viruses across six families but not for SARS-CoV-2 nucleocapsid, and a nucleoprotein-directed drug triggered interferon where a polymerase-directed drug did not.", "summary_150": "Nilsson-Payant and colleagues used recombinant influenza A and Sendai viruses carrying microRNA target sites downstream of the nucleoprotein open reading frame, which degrades nucleoprotein messenger RNA without targeting genomic RNA. Silencing abolished detectable viral protein and full-length genome replication while strongly inducing interferon-stimulated genes relative to the viral material present. Sequencing showed terminal read enrichment and increased noncanonical junctions, and Northern blotting showed accumulation of mini-viral RNA tracking with interferon beta. Titrating nucleoprotein against fixed polymerase in a reconstituted replication complex reproduced the inverse relationship between full-length product and mini-viral RNA, and reporter cells lacking RIG-I or MAVS lost the response while MDA5-deficient cells did not. Knockdown of nucleoprotein in seven negative-sense viruses across six families consistently paired lost replication with IFIT1 induction, whereas SARS-CoV-2 nucleocapsid knockdown reduced replication without inducing interferon. The authors propose nucleoprotein as a drug target that would also engage host defences.", "citation": "Nilsson-Payant BE, Blanco-Melo D, Uhl S, Escudero-Pérez B, Olschewski S, Thibault P, Panis M, Rosenthal M, Muñoz-Fontela C, Lee B, tenOever BR. Reduced nucleoprotein availability impairs negative-sense RNA virus replication and promotes host recognition. Journal of Virology. 2021. Volume 95, issue 9, article e02274-20. DOI 10.1128/jvi.02274-20. PMID 33568513. PMCID PMC8104106. Nilsson-Payant and Blanco-Melo contributed equally, with author order determined by drawing straws.", "sections": { "Citation": "Nilsson-Payant BE, Blanco-Melo D, Uhl S, Escudero-Pérez B, Olschewski S, Thibault P, Panis M, Rosenthal M, Muñoz-Fontela C, Lee B, tenOever BR. Reduced nucleoprotein availability impairs negative-sense RNA virus replication and promotes host recognition. Journal of Virology. 2021. Volume 95, issue 9, article e02274-20.\n\nDOI 10.1128/jvi.02274-20. PMID 33568513. PMCID PMC8104106.\n\nNilsson-Payant and Blanco-Melo contributed equally, with author order determined by drawing straws.", "One-sentence contribution": "Restricting nucleoprotein availability in negative-sense RNA virus infections both blocks full-length genome replication and increases production of aberrant replication products that are sensed through RIG-I and MAVS, so a strong interferon response accompanies rather than follows successful infection.", "Executive summary": "Negative-sense RNA viruses carry their own polymerase and wind their genomes around a nucleoprotein scaffold to form ribonucleoprotein complexes. Nucleoprotein was known to regulate the balance between transcription and genome replication and to shield viral RNA from host nucleases to differing degrees across virus families, but its role in preventing host detection during infection had not been separated from its role in replication. The authors used recombinant influenza A virus and Sendai virus carrying microRNA target sites in the untranslated region downstream of the nucleoprotein open reading frame, an arrangement that degrades nucleoprotein messenger RNA without targeting genomic viral RNA. Silencing nucleoprotein abolished detectable viral protein and genome replication yet produced a strongly elevated interferon response relative to the amount of virus present. Sequencing showed reduced genome coverage together with read enrichment at segment termini and increased noncanonical junction reads for influenza, and 3 prime terminal enrichment indicative of copy-back products for Sendai. Northern blotting revealed accumulation of mini-viral RNA that tracked with interferon beta induction, a relationship reproduced in a reconstituted replication complex where nucleoprotein was titrated against fixed polymerase. Reporter assays in knockout cells placed the response through RIG-I and MAVS rather than MDA5. Knockdown of nucleoprotein across seven negative-sense RNA viruses spanning six families gave the same pairing of lost replication with induced IFIT1, while equivalent knockdown of SARS-CoV-2 nucleocapsid reduced replication without inducing interferon. Nucleozin, which targets nucleoprotein, elicited interferon while the polymerase inhibitor baloxavir marboxil did not.", "Scientific context": "Nucleoprotein had two established roles that had largely been studied separately. As a replication factor it acts as an elongation factor for the polymerase, and for influenza it is dispensable for templates up to about 76 nucleotides while supporting diminished synthesis on templates up to about 125 nucleotides, which is understood to prioritise viral protein synthesis early and to delay genome replication until enough protein is present to package new material. For paramyxoviruses and rhabdoviruses the polymerase favours transcription when nucleoprotein is scarce and replication when it is abundant. As a protective scaffold, its effectiveness varies. Ribonucleoproteins of human parainfluenza virus 5 and Rift Valley fever virus resist ribonucleases, vesicular stomatitis virus sequesters RNA in a deep cavity, rabies virus ribonucleoproteins are only partially resistant, influenza genomes contain both shielded and nucleoprotein-free structured regions, and Ebola virus nucleoprotein appears less protective. Separately, work by other groups had established defective viral genomes as important agonists, including mini-viral RNAs of under 100 nucleotides consisting only of the terminal promoter regions, which are recognised by RIG-I and drive exaggerated interferon responses, and which accumulate when polymerase is in relative excess. What had not been tested was whether deliberately lowering nucleoprotein, rather than raising polymerase, produces the same outcome, and whether the relationship holds across the phylum.", "Central question": "Does nucleoprotein availability govern host detection of negative-sense RNA virus infection independently of its role in supporting replication, and if so, does limiting nucleoprotein generate the aberrant replication products that pattern recognition receptors sense?", "Experimental strategy": "The design turns on separating protein availability from genome availability. Recombinant influenza A and Sendai viruses carry a cassette of microRNA target sites downstream of the nucleoprotein open reading frame, positioned so that nucleoprotein messenger RNA is degraded in cells expressing those microRNAs while genomic viral RNA is not targeted. Matched viruses with nonfunctional target sites provide the control, so the comparison is between two infections that differ in nucleoprotein supply alone. Three layers of evidence are then stacked. Sequencing of viral reads reports genome coverage, terminal enrichment, and noncanonical junctions as proxies for defective genome production, with the authors noting that junction-spanning reads undercount the true defective population. Northern blotting against the conserved 5 prime promoter detects all replication products including species smaller than the 106 nucleotide U6 loading control, which allows mini-viral RNA to be seen directly alongside interferon beta induction measured on the same samples. To remove any strain-specific explanation, the influenza replication complex is reconstituted from plasmids with a truncated 200 nucleotide segment 6 template, fixed polymerase, and a titration of nucleoprotein, with a catalytically inactive polymerase as control, so the relationship between nucleoprotein dose, product size, and interferon can be read in isolation from infection. Sensing is then assigned genetically using A549 reporter cells deficient in RIG-I, MDA5, or MAVS. Generality is tested by small interfering RNA knockdown of nucleoprotein across seven negative-sense viruses covering Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Filoviridae and Arenaviridae, with SARS-CoV-2 nucleocapsid included as a positive-sense counterpoint. Finally the principle is tested pharmacologically by comparing a nucleoprotein-directed inhibitor with a polymerase-directed one.", "Key findings": "1. Targeting nucleoprotein messenger RNA abolished detectable viral protein through late time points for both influenza A virus and Sendai virus, yet IFIT1 protein rose markedly relative to the nontargeted controls (Figures 1C and 1D).\n\n2. Messenger RNA sequencing showed induction of type I interferon response genes including the receptors DDX58, IFIH1 and TLR3, the transcription factors IRF1 and IRF7, and effectors including BST2, IFIT1 to IFIT3, OAS1 and MX1 (Figures 1E and 1F). For Sendai virus the absolute induction was equal or slightly lower than control, but viral transcripts were reduced roughly 300-fold, so the response is large relative to the replication present. Gene set enrichment confirmed antiviral categories (Figure 1G).\n\n3. Viral read coverage and total relative viral RNA were significantly reduced for both targeted viruses (Figures 2A, 2B, 2E and 2F). For influenza, coverage of segments 1 to 3 and segment 8 was enriched at both termini, a pattern characteristic of defective interfering particles (Figures 2A and 2C), and noncanonical junction reads increased significantly (Figure 2D). For Sendai virus, reads were enriched at the 3 prime terminus of the antigenome, which the authors read as copy-back defective genomes typical of paramyxoviruses (Figure 2G).\n\n4. Northern blotting against the conserved 5 prime promoter showed steady accumulation of full-length viral RNA and small viral RNA in control infections. With nucleoprotein targeted, full-length viral RNA was undetectable while species larger than small viral RNA and smaller than the 106 nucleotide loading control accumulated strongly, matching the published description of mini-viral RNA (Figure 3A). Their appearance as early as three hours post-infection coincided with strong interferon beta messenger RNA induction (Figure 3B).\n\n5. In the reconstituted system, increasing nucleoprotein against fixed polymerase increased full-length viral RNA and decreased mini-viral RNA, while catalytically inactive polymerase or absent nucleoprotein gave no product at all, consistent with the published requirement for nucleoprotein on templates above roughly 76 nucleotides (Figure 3C). Interferon beta induction tracked with the presence of mini-viral RNA (Figure 3D). Because this system contains no NS1, the authors take it as showing that loss of the interferon antagonist is not what produces the response, while acknowledging that in infection its absence contributes.\n\n6. In interferon-stimulated response element reporter cells, the response to nucleoprotein-targeted influenza was strong in wild-type cells, lost in cells lacking RIG-I and lost in cells lacking MAVS, and not significantly affected by loss of MDA5 (Figure 3E).\n\n7. Small interfering RNA knockdown of nucleoprotein reduced the fraction of infected cells for influenza A virus, human parainfluenza virus 3, vesicular stomatitis virus and Ebola virus, with respiratory syncytial virus and Lassa virus not significantly changed by that measure, while mean viral protein fluorescence intensity fell significantly for all (Figures 4A and 4B). Immunoblotting showed loss of viral protein together with IFIT1 induction for all seven negative-sense viruses tested (Figures 4C to 4I).\n\n8. Knockdown of the SARS-CoV-2 subgenomic nucleocapsid transcript reduced viral replication, confirmed as a significant drop in the percentage of viral reads, without inducing IFIT1 (Figures 4J and 4K). The authors offer two candidate explanations, that positive-sense genomes are noninflammatory because they are capped and polyadenylated, or that coronavirus replication is sequestered in lipid-enclosed vesicles, and do not distinguish between them.\n\n9. Nucleozin, which targets nucleoprotein, and baloxavir marboxil, which targets the polymerase PA subunit, both blocked influenza replication in a concentration-dependent manner, but only nucleozin also induced IFIT1 (Figure 3F). The authors propose on this basis that nucleoprotein-directed drugs could offer bystander priming of neighbouring cells, which is an extrapolation from cell culture.", "Mechanistic model": "The mechanism the data support runs as follows. Nucleoprotein acts as an elongation factor, so when it is scarce the polymerase can initiate but cannot processively copy full-length templates. Short products, in particular mini-viral RNAs under about 100 nucleotides, fall below the length at which nucleoprotein is required and are therefore preferentially synthesised and amplified under exactly the conditions that prevent full-length replication. These short products carry 5 prime triphosphate promoter ends and are potent agonists, and the reporter genetics place their detection through RIG-I signalling to MAVS. The net result is an inverted relationship in which less virus produces more interferon. Several elements remain interpretation rather than demonstration. The authors state explicitly that it is unclear how much of the host response in their experiments is driven by mini-viral RNA as opposed to longer defective genomes, and they note that their junction-read quantification substantially undercounts total defective genomes because a read can only be classified as defective when it spans a noncanonical junction. Their extension of the model to explain why high multiplicity passage yields defective interfering particles, through nucleoprotein demand outstripping supply late in infection or under host shutoff, is an interpretation offered in discussion rather than a result. The absence of an interferon response after SARS-CoV-2 nucleocapsid knockdown is presented as a contrast whose cause is not resolved. The evolutionary argument in the discussion, that the fragility of the nucleoprotein balance may explain why negative-sense RNA viruses are less diverse than positive-sense ones, is explicitly speculative.", "Conceptual or technical advance": "The work reframes nucleoprotein as a single control point where replication competence and immune invisibility are coupled, so that any perturbation reducing it necessarily trades one for a worse outcome in the other. That has a direct practical consequence. Two drugs that block influenza replication equally well differ in whether they also engage host defences, which suggests that the target chosen determines whether an antiviral additionally primes uninfected cells, and the authors note that nucleoprotein has been comparatively neglected as a target for clinically important negative-sense viruses. The microRNA targeting arrangement also supplies a way to remove a viral protein during genuine infection without altering the genome that the polymerase copies, which is what allows protein supply and genome supply to be separated. The demonstration that the same pairing holds across six negative-sense families while failing for a coronavirus nucleocapsid sets a boundary on the principle that is useful for vaccine and adjuvant design.", "Relationship to the broader research program": "The recombinant influenza and Sendai viruses used here come from the laboratory's established practice of inserting microRNA target sites into viral genomes to control the expression of individual viral gene products, an approach developed earlier for species-restricted and tissue-restricted attenuation and used elsewhere in the corpus as a conditional genetics tool. Marked as category 3 synthesis, the corpus-level pattern is that the same insertion strategy recurs as an instrument across very different questions, here to titrate a structural protein during infection, elsewhere to silence a herpesvirus gene only in one lineage. A second recurring thread is the treatment of aberrant viral RNA as the actual substrate of innate detection, which connects this study to the laboratory's longstanding interest in what viral RNA species the cell sees and in small RNAs generated during influenza replication. The inclusion of SARS-CoV-2 alongside the negative-sense panel reflects the period in which the work was completed and the laboratory's parallel coronavirus programme.", "Related publications": "- Aguado et al. 2018 (PNAS), methodological foundation. Same laboratory. Cited as the source of the IAV-NPT, IAV-NPC, SeV-NT and SeV-NC recombinant viruses and of the microRNA silencing cassette used throughout, and of their rescue procedure.\n- Perez et al. 2010 (PNAS), predecessor. Same laboratory. Characterised influenza A virus-generated small RNAs and their role in the switch from transcription to replication, the small viral RNA species visible alongside mini-viral RNA in the Northern blots here.\n- Perez et al. 2012 (Journal of Virology), predecessor. Same laboratory. Small-RNA enhancement of viral polymerase activity, part of the same line of work on influenza replication products.\n- Te Velthuis et al. 2018, predecessor. Established mini-viral RNA as a RIG-I agonist generated when polymerase is in relative excess, the prior observation that the present work approaches from the opposite direction by limiting nucleoprotein.\n- Benitez et al. 2015, conceptual extension. Same laboratory. In vivo RNA interference screening identifying MDA5, cited here in the context of pattern recognition receptor contributions to influenza sensing.\n- Blanco-Melo et al. 2020 (Cell), companion. Same laboratory with overlapping authorship, and the source of the A549-ACE2 system used for the SARS-CoV-2 arm.", "Limitations and boundaries": "Nearly all of the work is in A549 lung epithelial cells or in transfected HEK-293T cells, with no primary cells, airway models, or animals, so the consequences of limited nucleoprotein in tissue or in an infected host are untested. Nucleoprotein levels are reduced by microRNA targeting or by small interfering RNA rather than titrated to defined amounts during infection, so the study describes a scarcity regime rather than a dose relationship, with the sole exception of the plasmid-based reconstitution, which uses a truncated 200 nucleotide template and therefore does not model a real genome. Quantification of defective genomes by junction-spanning reads is acknowledged by the authors to be a substantial undercount, and the relative contribution of mini-viral RNA against longer defective genomes to the interferon response is explicitly left unresolved. Most sequencing rests on two or three biological replicates at single time points and high multiplicity. The cross-family survey uses different reporter viruses, different readouts of replication, and a single knockdown condition per virus, and for respiratory syncytial virus and Lassa virus the percentage of infected cells did not change significantly even though protein intensity did. Sensing was assigned in one reporter cell line for influenza only, so RIG-I dependence is not established for the other viruses. The negative result with SARS-CoV-2 rests on one siRNA against the subgenomic nucleocapsid transcript in one cell line. The proposed therapeutic advantage of nucleoprotein-directed inhibitors rests on a single comparison of two compounds in cell culture with IFIT1 as the readout, with no demonstration of bystander protection or of benefit in vivo. The paper states that it shows for the first time that these findings apply broadly across negative-sense RNA viruses, and that priority claim is the paper's own.", "Audience summaries": "### 25 words\n\nStarving a negative-sense RNA virus of its nucleoprotein stops it replicating but makes it far more visible, because the polymerase then churns out short immune-triggering fragments.\n\n### 75 words\n\nNegative-sense RNA viruses wrap their genomes in nucleoprotein, which the polymerase also needs to copy full-length templates. Silencing nucleoprotein in influenza and Sendai virus infections blocked replication yet produced a much stronger interferon response, because the polymerase generated short defective genomes that RIG-I detects. The same pairing held for seven viruses across six families but not for SARS-CoV-2 nucleocapsid, and a nucleoprotein-directed drug triggered interferon where a polymerase-directed drug did not.\n\n### 150 words\n\nNilsson-Payant and colleagues used recombinant influenza A and Sendai viruses carrying microRNA target sites downstream of the nucleoprotein open reading frame, which degrades nucleoprotein messenger RNA without targeting genomic RNA. Silencing abolished detectable viral protein and full-length genome replication while strongly inducing interferon-stimulated genes relative to the viral material present. Sequencing showed terminal read enrichment and increased noncanonical junctions, and Northern blotting showed accumulation of mini-viral RNA tracking with interferon beta. Titrating nucleoprotein against fixed polymerase in a reconstituted replication complex reproduced the inverse relationship between full-length product and mini-viral RNA, and reporter cells lacking RIG-I or MAVS lost the response while MDA5-deficient cells did not. Knockdown of nucleoprotein in seven negative-sense viruses across six families consistently paired lost replication with IFIT1 induction, whereas SARS-CoV-2 nucleocapsid knockdown reduced replication without inducing interferon. The authors propose nucleoprotein as a drug target that would also engage host defences." }, "discoveries": [ "claim-03" ], "relationships": [ { "from": "2021-nilsson-payant-reduced-nucleoprotein-availability", "to": "2012-perez-a-small-rna-enhancer-of-viral-poly", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-nilsson-payant-reduced-nucleoprotein-availability" }, { "from": "2021-nilsson-payant-reduced-nucleoprotein-availability", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "companion", "evidence": "stated in the Related publications section of 2021-nilsson-payant-reduced-nucleoprotein-availability" } ], "canonical_url": "https://tenoeverlab.us/publications/2021-nilsson-payant-reduced-nucleoprotein-availability/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sars-cov-2", "vsv", "sendai-virus", "hpiv3", "rsv", "ebola-virus", "lassa-virus", "measles-virus" ], "technologies": [ "bulk-rna-seq", "reverse-genetics", "sirna-knockdown", "flow-cytometry", "mirna-target-site-insertion", "luciferase-promoter-reporter", "immunoblotting", "northern-blot", "in-vitro-reconstitution", "splice-junction-analysis" ] } }, { "id": "2021-nilsson-payant-the-nf-b-transcriptional-footprint", "slug": "2021-nilsson-payant-the-nf-b-transcriptional-footprint", "url": "/publications/2021-nilsson-payant-the-nf-b-transcriptional-footprint/", "title": "The NF-κB Transcriptional Footprint Is Essential for SARS-CoV-2 Replication", "authors": [ "Benjamin E. Nilsson-Payant", "Skyler Uhl", "Adrien Grimont", "Ashley S. Doane", "Phillip Cohen", "Roosheel S. Patel", "Christina A. Higgins", "Joshua A. Acklin", "Yaron Bram", "Vasuretha Chandar", "Daniel Blanco-Melo", "Maryline Panis", "Jean K. Lim", "Olivier Elemento", "Robert E. Schwartz", "Brad R. Rosenberg", "Rohit Chandwani", "Benjamin R. tenOever" ], "author_count": 18, "first_author": "Benjamin E. Nilsson-Payant", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Rohit Chandwani", "Benjamin R. tenOever" ], "tenoever_position": 18, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2021, "journal": "Journal of Virology", "volume": "95", "issue": "23", "pages": "e01257-21", "doi": "10.1128/jvi.01257-21", "doi_url": "https://doi.org/10.1128/jvi.01257-21", "pmid": "34523966", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/34523966/", "pmcid": "PMC8577386", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8577386/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8577386/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling", "pandemic-host-response" ], "themes": [ "calibration-of-interferon-in-vivo", "imbalanced-host-response" ], "pathogens": [ "SARS-CoV-2" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "human" ], "technologies": [ "bulk RNA sequencing", "single-cell RNA sequencing", "ATAC sequencing", "transcription factor motif accessibility analysis", "small interfering RNA silencing", "chimeric VPR transcriptional activators", "small-molecule inhibitor dose response", "multiplexed ELISA", "quantitative RT-PCR", "immunofluorescence microscopy", "western blotting" ], "biological_systems": [ "A549-ACE2 cells", "HeLa-ACE2 cells", "RELA knockout HeLa-ACE2 cells", "Vero E6 cells" ], "key_concepts": [ "NF-κB signalling", "type I interferon antagonism", "imbalanced host response", "proviral host dependency", "enhancer remodelling", "chromatin accessibility", "infected versus bystander cells", "proinflammatory cytokine induction", "transcription factor motif enrichment" ], "keywords": [ "SARS-CoV-2", "NF-κB", "RelA", "p65", "NF-κB1", "p50", "type I interferon", "ATAC-seq", "single-cell RNA-seq", "BAY11-7082", "MG115", "A549-ACE2", "COVID-19 inflammation" ], "one_sentence_contribution": "SARS-CoV-2 infection of human lung epithelial cells engages NF-κB at chromatin, transcriptional, protein and post-translational levels without engaging the type I interferon transcription factors, and loss of p65 or p50 abolishes viral replication in a manner rescued by reconstituting RelA transcriptional activity.", "summary_25": "SARS-CoV-2 switches on the inflammatory factor NF-κB rather than the antiviral interferon programme, and the virus needs that NF-κB activity to replicate in human lung cells.", "summary_75": "Severe COVID-19 combines weak interferon responses with strong inflammation. In human lung epithelial cells, SARS-CoV-2 infection turned on NF-κB across chromatin, gene expression and protein readouts while leaving interferon transcription factors inactive. Removing either NF-κB subunit stopped the virus making protein, and restoring NF-κB-driven transcription with an engineered activator restored infection. Several drugs targeting the pathway also suppressed the virus. The inflammation therefore appears to reflect something the virus requires, not something it failed to block.", "summary_150": "High-resolution kinetics of SARS-CoV-2 infection in ACE2-expressing A549 cells showed a host response beginning between 6 and 9 hours, trailing peak viral RNA by about 3 hours, and dominated by tumour necrosis factor alpha signalling through NF-κB with no significant interferon signature and no STAT1 or IRF3 phosphorylation. Single-cell sequencing placed that signature predominantly in infected rather than bystander cells. ATAC sequencing showed remodelling concentrated at distal regulatory elements, with opening sites enriched for REL, RELA and NFKB1 motifs and not IRF3 or IRF7, and with previously poised enhancers gaining accessibility as previously active ones lost it. Silencing RelA reduced and silencing NF-κB1 eliminated nucleocapsid protein, and infection of RELA knockout cells was rescued by a RelA DNA-binding domain fused to a VPR activator, whereas an equivalent IRF3 construct restricted the virus. Which NF-κB target genes are required was not determined, and the work is in vitro.", "citation": "Nilsson-Payant BE, Uhl S, Grimont A, Doane AS, Cohen P, Patel RS, Higgins CA, Acklin JA, Bram Y, Chandar V, Blanco-Melo D, Panis M, Lim JK, Elemento O, Schwartz RE, Rosenberg BR, Chandwani R, tenOever BR. The NF-κB Transcriptional Footprint Is Essential for SARS-CoV-2 Replication. Journal of Virology. 2021. Volume 95, issue 23, article e01257-21. DOI 10.1128/jvi.01257-21. PMID 34523966. PMCID PMC8577386.", "sections": { "Citation": "Nilsson-Payant BE, Uhl S, Grimont A, Doane AS, Cohen P, Patel RS, Higgins CA, Acklin JA, Bram Y, Chandar V, Blanco-Melo D, Panis M, Lim JK, Elemento O, Schwartz RE, Rosenberg BR, Chandwani R, tenOever BR. The NF-κB Transcriptional Footprint Is Essential for SARS-CoV-2 Replication. Journal of Virology. 2021. Volume 95, issue 23, article e01257-21.\n\nDOI 10.1128/jvi.01257-21. PMID 34523966. PMCID PMC8577386.", "One-sentence contribution": "SARS-CoV-2 infection of human lung epithelial cells engages NF-κB at chromatin, transcriptional, protein and post-translational levels without engaging the type I interferon transcription factors, and loss of p65 or p50 abolishes viral replication in a manner rescued by reconstituting RelA transcriptional activity.", "Executive summary": "COVID-19 is characterised by a blunted type I interferon response alongside vigorous cytokine production, an imbalance that had been documented but not explained. Working in clonal A549 lung epithelial cells engineered to express ACE2, the authors mapped the early transcriptional response to SARS-CoV-2 at high temporal resolution and found that the dominant signature from 9 hours after infection onward was tumour necrosis factor alpha signalling through NF-κB, with no significant type I interferon signature and, at the protein level, no phosphorylation of STAT1 or IRF3. Single-cell sequencing separated infected from bystander cells and placed the NF-κB signature predominantly in the infected population. Chromatin accessibility profiling showed that infection opens regions enriched for REL, RELA and NFKB1 motifs and not for IRF3 or IRF7 motifs, with the largest changes occurring at distal regulatory elements rather than promoters. Silencing RelA reduced viral nucleocapsid protein and silencing NF-κB1 abolished it, and infection of RELA knockout cells was rescued by a chimeric RelA DNA-binding domain fused to a VPR transcriptional activator. Four small molecules targeting different steps of NF-κB activation reduced infection. The authors conclude that the inflammatory profile of SARS-CoV-2 infection reflects a viral requirement for NF-κB-driven transcription rather than a failure of viral antagonism.", "Scientific context": "The antiviral response is described in the paper as two coordinated strategies, direct restriction through type I interferon and interferon-stimulated genes, and recruitment of immune cells through chemokines. Induction of type I interferon requires concurrent activation of interferon regulatory factors, notably IRF3 and IRF7, together with NF-κB members RelA and p50, while NF-κB alone is sufficient for a large part of the proinflammatory cytokine and chemokine programme. NF-κB differs from the IRFs in that it is activated indirectly, through phosphorylation and degradation of an inhibitor, and in that many cellular stresses beyond pattern recognition receptor engagement can trigger it.\n\nAgainst that background, SARS-CoV-2 had been reported by the same laboratory and others to inhibit type I interferon signalling selectively while allowing chemokine production to proceed. Many SARS-CoV-2 gene products had been implicated in suppressing the interferon response. What was unresolved is why the cytokine arm remains so active when the virus is evidently capable of dampening host transcriptional responses, and whether that activity is simply an escape from viral control or serves the virus.", "Central question": "What is the molecular basis of the imbalanced host response to SARS-CoV-2, in which strong cytokine production coexists with a blunted type I interferon response, and does the transcription factor responsible for the cytokine arm serve the virus rather than the host.", "Experimental strategy": "The design moves from description to dependency in three steps, each addressing a limitation of the step before.\n\nDescriptive transcriptomics at high temporal resolution in a clonal ACE2-expressing A549 line establishes the timing and composition of the host response and relates it to viral RNA accumulation, viral protein and double-stranded RNA production. Running the kinetics at several multiplicities of infection, and then repeating at a high multiplicity to synchronise the population, separates responses arising in infected cells from those arising in uninfected bystanders, a confound the authors note explicitly when comparing the two datasets.\n\nSingle-cell sequencing then resolves that confound directly, using viral transcript content to classify cells as infected or bystander so the NF-κB signature can be assigned to one population or the other. Chromatin accessibility profiling adds an orthogonal layer, because transcription factor motif enrichment among newly accessible sites reports which factors are acting, independent of the downstream gene expression readout, and distinguishes promoter from distal enhancer remodelling.\n\nPerturbation then tests necessity. Silencing RelA and NF-κB1 separately asks whether either subunit is required, with silencing of the viral nucleocapsid transcript as a positive control for the assay. Because silencing and knockout can have pleiotropic effects, the rescue experiment is the critical control, using a chimeric factor comprising the RelA DNA-binding domain fused to the VPR activator to restore transcription from RelA-accessible enhancers in RELA knockout cells. Parallel IRF3-VPR and GFP-VPR constructs serve as specificity controls and, in the IRF3 case, as a check that driving the alternative arm is restrictive rather than permissive. Finally, four chemically distinct inhibitors acting at different steps of the pathway test whether the dependency is pharmacologically accessible, with paired viability measurement to separate antiviral effect from cytotoxicity.", "Key findings": "1. The host transcriptional response tracks viral load and trails it. Differential expression began at 8 hours after infection at a multiplicity of 1 and by 16 hours was robust at all multiplicities, plateauing at 36 hours (Figure 1A), correlating with viral read fraction (Figure 1B) and with nucleocapsid and spike protein (Figure 1C). At high multiplicity the response began between 6 and 9 hours and trailed peak viral replication by roughly 3 hours (Figure 1D and 1E), corroborated by quantitative RT-PCR for subgenomic nucleocapsid and genomic envelope RNA (Figure 1F). Double-stranded RNA was readily detected by immunofluorescence (Figure 1H).\n\n2. The dominant early signature is NF-κB and not interferon. Gene set enrichment identified tumour necrosis factor alpha signalling via NF-κB as the most upregulated set from 9 hours onward (Figure 2A), with induction of CXCL8, CXCL10, CXCL11, CCL20, IL1A and IL6 (Figure 2B and 2C). Only a small subset of interferon-related genes rose, and only at the latest time point (Figure 2D). Quantitative RT-PCR showed NFKBIA induction without MX1 induction (Figure 2E). At the protein level, MX1 was absent and neither STAT1 nor IRF3 was phosphorylated, while IκBα and RelA were phosphorylated (Figure 2F). Secretion of CXCL1, CXCL2, CXCL8, CCL2, CCL20 and IL-6 was confirmed by ELISA (Figure 2G).\n\n3. The NF-κB signature is concentrated in infected cells. Single-cell sequencing classified cells by viral transcript content and confirmed the reported loss of host messenger RNA in infected cells (Figure 3A to 3C). Tumour necrosis factor alpha signalling via NF-κB was enriched in both populations but dominated the infected transcriptome (Figure 3D and 3E). The authors read this as indicating that virus replication itself, rather than paracrine signalling, drives the response.\n\n4. Infection remodels chromatin toward NF-κB-responsive elements. Accessibility increased at CXCL2, NFKBIA and TNF loci (Figure 4A), with the largest changes 20 to 200 kilobases from transcription start sites rather than at promoters (Figure 4B). Newly accessible sites corresponded to previously poised enhancers and newly inaccessible sites to previously active enhancers based on A549 ENCODE histone marks (Figure 4C to 4E). The authors interpret this as repression of normally active enhancers alongside activation of normally poised ones.\n\n5. Motif analysis of opening sites showed enrichment for REL, NFKB1 and RELA and not for IRF3 or IRF7 (Figure 4F). The largest accessibility gains corresponded to NF-κB family binding sites and the largest losses to TEAD pathway sites (Figure 4G), and these changes had a corresponding transcriptional impact (Figure 4H). Enrichment for NF-κB associated genes and enhancers, to the exclusion of interferon signatures, was seen in both the expression and accessibility data (Figure 4I to 4L).\n\n6. NF-κB subunits are required for viral protein production. Silencing RelA significantly reduced nucleocapsid protein and silencing NF-κB1 eliminated detectable nucleocapsid, comparable to directly targeting the viral subgenomic nucleocapsid transcript (Figure 5A). Immunofluorescence quantification across eight biological replicates confirmed a significant loss of nucleocapsid-positive cells after silencing NF-κB1, RelA or nucleocapsid (Figure 5B).\n\n7. The requirement is for NF-κB transcriptional output. RELA knockout HeLa-ACE2 cells supported far less viral protein production than wild-type, and this was rescued by transient expression of a RelA DNA-binding domain fused to the VPR activator (Figure 5C). An IRF3-VPR construct instead inhibited replication and induced IFIT1, consistent with known interferon sensitivity of SARS-CoV-2.\n\n8. Pharmacological inhibition of NF-κB reduces infection. BAY11-7082, MG115, parthenolide and p-xyleneselenocyanate each reduced infected cell number, with no or minimal cytotoxicity at low concentrations (Figure 5D). BAY11-7082 and MG115 reduced viral protein and RNA, with near complete loss under MG115 (Figure 5E and 5F), reduced viral read fraction and NF-κB target gene expression including CXCL2, NFKBIA, JUN and JUNB (Figure 5G and 5H), and BAY11-7082 reduced secreted CXCL5, CXCL8, CCL2 and IL-6 (Figure 5I).", "Mechanistic model": "The study establishes a requirement but does not establish the mechanism of that requirement, and the authors say so directly. They state that it remains difficult to deconvolute which NF-κB target gene or genes produce the phenotype, and they note that their data do not exclude an IKK-mediated post-translational modification of viral proteins operating alongside the transcriptional requirement.\n\nWhat the data constrain is the following. Virus replication generates signals sufficient to activate IKK and drive IκBα phosphorylation and NF-κB nuclear translocation, since the signature is concentrated in infected cells and its kinetics depend on viral load. NF-κB engages cognate distal enhancers and drives a cytokine programme, while the IRF arm is not engaged at motif, transcript or protein level. Loss of either p65 or p50 blocks viral protein accumulation, and restoring transcription from RelA-accessible enhancers with a heterologous activator restores replication, which places the requirement at the level of NF-κB-driven gene expression rather than at the level of the NF-κB protein itself performing some non-transcriptional function.\n\nWhat the data do not constrain is the identity of the required gene or genes, and the specific viral trigger for IKK activation. On the latter the authors offer a proposal rather than a demonstration, suggesting that accumulating pathogen-associated molecular patterns such as viral double-stranded RNA or misfolded proteins induce a cellular stress response that converges on IκBα degradation. They also note that a strong NF-κB response carries costs for the virus by recruiting immunity, so the dependency they describe is presented as explaining why the virus tolerates that cost.", "Conceptual or technical advance": "The work reframes the inflammatory profile of COVID-19 as a consequence of a viral dependency rather than as a failure of viral immune evasion, which makes the cytokine arm and the replication arm two readouts of the same requirement and predicts that blocking one blocks the other. The chimeric transcription factor rescue is the element that makes the claim interpretable, because it separates loss of an NF-κB protein from loss of NF-κB-driven transcription. Practically, the observation that four mechanistically distinct NF-κB inhibitors reduce replication in vitro identifies a host pathway whose targeting would in principle suppress both replication and the inflammatory response at once, while the authors themselves note the absence of FDA-approved NF-κB inhibitors and the pathway's central role in cell survival and proliferation as obstacles.", "Relationship to the broader research program": "The study builds directly on the laboratory's earlier characterisation of the imbalanced host response to SARS-CoV-2, cited here as reference 18, which reported suppression of type I interferon alongside preserved chemokine production. The present work supplies a transcription factor level explanation for that pattern. The A549-ACE2 system, the paired RNA sequencing and quantitative RT-PCR readouts and the emphasis on separating infected from bystander responses recur across the laboratory's SARS-CoV-2 work.\n\nCategory 3 synthesis. The discussion notes that influenza A virus has also been reported to depend on NF-κB, with the mechanism unclear, which positions this paper within a broader recurring question in the corpus about whether respiratory RNA viruses co-opt inflammatory transcription for their own replication. Establishing that as a cross-paper claim requires the influenza records alongside this one.", "Related publications": "- Blanco-Melo and colleagues, 2020, predecessor. Cited here as reference 18 for the imbalanced host response to SARS-CoV-2, the observation this study seeks to explain mechanistically, and as the source of the plaque assay protocol used here.\n- Nilsson-Payant and colleagues, companion work on the A549-ACE2 system, methodological foundation. The ACE2-expressing A549 line used throughout is cited to prior description by this group.", "Limitations and boundaries": "The mechanistic claim is bounded to transformed human cell lines, principally clonal A549 cells engineered to overexpress ACE2, with the rescue experiment in HeLa cells similarly engineered, and no primary human airway or organoid system is used here. The authors state in the discussion that they were unable to recapitulate the in vivo NF-κB inhibitor effect reported for influenza when they tested SARS-CoV-2 in their hamster model, so the requirement is demonstrated in vitro only. Only one viral isolate is used, USA-WA1/2020, and no variants are tested. The required NF-κB target gene or genes are not identified, and the authors note that an IKK-mediated post-translational effect on viral proteins is not excluded. The trigger for NF-κB activation is proposed rather than shown, and the paper offers no experiment discriminating double-stranded RNA sensing from misfolded protein stress or other stress inputs. The small molecules used are broadly acting rather than NF-κB specific, and MG115 in particular is a proteasome inhibitor with effects well beyond IκBα stability, so the pharmacology supports the genetic result rather than standing alone. Time points are largely 24 hours after infection for the perturbation experiments, which does not address later or persistent infection. Readouts of replication are principally viral protein, viral RNA and infected cell fraction rather than infectious titre.", "Audience summaries": "### 25 words\n\nSARS-CoV-2 switches on the inflammatory factor NF-κB rather than the antiviral interferon programme, and the virus needs that NF-κB activity to replicate in human lung cells.\n\n### 75 words\n\nSevere COVID-19 combines weak interferon responses with strong inflammation. In human lung epithelial cells, SARS-CoV-2 infection turned on NF-κB across chromatin, gene expression and protein readouts while leaving interferon transcription factors inactive. Removing either NF-κB subunit stopped the virus making protein, and restoring NF-κB-driven transcription with an engineered activator restored infection. Several drugs targeting the pathway also suppressed the virus. The inflammation therefore appears to reflect something the virus requires, not something it failed to block.\n\n### 150 words\n\nHigh-resolution kinetics of SARS-CoV-2 infection in ACE2-expressing A549 cells showed a host response beginning between 6 and 9 hours, trailing peak viral RNA by about 3 hours, and dominated by tumour necrosis factor alpha signalling through NF-κB with no significant interferon signature and no STAT1 or IRF3 phosphorylation. Single-cell sequencing placed that signature predominantly in infected rather than bystander cells. ATAC sequencing showed remodelling concentrated at distal regulatory elements, with opening sites enriched for REL, RELA and NFKB1 motifs and not IRF3 or IRF7, and with previously poised enhancers gaining accessibility as previously active ones lost it. Silencing RelA reduced and silencing NF-κB1 eliminated nucleocapsid protein, and infection of RELA knockout cells was rescued by a RelA DNA-binding domain fused to a VPR activator, whereas an equivalent IRF3 construct restricted the virus. Which NF-κB target genes are required was not determined, and the work is in vitro." }, "discoveries": [ "claim-12" ], "relationships": [ { "from": "2021-nilsson-payant-the-nf-b-transcriptional-footprint", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2021-nilsson-payant-the-nf-b-transcriptional-footprint" } ], "canonical_url": "https://tenoeverlab.us/publications/2021-nilsson-payant-the-nf-b-transcriptional-footprint/", "controlled_vocabulary": { "pathogens": [ "sars-cov-2" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "immunofluorescence-microscopy", "sirna-knockdown", "immunoblotting", "multiplex-cytokine-assay", "small-molecule-inhibitor-profiling", "single-cell-rna-seq", "atac-seq", "engineered-tf-constructs", "tf-activity-inference" ] } }, { "id": "2021-si-a-human-airway-on-a-chip-for-the-r", "slug": "2021-si-a-human-airway-on-a-chip-for-the-r", "url": "/publications/2021-si-a-human-airway-on-a-chip-for-the-r/", "title": "A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics", "authors": [ "Longlong Si", "Haiqing Bai", "Melissa Rodas", "Wuji Cao", "Crystal Yuri Oh", "Amanda Jiang", "Rasmus Moller", "Daisy Hoagland", "Kohei Oishi", "Shu Horiuchi", "Skyler Uhl", "Daniel Blanco-Melo", "Randy A. Albrecht", "Wen-Chun Liu", "Tristan Jordan", "Benjamin E. Nilsson-Payant", "Ilona Golynker", "Justin Frere", "James Logue", "Robert Haupt", "Marisa McGrath", "Stuart Weston", "Tian Zhang", "Roberto Plebani", "Mercy Soong", "Atiq Nurani", "Seong Min Kim", "Danni Y. Zhu", "Kambez H. Benam", "Girija Goyal", "Sarah E. Gilpin", "Rachelle Prantil-Baun", "Steven P. Gygi", "Rani K. Powers", "Kenneth E. Carlson", "Matthew Frieman", "Benjamin R. tenOever", "Donald E. Ingber" ], "author_count": 38, "first_author": "Longlong Si", "senior_authors": [ "Donald E. Ingber" ], "corresponding_authors": [ "Donald E. Ingber" ], "tenoever_position": 37, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2021, "journal": "Nature Biomedical Engineering", "volume": "5", "issue": "8", "pages": "815-829", "doi": "10.1038/s41551-021-00718-9", "doi_url": "https://doi.org/10.1038/s41551-021-00718-9", "pmid": "33941899", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/33941899/", "pmcid": "PMC8387338", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387338/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387338/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "host-factors-and-druggable-signaling", "models-for-pandemic-virology" ], "pathogens": [ "influenza A virus", "SARS-CoV-2", "vesicular stomatitis virus" ], "viral_families": [ "Orthomyxoviridae", "Coronaviridae", "Rhabdoviridae" ], "host_species": [ "human", "golden hamster" ], "technologies": [ "organ-on-a-chip microfluidics", "air-liquid interface culture", "pseudotyped virus entry assay", "immunofluorescence confocal microscopy", "barrier permeability measurement", "cytokine multiplex assay", "quantitative mass spectrometry proteomics", "RNA sequencing", "RT-qPCR", "plaque assay", "pharmacokinetic analysis", "hamster challenge and transmission models" ], "biological_systems": [ "microfluidic human bronchial airway chip", "primary human bronchial airway basal stem cells", "primary human pulmonary microvascular endothelium", "primary human neutrophils", "Huh-7 cells", "Vero E6 cells", "ACE2-expressing A549 cells", "golden hamster" ], "key_concepts": [ "drug repurposing", "clinically relevant drug exposure under flow", "strain-dependent virulence", "neutrophil recruitment and transmigration", "serine protease priming of hemagglutinin", "therapeutic time window", "viral entry inhibition", "preclinical model fidelity", "prophylaxis versus treatment" ], "keywords": [ "airway chip", "organ chip", "influenza", "SARS-CoV-2", "amodiaquine", "nafamostat", "oseltamivir", "hydroxychloroquine", "pseudoparticle", "hamster" ], "one_sentence_contribution": "A microfluidic bronchial airway chip lined with differentiated human airway epithelium and pulmonary endothelium reproduces strain-dependent influenza virulence, cytokine output and neutrophil recruitment, and when drugs are delivered at clinically achievable blood concentrations under flow it separates candidates that work in cell lines from those that also work in hamsters challenged with SARS-CoV-2.", "summary_25": "A perfused chip of human airway tissue reproduced influenza severity differences and immune cell recruitment, and picked out amodiaquine, which protected hamsters from SARS-CoV-2 where hydroxychloroquine failed.", "summary_75": "Drug candidates nominated in cell lines often fail in people. A microfluidic chip lined with differentiated human airway epithelium over pulmonary endothelium reproduced strain differences in influenza severity, cytokine output and neutrophil recruitment, and matched the clinical effect and two-day treatment window of oseltamivir, which a protease inhibitor doubled. Tested at human blood concentrations under flow, hydroxychloroquine and chloroquine failed against pseudotyped SARS-CoV-2 while amodiaquine worked, and amodiaquine went on to protect hamsters from infection.", "summary_150": "The assays used to nominate repurposing candidates use cell lines with impaired interferon responses, no airway architecture, no circulating immune cells and static drug exposure. This study used a two-channel chip with primary human bronchial epithelium differentiated at an air liquid interface above perfused pulmonary endothelium. It reproduced replication differences among six influenza strains, greater barrier damage from H3N2 than H1N1, cytokine output that tracked clinical severity rather than viral load for H5N1, and neutrophil adhesion, transmigration and virus clearance. Oseltamivir acid delivered at blood-like concentrations reproduced its clinical efficacy and its two-day window, which the protease inhibitor nafamostat extended to four days by blocking hemagglutinin cleavage. Eight drugs active against spike-pseudotyped particles in a liver cell line were retested on chip at human maximum plasma concentrations, and only amodiaquine, toremifene and clomiphene remained active. Amodiaquine reduced native SARS-CoV-2 in hamsters prophylactically, therapeutically and in transmission, while hydroxychloroquine did not.", "citation": "Si L, Bai H, Rodas M, Cao W, Oh CY, Jiang A, Moller R, Hoagland D, Oishi K, Horiuchi S, Uhl S, Blanco-Melo D, Albrecht RA, Liu WC, Jordan T, Nilsson-Payant BE, Golynker I, Frere J, Logue J, Haupt R, McGrath M, Weston S, Zhang T, Plebani R, Soong M, Nurani A, Kim SM, Zhu DY, Benam KH, Goyal G, Gilpin SE, Prantil-Baun R, Gygi SP, Powers RK, Carlson KE, Frieman M, tenOever BR, Ingber DE. A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics. Nature Biomedical Engineering. 2021. Volume 5, issue 8, pages 815-829. DOI 10.1038/s41551-021-00718-9. ", "sections": { "Citation": "Si L, Bai H, Rodas M, Cao W, Oh CY, Jiang A, Moller R, Hoagland D, Oishi K, Horiuchi S, Uhl S, Blanco-Melo D, Albrecht RA, Liu WC, Jordan T, Nilsson-Payant BE, Golynker I, Frere J, Logue J, Haupt R, McGrath M, Weston S, Zhang T, Plebani R, Soong M, Nurani A, Kim SM, Zhu DY, Benam KH, Goyal G, Gilpin SE, Prantil-Baun R, Gygi SP, Powers RK, Carlson KE, Frieman M, tenOever BR, Ingber DE. A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics. Nature Biomedical Engineering. 2021. Volume 5, issue 8, pages 815-829.\n\nDOI 10.1038/s41551-021-00718-9. PMID 33941899. PMCID PMC8387338.", "One-sentence contribution": "A microfluidic bronchial airway chip lined with differentiated human airway epithelium and pulmonary endothelium reproduces strain-dependent influenza virulence, cytokine output and neutrophil recruitment, and when drugs are delivered at clinically achievable blood concentrations under flow it separates candidates that work in cell lines from those that also work in hamsters challenged with SARS-CoV-2.", "Executive summary": "Repurposing approved drugs is one of the fastest routes to a therapeutic during a pandemic, but the cell line assays that generate candidates do not reproduce airway tissue architecture, interferon competence, immune cell recruitment or the time-varying drug exposures that follow oral dosing. This work applies a two-channel microfluidic chip in which primary human bronchial basal stem cells differentiate at an air liquid interface above primary pulmonary endothelium under continuous perfusion. Chips supported replication of six influenza A strains with the relative differences seen clinically, recruited perfused human neutrophils that transmigrated and cleared infected cells, and produced cytokine profiles that scaled with strain virulence rather than with viral load. Oseltamivir acid delivered through the vascular channel reproduced both the efficacy and the two-day treatment window seen in patients, and the protease inhibitor nafamostat delivered to the airway channel extended that window to four days. The group then pivoted to SARS-CoV-2, first using spike-pseudotyped particles under biosafety level 2 conditions. Eight drugs active against pseudoparticle entry in a liver cell line were retested on chips at their reported human maximum plasma concentrations under flow, and only amodiaquine, toremifene and clomiphene remained active, while hydroxychloroquine, chloroquine and arbidol did not. Amodiaquine subsequently reduced native SARS-CoV-2 in hamsters given prophylactically, therapeutically and in a transmission setting, whereas hydroxychloroquine did not.", "Scientific context": "The paper states its gap in terms of the mismatch between the assays used to nominate repurposing candidates and the human tissue those candidates must act in. Cell lines frequently carry defects in interferon responses, because the cell cycle arrest that interferon imposes selects against competence during continuous passage. Neither cell lines nor conventionally cultured primary airway cells form the mucociliary pseudostratified epithelium of the living airway. Explanted human respiratory tissue avoids that problem but is scarce and short lived. Organoids give a more functional epithelium but do not permit an air liquid interface, epithelial to endothelial cross-talk, mucociliary clearance or recruitment of circulating immune cells. Across all of these, and in Transwell co-cultures that do support an air liquid interface, drugs are applied statically, so the exposure profile that a patient experiences after oral dosing is not represented. The authors place their work explicitly against the confusion over hydroxychloroquine and chloroquine in early 2020, where cell line activity was not borne out clinically. They note the programme was funded by DARPA and the National Institutes of Health two years before the pandemic in anticipation of biothreat challenges, and that their COVID-19 work began on 13 January 2020, one day after the viral genome sequence was released.", "Central question": "Can a perfused human airway chip that reproduces airway tissue structure, innate immune responses and clinically relevant drug exposure predict which approved drugs will work against respiratory viruses better than static cell line assays do.", "Experimental strategy": "Validation precedes application throughout, and the logic is to establish clinical mimicry with a virus and a drug whose human behaviour is already known before asking the model anything new. The chip places differentiated airway epithelium at an air liquid interface on one face of a porous membrane and primary pulmonary microvascular endothelium under flow on the other, with pore size chosen to permit immune cell transmigration. Fidelity was assessed at several levels, cell type composition and junctional structure, protease and receptor expression against a commonly used cell line, replication kinetics of six influenza strains spanning three subtypes and both clinical isolates and laboratory strains, barrier permeability, cytokine output from the vascular effluent, and recruitment and transmigration of perfused primary human neutrophils, with donor variability tested across five epithelial donors. Drugs are then delivered by the route that matches their clinical use, oseltamivir acid through the vascular channel to mimic blood levels after oral dosing and hepatic conversion, protease inhibitors into the airway channel to mimic inhaled delivery, and always at concentrations set by published human maximum plasma concentration values rather than by what is convenient in vitro. Treatment window is tested by adding drug at times spanning twenty-four hours before to ninety-six hours after infection. For SARS-CoV-2 the work proceeds in stages defined by containment, spike-pseudotyped luciferase particles at biosafety level 2 for entry inhibition with vesicular stomatitis virus glycoprotein particles as a specificity and toxicity control, then native virus in cell lines, then hamsters in higher containment for prophylaxis, therapy and animal to animal transmission, with pharmacokinetics measured in hamsters to set dosing and with oral and subcutaneous routes compared. Proteomics was used to ask why one antimalarial behaved differently from its close relatives.", "Key findings": "1. Differentiated airway epithelium on chip formed a pseudostratified mucociliary layer with basal and ciliated cells in proportions resembling the human airway, continuous tight junctions and an endothelium with adherens junctions, and expressed TMPRSS2, TMPRSS4, TMPRSS11D and TMPRSS11E at higher levels than MDCK cells, with ACE2 messenger RNA and protein rising on differentiation (Figures 1b to 1f, Supplementary Figure 1).\n2. Influenza infection introduced into the air channel infected the epithelium, disrupted tight junctions, caused cilia loss and increased barrier permeability, and disrupted endothelial adherens junctions without detectably infecting the endothelium, which the authors align with vascular leakage observed in patients (Figures 1c and 1g, Supplementary Figure 2a). Undifferentiated basal epithelium was much less susceptible.\n3. Across six strains, H1N1 and H3N2 propagated by three to four logs over twenty-four to forty-eight hours while H5N1 grew more slowly, and the two H3N2 strains replicated roughly tenfold better than the H1N1 strains and caused more barrier disruption and cilia loss (Figure 2a, Figure 1g, Supplementary Figure 2b). Five epithelial donors gave similar infectivity (Supplementary Figure 2c).\n4. Perfused primary human neutrophils adhered to the activated endothelium within minutes, transmigrated into the epithelium over hours, targeted nucleoprotein-positive cells and cleared virus over one to two days, with more recruitment for H3N2 than H1N1 and reduced titres for both when neutrophils were present (Figures 2b to 2d, Supplementary Figure 3a). Neutrophils also increased cytokine production and barrier damage (Supplementary Figures 3b and 3c), and the authors note that neutrophils can protect or harm depending on context.\n5. Three clinical isolates of differing virulence produced cytokine and chemokine levels that ranked with clinical severity, H5N1 highest despite the lowest replication (Figure 2e). This dissociation between viral load and inflammatory output is the clearest demonstration that the chip reports something beyond replication.\n6. Oseltamivir acid at 1 micromolar in the vascular channel reduced progeny titre, preserved barrier function and tight junctions, and lowered cytokine output (Figures 3a to 3d), an effect the authors compare with the approximately one log reduction reported in a randomised trial.\n7. Nafamostat and Trasylol delivered to the airway channel reduced H1N1 and H3N2 titres, preserved barrier and junction integrity, lowered cytokines, and blocked cleavage of hemagglutinin into HA1 and HA2 (Figure 3e, Supplementary Figures 4a to 4e).\n8. In a time-course experiment, oseltamivir worked only when added within forty-eight hours of infection, matching its clinical recommendation, and combining it with nafamostat extended effective treatment to ninety-six hours (Figure 3f). The authors describe the combined effect at later times as synergistic. The observation is a doubling of the effective window in this model.\n9. Eight approved drugs inhibited spike-pseudotyped particle entry in Huh-7 cells in a dose-dependent manner without detectable toxicity (Figure 4a). The authors note that Huh-7 cells express little ACE2, lack TMPRSS2 and derive from liver.\n10. On chips pretreated for twenty-four hours at published human maximum plasma concentrations and maintained under flow, only amodiaquine, toremifene and clomiphene reduced entry, by 59.1, 51.1 and 28.1 percent, while hydroxychloroquine, chloroquine, arbidol, verapamil and amiodarone did not (Figure 4b, Table 1). The authors point out that the three that failed on chip also failed in clinical trials. The active amodiaquine metabolite desethylamodiaquine gave about 60 percent inhibition at a clinically relevant concentration (Supplementary Figure 8).\n11. Quantitative mass spectrometry showed that amodiaquine perturbed the airway epithelial proteome more broadly and differently than chloroquine or hydroxychloroquine, with the most affected proteins related to cilium regulation and lysosomal function (Figures 5a to 5c). The authors offer this as something that may be responsible for the greater effect on entry, which is a proposal rather than a mechanism established here.\n12. Against native SARS-CoV-2, amodiaquine and desethylamodiaquine inhibited Vero E6 infection with half-maximal inhibitory concentrations of 7.5 and 9.9 micromolar (Supplementary Figure 9), and amodiaquine at 10 micromolar reduced viral load by about three orders of magnitude in ACE2-expressing A549 cells (Figure 6a).\n13. In hamsters, subcutaneous amodiaquine begun one day before intranasal challenge reduced lung subgenomic nucleocapsid RNA by roughly 70 percent at day three with reduced nucleocapsid staining in lung sections (Figures 6b and 6c). In a co-caging transmission model where all vehicle animals became infected within two days, amodiaquine reduced nucleocapsid RNA by 90 percent and titres by more than one log (Figure 6d).\n14. Oral amodiaquine at 75 milligrams per kilogram gave pharmacokinetics comparable to subcutaneous dosing with higher tissue than plasma levels, prevented infection to a similar degree, and in the same model hydroxychloroquine at a dose reported to give clinically relevant lung exposure had no significant effect (Figure 6e, Supplementary Figure 11). RNA sequencing of treated infected hamsters showed downregulation of inflammatory gene sets including TNF alpha and NF kappa B signalling, IL-6 JAK STAT3 and interferon gamma (Supplementary Figure 12).\n15. Treatment begun one day after infection gave about 70 percent inhibition at day three and no detectable nucleocapsid transcript in lung at day seven (Figure 6f).", "Mechanistic model": "The study is a model validation and drug discovery paper rather than a mechanistic one, and it does not establish how amodiaquine acts against SARS-CoV-2. For nafamostat the mechanism is supported directly, since the protease inhibitors blocked cleavage of hemagglutinin precursor into its subunits by TMPRSS11D and TMPRSS2, which is required for entry, and the chip expresses those proteases at levels above the cell lines conventionally used.\n\nFor amodiaquine the paper establishes an effect on entry of spike-pseudotyped particles and on infection by native virus in cells and animals, but not the molecular target. The proteomic comparison with chloroquine and hydroxychloroquine shows divergent host proteome effects concentrated on cilium regulation and lysosomal proteins, and the authors write that this may be responsible for the greater effect on entry. That is a proposal, and since the pseudoparticle system reports only entry the activity against native virus could involve additional steps not tested here.\n\nThe claim that carries the most weight, that the chip predicts clinical outcome better than static cell line assays, is supported by concordance rather than by a mechanism. Drugs that failed on chip also failed in trials, drugs that succeeded on chip succeeded in hamsters. The authors are explicit that a caveat applies, namely that drug absorption into the device material and protein binding were not quantified in this study, so the concentration actually reaching the cells is not known. They also note that full pharmacokinetic profiles rather than fixed maximum concentrations could be recapitulated in future work.", "Conceptual or technical advance": "The work demonstrates that an airway chip can reproduce several features of human influenza that had not been available together in one in vitro system, notably strain-dependent virulence ranking, the dissociation between viral load and inflammatory output seen with H5N1, endothelial disruption without endothelial infection, and the full sequence of neutrophil adhesion, transmigration and clearance under flow. It then shows that the model discriminates among drugs in a way that static assays do not, which is the practical contribution. The combination finding, that a serine protease inhibitor doubles the window during which a neuraminidase inhibitor remains useful, is directly actionable because most patients present late. On the coronavirus side, the study establishes a staged discovery route that begins under biosafety level 2 with pseudotyped particles, which lowered the barrier to entry for laboratories without high containment during the early pandemic, and it nominated amodiaquine on evidence that subsequently supported the initiation of Phase 2 trials in Africa by Medicines for Malaria Venture with Wits University and by the ANTICOV programme, a development the authors report.", "Relationship to the broader research program": "The tenOever laboratory's contribution, as stated in the author contributions, was to develop the hamster COVID-19 infection model and to test drug efficacy against native SARS-CoV-2 in vivo, together with colleagues at Mount Sinai. The study was conceived by Si, Bai and Ingber at the Wyss Institute, who also developed the discovery pipeline, with the Frieman group at Maryland testing amodiaquine and its metabolite against native virus. The connection to the tenOever programme runs through the golden hamster model of SARS-CoV-2, which that laboratory developed and applied across its own pandemic-era work, and through the transcriptional readout of the host inflammatory response, which is the laboratory's characteristic way of scoring infection. Reading this paper alongside the laboratory's own SARS-CoV-2 studies shows the same animal model and the same host response framing serving as a shared platform for collaborations with groups bringing different front ends, which is a category 3 synthesis available from the corpus rather than a claim made here.", "Related publications": "- Si and colleagues, 2020 preprint, predecessor to this article, from the same collaboration. The bioRxiv report of the drug repurposing findings, published on 13 April 2020, which the authors state contributed in part to the initiation of amodiaquine clinical trials.\n- Benam and colleagues, cited as reference 17, methodological foundation, from the Ingber laboratory. The earlier human airway chip, from which this device differs in membrane chemistry, material properties and pore size, the last change permitting immune cell transmigration.\n- Thacker and colleagues, 2020, and Zhang and colleagues, 2020, companion, from other laboratories. Contemporaneous organ chip studies of SARS-CoV-2 infection that the authors distinguish from this work on the grounds that they used alveolar epithelium or cell lines rather than airway epithelium and did not address drug repurposing.", "Limitations and boundaries": "The SARS-CoV-2 chip experiments used spike-pseudotyped particles, which report entry only and do not replicate, so the chip data speak to prophylaxis against initial infection rather than to therapy, and the authors say that integrating these chips into higher containment laboratories will be necessary to study native virus on chip. The authors state that they did not quantify drug absorption into the device or protein binding, so nominal and delivered concentrations may differ, and that fixed maximum plasma concentrations were used rather than full pharmacokinetic profiles. Amodiaquine carries a black-box warning in the United States for rare agranulocytosis and liver damage with high or prolonged dosing, which the authors raise alongside their suggestion that a short course could be considered, and no safety or efficacy claim in humans follows from the animal data presented. Hamster group sizes were small, between three and eight animals per condition. Endpoints in animals were subgenomic nucleocapsid RNA, plaque titre and histology rather than survival or clinical scoring. The influenza work uses one chip format, a limited number of strains and healthy donor cells, with no comorbidity, age or prior immunity represented, and the model lacks adaptive immunity, alveolar tissue and systemic circulation. The neutrophil experiments use cells from blood donors perfused at physiological concentration for a short period and do not model sustained recruitment. The proteomic explanation for why amodiaquine differs from its relatives is a correlation with pathway annotation, not a demonstrated mechanism. Finally, the concordance argument, that chip results match clinical trial outcomes, rests on a small number of drugs chosen for testing on the basis of prior reports.", "Audience summaries": "### 25 words\n\nA perfused chip of human airway tissue reproduced influenza severity differences and immune cell recruitment, and picked out amodiaquine, which protected hamsters from SARS-CoV-2 where hydroxychloroquine failed.\n\n### 75 words\n\nDrug candidates nominated in cell lines often fail in people. A microfluidic chip lined with differentiated human airway epithelium over pulmonary endothelium reproduced strain differences in influenza severity, cytokine output and neutrophil recruitment, and matched the clinical effect and two-day treatment window of oseltamivir, which a protease inhibitor doubled. Tested at human blood concentrations under flow, hydroxychloroquine and chloroquine failed against pseudotyped SARS-CoV-2 while amodiaquine worked, and amodiaquine went on to protect hamsters from infection.\n\n### 150 words\n\nThe assays used to nominate repurposing candidates use cell lines with impaired interferon responses, no airway architecture, no circulating immune cells and static drug exposure. This study used a two-channel chip with primary human bronchial epithelium differentiated at an air liquid interface above perfused pulmonary endothelium. It reproduced replication differences among six influenza strains, greater barrier damage from H3N2 than H1N1, cytokine output that tracked clinical severity rather than viral load for H5N1, and neutrophil adhesion, transmigration and virus clearance. Oseltamivir acid delivered at blood-like concentrations reproduced its clinical efficacy and its two-day window, which the protease inhibitor nafamostat extended to four days by blocking hemagglutinin cleavage. Eight drugs active against spike-pseudotyped particles in a liver cell line were retested on chip at human maximum plasma concentrations, and only amodiaquine, toremifene and clomiphene remained active. Amodiaquine reduced native SARS-CoV-2 in hamsters prophylactically, therapeutically and in transmission, while hydroxychloroquine did not." }, "discoveries": [], "relationships": [], "canonical_url": "https://tenoeverlab.us/publications/2021-si-a-human-airway-on-a-chip-for-the-r/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sars-cov-2", "vsv" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "immunofluorescence-microscopy", "multiplex-cytokine-assay", "animal-transmission-model", "mass-spectrometry-proteomics", "pseudotyped-entry-reporter", "ali-culture", "barrier-permeability", "organ-on-chip", "pharmacokinetics" ] } }, { "id": "2022-frere-sars-cov-2-infection-in-hamsters-a", "slug": "2022-frere-sars-cov-2-infection-in-hamsters-a", "url": "/publications/2022-frere-sars-cov-2-infection-in-hamsters-a/", "title": "SARS-CoV-2 infection in hamsters and humans results in lasting and unique systemic perturbations after recovery", "authors": [ "Justin J. Frere", "Randal A. Serafini", "Kerri D. Pryce", "Marianna Zazhytska", "Kohei Oishi", "Ilona Golynker", "Maryline Panis", "Jeffrey Zimering", "Shu Horiuchi", "Daisy A. Hoagland", "Rasmus Møller", "Anne Ruiz", "Albana Kodra", "Jonathan B. Overdevest", "Peter D. Canoll", "Alain C. Borczuk", "Vasuretha Chandar", "Yaron Bram", "Robert Schwartz", "Stavros Lomvardas", "Venetia Zachariou", "Benjamin R. tenOever" ], "author_count": 22, "first_author": "Justin J. Frere", "senior_authors": [ "Venetia Zachariou", "Benjamin R. tenOever" ], "corresponding_authors": [ "Venetia Zachariou", "Benjamin R. tenOever" ], "tenoever_position": 22, "tenoever_role": "senior", "contribution_character": "co-led", "year": 2022, "journal": "Science Translational Medicine", "volume": "14", "issue": "664", "pages": "eabq3059", "doi": "10.1126/scitranslmed.abq3059", "doi_url": "https://doi.org/10.1126/scitranslmed.abq3059", "pmid": "35857629", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/35857629/", "pmcid": "PMC9210449", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9210449/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9210449/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "post-acute-sequelae" ], "pathogens": [ "SARS-CoV-2", "influenza A virus" ], "viral_families": [ "Coronaviridae", "Orthomyxoviridae" ], "host_species": [ "golden hamster", "human" ], "technologies": [ "bulk RNA sequencing", "gene set enrichment analysis", "cell type deconvolution", "quantitative RT-PCR", "plaque assay", "histology and immunohistochemistry", "RNA in situ hybridization", "TUNEL staining", "buried food finding test", "marble burying assay" ], "biological_systems": [ "golden hamster lung heart and kidney", "hamster olfactory bulb and olfactory epithelium", "hamster striatum thalamus cerebellum medial prefrontal cortex and trigeminal ganglion", "post-mortem human olfactory bulb and olfactory epithelium" ], "key_concepts": [ "post-acute sequelae of COVID-19", "long COVID", "benchmarking against influenza", "persistent interferon signaling after viral clearance", "olfactory bulb inflammation", "microglial and myeloid activation", "peribronchiolar metaplasia", "renal tubular atrophy", "behavioral change after recovery" ], "keywords": [ "SARS-CoV-2", "long COVID", "golden hamster", "olfactory bulb", "olfactory epithelium", "interferon", "microglia", "influenza A virus", "behavior" ], "one_sentence_contribution": "Benchmarked against pandemic influenza in golden hamsters, SARS-CoV-2 uniquely sustains interferon signaling, chemokine production and myeloid activation in olfactory bulb and epithelium a month after clearance, alongside altered behavior and matching signatures in recovered human olfactory tissue.", "summary_25": "A month after clearing SARS-CoV-2, hamsters still show inflammation and myeloid activation in olfactory tissue and altered behavior, changes absent after influenza and mirrored in recovered people.", "summary_75": "To find what is particular to SARS-CoV-2, hamsters were infected with either that virus or pandemic influenza and followed for a month past recovery. Both left scarring in lung and kidney, worse after SARS-CoV-2. Only SARS-CoV-2 left the olfactory bulb and the olfactory lining inflamed, with activated immune cells and interferon signaling still running despite no detectable virus. Infected animals behaved differently, and olfactory tissue from people who had recovered showed comparable inflammatory programs.", "summary_150": "This study addresses the biology of post-acute sequelae of COVID-19 by comparing SARS-CoV-2 with 2009 pandemic influenza in the golden hamster at matched peak viral loads, profiling lung, heart, kidney and six nervous system regions at peak infection, one week after clearance, and 31 days. Acute responses were largely shared. At 31 days both viruses had left peribronchiolar metaplasia and renal tubular atrophy, more extensive after SARS-CoV-2, while lung transcriptomes had shifted to repair programs. Uniquely after SARS-CoV-2, the olfactory bulb retained interferon signatures, chemokine expression and microglial and macrophage activation with no detectable viral RNA and no increase in apoptosis, and the olfactory epithelium added T cell recruitment and activation. The phenotype occurred in both sexes. Hamsters showed transient anosmia acutely and reduced marble burying at 26 days. Post-mortem olfactory bulb and epithelium from donors recovered from documented COVID-19 showed correlated inflammatory enrichment, though from very few individuals.", "citation": "Frere JJ, Serafini RA, Pryce KD, Zazhytska M, Oishi K, Golynker I, Panis M, Zimering J, Horiuchi S, Hoagland DA, Møller R, Ruiz A, Kodra A, Overdevest JB, Canoll PD, Borczuk AC, Chandar V, Bram Y, Schwartz R, Lomvardas S, Zachariou V, tenOever BR. SARS-CoV-2 infection in hamsters and humans results in lasting and unique systemic perturbations after recovery. Science Translational Medicine. 2022. 14(664), eabq3059. DOI 10.1126/scitranslmed.abq3059. PMID 35857629. PMCID PMC9210449. Correspondence is addressed to Venetia Zachariou and Benjamin R. tenOever.", "sections": { "Citation": "Frere JJ, Serafini RA, Pryce KD, Zazhytska M, Oishi K, Golynker I, Panis M, Zimering J, Horiuchi S, Hoagland DA, Møller R, Ruiz A, Kodra A, Overdevest JB, Canoll PD, Borczuk AC, Chandar V, Bram Y, Schwartz R, Lomvardas S, Zachariou V, tenOever BR. SARS-CoV-2 infection in hamsters and humans results in lasting and unique systemic perturbations after recovery. Science Translational Medicine. 2022. 14(664), eabq3059.\n\nDOI 10.1126/scitranslmed.abq3059. PMID 35857629. PMCID PMC9210449.\n\nCorrespondence is addressed to Venetia Zachariou and Benjamin R. tenOever.", "One-sentence contribution": "Benchmarked against pandemic influenza in golden hamsters, SARS-CoV-2 uniquely sustains interferon signaling, chemokine production and myeloid activation in olfactory bulb and epithelium a month after clearance, alongside altered behavior and matching signatures in recovered human olfactory tissue.", "Executive summary": "Long COVID is defined clinically by symptoms persisting beyond four weeks, and its biological basis was unresolved. This study asks what is specific to SARS-CoV-2 by running a matched comparison against a pandemic influenza A virus in the same animal model at doses chosen to give comparable peak titers. Hamsters were profiled at peak infection, at one week after clearance, and at 31 days, across lung, heart, kidney, and six regions of the nervous system, with histology read by a board-certified pathologist and transcriptomes compared against human cadaver tissue. Acutely, the two viruses looked similar, with interferon and TNF signatures across tissues and comparable pulmonary infiltrates. After clearance the pictures diverged. Both viruses left peribronchiolar metaplasia and renal tubular atrophy, but the lesions were more extensive after SARS-CoV-2. In the nervous system, SARS-CoV-2 uniquely maintained interferon signatures in the olfactory bulb at 31 days, with elevated ISG15, MX2 and IRF7, chemokines including CXCL10 and CCL5, and enrichment for microglial and myeloid gene sets, in the absence of detectable viral RNA. The olfactory epithelium showed the same interferon signature plus T cell recruitment and activation. Hamsters showed transient anosmia at 3 days and reduced marble burying at 26 days. Post-mortem olfactory bulb and epithelium from donors who had recovered from documented COVID-19 more than a month before death showed correlated inflammatory programs.", "Scientific context": "By 2022 the acute biology of SARS-CoV-2 was reasonably well described. Infected cells fail to mount an efficient type I interferon response, much of the interferon that appears derives from uninfected phagocytic cells, and NF-kB-driven cytokine and chemokine production proceeds unchecked, drawing neutrophils and monocytes into an airway where antiviral defense is poorly engaged. Productive infection is largely confined to the respiratory tract in vivo, which the authors attribute not to tropism but to the systemic interferon response rendering distal tissues refractory, an interpretation supported by the observation that organoids and organotypic brain cultures are readily infected ex vivo while the same tissues are rarely infected in animals unless interferon biology is disrupted. What was missing was an account of the persistent phase. Post-acute sequelae of COVID-19 were well documented clinically, including breathlessness, fatigue, depression, anxiety, and impaired memory and concentration, but their cause was unknown, and there was no small animal system in which to study them. The golden hamster was already established as phenocopying acute COVID-19 without viral adaptation.", "Central question": "What distinguishes SARS-CoV-2 from another pandemic respiratory virus in the changes it leaves behind after infection is cleared, and can any of those lasting changes, in particular in the nervous system, be linked to functional and behavioral consequences and corroborated in humans who have recovered?", "Experimental strategy": "The central design choice is the comparator. By infecting parallel cohorts with the 2009 pandemic H1N1 influenza virus at a dose chosen to match SARS-CoV-2 peak titers, the study makes it possible to say which findings belong to SARS-CoV-2 rather than to severe respiratory viral infection generally, and the authors argue in the discussion that this benchmarking is a methodological requirement rather than an optional control. Three time points structure the work, 3 days for peak replication in both models, 14 days for the week after clearance, and 31 days, which corresponds to the clinical threshold for long COVID in a human patient. Tissue sampling is deliberately broad, taking peripheral organs where COVID-19 complications are reported alongside six nervous system regions chosen either because they were documented as virus positive in patients or because they govern sensory, motor, cognitive or affective functions altered in long COVID. Transcriptional profiling is paired throughout with histology, immunohistochemistry, in situ hybridization, and targeted quantitative PCR so that a signature can be checked against tissue. Because whole regions were sequenced, cell type inference is approached by deconvolution and by gene sets for neural and glial populations rather than by single-cell methods. Finally, behavior is assessed with a buried food test for olfaction and a marble burying assay for anxiety-like and repetitive behavior, and the animal findings are set against post-mortem olfactory tissue from donors with documented recovered COVID-19.", "Key findings": "1. Both viruses replicated in hamster lung with peak titers at 3 days, influenza at about 10^7 plaque-forming units per gram and SARS-CoV-2 at about 10^8 persisting to day 5, and neither yielded infectious virus at day 7, although viral RNA remained detectable (Fig. 1A and 1B).\n2. At 3 days both infections produced hypercellularity and infiltration of macrophages, neutrophils and T cells in lung, with neutrophils and macrophages predominating, and SARS-CoV-2 infiltration concentrated around bronchioles and larger airways (Fig. 1C to 1F). Kidney showed no infiltration at this point and heart showed some leukocytic infiltrate for both viruses.\n3. Acute lung transcriptomes were dominated by type I and type II interferon, TNF and IL-2 signatures for both viruses, corroborated by MX1 immunohistochemistry, and by 14 days showed minimal antiviral response (Fig. 1G and 1H).\n4. Acute signatures in lung, heart and kidney matched those in human cadaver tissue from individuals who died with high viral loads, supporting the model's clinical relevance (Fig. 2A to 2C).\n5. At 31 days neither virus showed interferon or chemokine enrichment in lung, heart or kidney (Fig. 2D to 2F). Instead lung showed repair and regeneration programs including axoneme assembly and ciliary biogenesis, and at 14 days SARS-CoV-2 uniquely showed negative enrichment of microtubular motor activity and axoneme assembly (Fig. 2I and 2J), which the authors read as more severe and longer-lasting ciliary damage.\n6. Histology at 31 days showed peribronchiolar metaplasia and enlarged airway spaces after both infections, more extensive after SARS-CoV-2 by morphometric quantification (Fig. 3A and 3C), and renal tubular atrophy with proteinaceous interstitial fluid, again greater after SARS-CoV-2 (Fig. 3B and 3D). Verhoeff Van Gieson staining showed no evident fibrosis or collagen deposition, and heart infiltration had fully resolved.\n7. Viral reads were detectable in nervous tissue of a subset of SARS-CoV-2 infected hamsters and in none of the influenza animals, with most reads mapping to the nucleocapsid transcript in one animal where all surveyed regions were positive (Fig. 4B). The authors interpret this as possible deposition of circulating subgenomic RNA rather than replication, and they do not claim neuroinvasion.\n8. Time course quantitative PCR showed subgenomic nucleocapsid transcript in olfactory bulb rising through 4 days in two of three animals and dissipating, with sporadic early positivity in striatum and cerebellum only. ISG15 generally tracked subgenomic RNA, with the exception that olfactory bulb ISG15 was newly elevated at 14 days with no subgenomic signal (Fig. S4).\n9. Across neural regions, both viruses produced acute interferon signatures and region-specific metabolic, synaptic and plasticity changes, some persisting past one month. Thalamus responded differently to the two viruses, which the authors describe as a hypoexcitable state after SARS-CoV-2 against a hyperexcitable state after influenza, and dendrite development genes were altered in thalamus after SARS-CoV-2 only.\n10. At 31 days the olfactory bulb of influenza-infected animals had returned to baseline while SARS-CoV-2 animals retained interferon signatures, elevated ISG15, MX2 and IRF7 confirmed by quantitative PCR, MX1 protein localized to the glomerular periphery, and elevated CXCL10 and CCL5 (Fig. 5A to 5F and Fig. S5A).\n11. Deconvolution and directed enrichment of olfactory bulb at 31 days showed microglial and myeloid activation specific to SARS-CoV-2, with negative enrichment of neuronal gene sets, and IBA1 staining showed myeloid cells with enlarged rounded bodies at the bulb periphery (Fig. 5G, Fig. S5C to S5F). CD3 staining was sparse across all groups, so T cells were judged not to contribute in this tissue.\n12. No viral transcripts were detectable in olfactory bulb at 31 days by quantitative PCR or in situ hybridization, and TUNEL staining showed no difference in apoptotic nuclei between groups at either time point (Fig. 5I, Fig. S6A and S6B). Persistent inflammation therefore occurs without detectable virus and without measurable local apoptosis.\n13. Olfactory epithelium at 31 days showed the same SARS-CoV-2-specific interferon signature plus chemotactic recruitment driven by CCL7, CXCL10, CCL5 and CCL11 and T cell recruitment and activation driven by antigen presentation and CD3 genes, along with negative enrichment for olfactory receptor genes in both infections (Fig. 5J and Fig. S6C).\n14. The olfactory phenotype was present in female as well as male hamsters, with elevated ISG15 and CCL5 at 24 days (Fig. S6E and S6F).\n15. SARS-CoV-2-infected hamsters took longer to find buried food at 3 days but not at 15 or 28 days, with no difference in a visible food control (Fig. 6A to 6C and Fig. S6J to S6L), and buried fewer marbles at 26 days than mock animals, while influenza animals performed like mock (Fig. 6D).\n16. Post-mortem olfactory bulb from two donors recovered from documented COVID-19, compared with one control, showed enriched complement and interferon programs, and olfactory epithelium from two further recovered donors against three controls showed chemotactic and T cell programs. Enrichment scores for the same gene sets correlated with the hamster data at 31 days (Fig. 7).", "Mechanistic model": "The study does not establish a mechanism for the persistent olfactory inflammation and it states this limitation directly, including that it does not demonstrate causality between brain inflammation and the behavioral changes measured. What the data constrain is the following. Sustained interferon and chemokine signaling in the olfactory bulb and epithelium at 31 days occurs when no infectious virus and no detectable viral RNA remain in those tissues, when animals have long since mounted an antibody response, and without a measurable increase in local apoptosis. The inflammatory cells involved are predominantly microglia and infiltrating macrophages in the bulb, with T cell involvement confined to the epithelium. Two explanations are offered by the authors as hypotheses and neither is tested here. One is that persistent defective viral genomes or residual viral debris in a tissue that was positive early continue to drive a host response. The other is that infection damages the olfactory epithelial barrier, allowing commensal microorganisms access to the bulb, an idea the authors support by pointing to reported thinning and sloughing of the epithelium. They also note that low-level persistence of viral material has been reported by others under some circumstances and should not be ruled out. The link from olfactory inflammation to behavior is an inference from the anatomical connection between the bulb and the limbic system together with prior literature associating bulb damage with depressive phenotypes, and the greater severity of SARS-CoV-2 damage relative to influenza is attributed speculatively to the larger amount of double-stranded RNA generated during subgenomic RNA production.", "Conceptual or technical advance": "The work provides a tractable small animal system in which the persistent phase of SARS-CoV-2 infection can be studied, with molecular, histological and behavioral readouts in the same animals, and it establishes a methodological standard for that work by requiring a benchmark virus. Several findings would have read as COVID-specific without that comparator and turn out not to be, including the acute interferon response across tissues, peribronchiolar metaplasia, renal tubular atrophy, and loss of olfactory receptor transcripts, all shared with influenza and differing in degree rather than in kind. What survives the comparison is narrower and therefore more informative, namely the sustained olfactory bulb and epithelium inflammation with myeloid activation, present in both sexes, occurring without detectable virus, and mirrored in human tissue collected long after recovery. That combination supplies a candidate biological correlate for long COVID symptomatology that can now be tested causally.", "Relationship to the broader research program": "The interpretive frame for the acute phase, in which SARS-CoV-2 produces a strong NF-kB-driven inflammatory response alongside a deficient type I interferon response and interferon arises largely from uninfected bystander cells, comes from earlier work by this laboratory and is cited throughout. The hamster model, the influenza benchmark, and the earlier characterization of SARS-CoV-2 tropism and ciliary loss in that model are likewise laboratory antecedents. What is new to the collaboration is the neuroscience arm, contributed with the Zachariou laboratory, and the olfactory system expertise contributed with the Lomvardas and Overdevest groups, whose work on the molecular basis of anosmia is cited for the loss of sustentacular cells and the negative enrichment of neuronal populations. Reading this paper together with other corpus entries on tissue-specific SARS-CoV-2 responses, including the ocular work, would be category 3 synthesis, and the common thread of a persistent inflammatory program decoupled from detectable virus should be assembled centrally rather than asserted here.", "Related publications": "- Blanco-Melo et al. 2020, imbalanced host response to SARS-CoV-2 drives development of COVID-19. Predecessor from the tenOever laboratory, source of the acute interferon-low and inflammation-high framing used throughout.\n- Hoagland et al. 2021, work from the same laboratory on the golden hamster model and interferon dynamics during SARS-CoV-2 infection. Methodological foundation, source of the hamster model parameters, dosing, and the nucleocapsid transcript release observation.\n- de Melo et al., anosmia in the golden hamster after SARS-CoV-2 infection. Predecessor from another group, replicated here in the buried food finding test.\n- Zazhytska and colleagues on the molecular basis of anosmia, with co-authors shared with this study. Companion, cited for loss of sustentacular cells and for olfactory receptor downregulation.\n- tenOever laboratory work on SARS-CoV-2 in human cadaver tissues, cited as the source of the matched human comparison data in Fig. 2C. Companion.", "Limitations and boundaries": "The authors are explicit about several boundaries. The different replication trajectories of the two viruses restricted the comparison to peak infection and to time points after clearance, since intermediate time points differ in replication and clearance rate and are hard to interpret. Causality between persistent brain inflammation and altered behavior is not demonstrated. The behavioral assays used were developed and characterized in mice and rats rather than hamsters, and the group sizes are smaller than standard for behavioral work because the experiments had to be performed inside high containment. The human comparison rests on very few donors, two recovered and one control for olfactory bulb and two recovered and three controls for epithelium, and it compares a moderate hamster infection with human cases that ended in death, a mismatch the authors call unavoidable because patients with moderate disease do not generally come to autopsy. Peripheral organ findings likely reflect a milder infection state than the human post-mortem literature on kidney involvement. Sequencing was performed on whole brain regions, so differentially expressed genes are summed across all cell populations present and cell type attribution rests on deconvolution rather than on single-cell measurement. The absence of detectable virus at 31 days is a limit of the assays used and the authors note that persistence under some circumstances has been reported by others. Finally, the study is confined to one SARS-CoV-2 isolate, one influenza comparator, one inoculation route, and animals that were not previously immune or vaccinated.", "Audience summaries": "### 25 words\n\nA month after clearing SARS-CoV-2, hamsters still show inflammation and myeloid activation in olfactory tissue and altered behavior, changes absent after influenza and mirrored in recovered people.\n\n### 75 words\n\nTo find what is particular to SARS-CoV-2, hamsters were infected with either that virus or pandemic influenza and followed for a month past recovery. Both left scarring in lung and kidney, worse after SARS-CoV-2. Only SARS-CoV-2 left the olfactory bulb and the olfactory lining inflamed, with activated immune cells and interferon signaling still running despite no detectable virus. Infected animals behaved differently, and olfactory tissue from people who had recovered showed comparable inflammatory programs.\n\n### 150 words\n\nThis study addresses the biology of post-acute sequelae of COVID-19 by comparing SARS-CoV-2 with 2009 pandemic influenza in the golden hamster at matched peak viral loads, profiling lung, heart, kidney and six nervous system regions at peak infection, one week after clearance, and 31 days. Acute responses were largely shared. At 31 days both viruses had left peribronchiolar metaplasia and renal tubular atrophy, more extensive after SARS-CoV-2, while lung transcriptomes had shifted to repair programs. Uniquely after SARS-CoV-2, the olfactory bulb retained interferon signatures, chemokine expression and microglial and macrophage activation with no detectable viral RNA and no increase in apoptosis, and the olfactory epithelium added T cell recruitment and activation. The phenotype occurred in both sexes. Hamsters showed transient anosmia acutely and reduced marble burying at 26 days. Post-mortem olfactory bulb and epithelium from donors recovered from documented COVID-19 showed correlated inflammatory enrichment, though from very few individuals." }, "discoveries": [ "claim-14" ], "relationships": [ { "from": "2022-frere-sars-cov-2-infection-in-hamsters-a", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2022-frere-sars-cov-2-infection-in-hamsters-a" }, { "from": "2022-frere-sars-cov-2-infection-in-hamsters-a", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2022-frere-sars-cov-2-infection-in-hamsters-a" }, { "from": "2022-zazhytska-non-cell-autonomous-disruption-of-", "to": "2022-frere-sars-cov-2-infection-in-hamsters-a", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2022-zazhytska-non-cell-autonomous-disruption-of-" }, { "from": "2022-zazhytska-non-cell-autonomous-disruption-of-", "to": "2022-frere-sars-cov-2-infection-in-hamsters-a", "relationship": "companion", "evidence": "stated in the Related publications section of 2022-zazhytska-non-cell-autonomous-disruption-of-" }, { "from": "2023-serafini-sars-cov-2-airway-infection-result", "to": "2022-frere-sars-cov-2-infection-in-hamsters-a", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2023-serafini-sars-cov-2-airway-infection-result" } ], "canonical_url": "https://tenoeverlab.us/publications/2022-frere-sars-cov-2-infection-in-hamsters-a/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sars-cov-2" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "pathway-enrichment-analysis", "histopathology", "rna-in-situ-hybridization", "cell-death-assays", "behavioural-and-sensory-testing", "deconvolution" ] } }, { "id": "2022-nilsson-payant-the-host-factor-anp32a-is-required", "slug": "2022-nilsson-payant-the-host-factor-anp32a-is-required", "url": "/publications/2022-nilsson-payant-the-host-factor-anp32a-is-required/", "title": "The Host Factor ANP32A Is Required for Influenza A Virus vRNA and cRNA Synthesis", "authors": [ "Benjamin E. Nilsson-Payant", "Benjamin R. tenOever", "Aartjan J. W. te Velthuis" ], "author_count": 3, "first_author": "Benjamin E. Nilsson-Payant", "senior_authors": [ "Aartjan J. W. te Velthuis" ], "corresponding_authors": [ "Benjamin E. Nilsson-Payant", "Aartjan J. W. te Velthuis" ], "tenoever_position": 2, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2022, "journal": "Journal of Virology", "volume": "96", "issue": "4", "pages": "e02092-21", "doi": "10.1128/jvi.02092-21", "doi_url": "https://doi.org/10.1128/jvi.02092-21", "pmid": "34935435", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/34935435/", "pmcid": "PMC8865535", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8865535/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8865535/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "influenza-genome-regulation" ], "themes": [ "polymerase-nucleoprotein-host-factors" ], "pathogens": [ "influenza A virus" ], "viral_families": [ "Orthomyxoviridae" ], "host_species": [ "human", "chicken", "dog" ], "technologies": [ "minigenome assay", "influenza reverse genetics", "primer extension", "site-directed promoter mutagenesis", "single-molecule FRET", "RNA immunoprecipitation", "siRNA knockdown", "plaque assay", "cycloheximide and actinomycin D blocks" ], "biological_systems": [ "HEK-293T cells", "A549 cells", "DF-1 chicken embryonic fibroblasts", "MDCK cells", "recombinant purified influenza RNA polymerase" ], "key_concepts": [ "ANP32A", "host range restriction", "PB2 627 polymorphism", "vRNA synthesis", "cRNA synthesis", "replicase complex assembly", "encapsidating polymerase", "low-complexity acidic region", "viral promoter mutation", "primary transcription" ], "keywords": [ "influenza A virus", "ANP32A", "RNA polymerase", "PB2-E627K", "genome replication", "cRNA", "vRNA", "host adaptation", "minigenome", "smFRET" ], "one_sentence_contribution": "Uncoupling the two steps of influenza A virus genome replication with viral promoter mutations shows that ANP32A is required for synthesis of both the complementary RNA intermediate and progeny genomic RNA, and that it acts on the actively replicating polymerase rather than the encapsidating one.", "summary_25": "Influenza copies its genome in two steps. Blocking each step separately with promoter mutations shows the host protein ANP32A is needed for both, not just one.", "summary_75": "Influenza A virus replicates its genome by first making a complementary copy and then copying that back into genomes, and the host protein ANP32A is essential for this and sets a barrier to avian viruses in mammalian cells. It had been proposed that ANP32A acts only at the second step. Using viral promoter mutations that permit one step at a time, this study shows both steps require ANP32A, acting on the actively synthesizing polymerase.", "summary_150": "Influenza A virus genome replication proceeds through a complementary RNA intermediate, and each nascent product is encapsidated by an additional polymerase, so more than one polymerase participates. The host factor ANP32A is essential for replication, bridges an RNA-bound and an RNA-free polymerase in published structures, and differs between birds and mammals in ways that restrict avian polymerases. Work with purified protein had suggested ANP32A is needed only for synthesis of genomic RNA from the intermediate. Using mutations in the viral promoters that block initiation on the resulting product, this study restricts minigenomes to one replication step at a time and finds that an avian-like PB2 627E polymerase is impaired at both steps and rescued by chicken ANP32A at both. Single-molecule FRET shows the 627 position does not affect promoter binding, and pre-expression experiments place the requirement on the replicating rather than the encapsidating polymerase.", "citation": "Nilsson-Payant BE, tenOever BR, te Velthuis AJW. The Host Factor ANP32A Is Required for Influenza A Virus vRNA and cRNA Synthesis. Journal of Virology. 2022. Volume 96, Issue 4, article e02092-21. DOI 10.1128/jvi.02092-21. PMID 34935435. PMCID PMC8865535.", "sections": { "Citation": "Nilsson-Payant BE, tenOever BR, te Velthuis AJW. The Host Factor ANP32A Is Required for Influenza A Virus vRNA and cRNA Synthesis. Journal of Virology. 2022. Volume 96, Issue 4, article e02092-21.\n\nDOI 10.1128/jvi.02092-21. PMID 34935435. PMCID PMC8865535.", "One-sentence contribution": "Uncoupling the two steps of influenza A virus genome replication with viral promoter mutations shows that ANP32A is required for synthesis of both the complementary RNA intermediate and progeny genomic RNA, and that it acts on the actively replicating polymerase rather than the encapsidating one.", "Executive summary": "Influenza A virus replicates its segmented negative-sense genome in two steps, first copying genomic RNA into a complementary intermediate and then copying that intermediate back into progeny genomes. Each nascent product must be encapsidated by an additional polymerase, so at least two polymerase complexes participate. The host protein ANP32A is essential for replication and bridges an RNA-bound polymerase to an RNA-free one, and differences between avian and mammalian ANP32A are sufficient to restrict avian polymerases in mammalian cells. Based on work with purified protein showing enhancement of genomic RNA synthesis, it had been proposed that ANP32A is needed only for the second step. The paper notes that this does not fit recent structural evidence, since both nascent species are encapsidated and it seems unlikely that separate encapsidation complexes evolved for each.\n\nThe study resolves this by making the two steps experimentally separable. Mutations in the viral promoters block initiation on the resulting product, so a minigenome can be restricted to primary complementary RNA synthesis or to genomic RNA synthesis alone. Combined with an avian-like polymerase carrying PB2 627E and with chicken ANP32A supplied in trans, this permits each step to be tested independently.\n\nBoth steps required ANP32A. The avian-like polymerase was impaired for each and was rescued by chicken ANP32A in each case. Single-molecule FRET showed the 627 position does not affect promoter binding, and an infection experiment with pre-expressed catalytically inactive or active polymerase placed the requirement at the replicating polymerase rather than at the encapsidating one.", "Scientific context": "Influenza A viruses circulate mainly in wild aquatic birds and infect humans only occasionally, in part because avian viruses cannot replicate efficiently in mammalian cells without overcoming host barriers. Two adaptations are emphasized, a shift in receptor binding preference and restoration of binding between the viral polymerase and the host factor ANP32A. A single substitution in PB2, glutamic acid to lysine at position 627, is sufficient to restore avian polymerase activity in mammalian cells, and expressing avian ANP32A alone achieves the same thing, which the paper reads as indicating that the PB2 substitution compensates for an impaired interaction with mammalian ANP32A.\n\nANP32A has an N-terminal leucine-rich repeat domain and a C-terminal low-complexity acidic region. Avian and mammalian homologues are similar in sequence, but the avian gene carries an exon duplication that yields, through alternative splicing, isoforms with extra sequence. Restoration of avian polymerase activity by avian ANP32A depends on that duplication, and the differential interaction has been mapped to the low-complexity acidic region contacting the flexible PB2 627-domain. ANP32B supports mammalian-adapted polymerase function but does not restore avian polymerase replication.\n\nCryo-electron microscopy of an influenza C virus polymerase dimer showed ANP32A bridging an RNA-bound polymerase and an RNA-free polymerase, suggesting it mediates assembly of the replicase complex. ANP32A appears to be required for genome replication but not primary transcription. Work with recombinant purified protein showed human ANP32A enhancing genomic RNA synthesis by human-adapted polymerases, and from that it was proposed that ANP32A is required only for the step producing genomic RNA from the complementary intermediate and not the reverse. The paper states directly that this view does not match the molecular evidence, because both species are encapsidated and separate encapsidation machinery for each seems unlikely. Several competing explanations for avian polymerase restriction in mammalian cells also remained in play, including destabilized polymerase and nucleoprotein interactions, reduced promoter binding, differential importin-alpha interactions, and unstable complementary ribonucleoprotein structures.", "Central question": "At which stage or stages of influenza A virus genome replication is ANP32A required, and does it act on the polymerase that is actively synthesizing RNA or on the polymerase that encapsidates the nascent product?", "Experimental strategy": "The central difficulty is that in an infection the two replication steps are obligately coupled, since complementary RNA must be made before genomic RNA can be made from it. The strategy is to break that coupling genetically at the level of the promoter.\n\nTwo promoter manipulations do the work. A G5U change in the 5-prime genomic promoter prevents internal initiation on the resulting complementary RNA product, permitting primary messenger RNA and primary complementary RNA synthesis while blocking subsequent genomic RNA synthesis. A G2C and C9G pair in the 5-prime complementary promoter prevents 3-prime terminal initiation on the resulting genomic product, abolishing further complementary RNA synthesis without blocking transcription. Each isolates one step. A separate G3A and C8U pair in the 3-prime genomic promoter, previously reported to improve avian polymerase activity, is included to test whether promoter strength alone explains the restriction.\n\nAvian-like polymerase is produced not by using an avian virus but by introducing a single PB2 K627E substitution into a mammalian-adapted background, which keeps every other viral component constant. Chicken ANP32A supplied in trans is the rescue arm, with human ANP32A and green fluorescent protein as controls, so the species difference in one host protein is the only variable.\n\nSeveral alternative explanations are tested and excluded rather than argued away. Cycloheximide restricts analysis to primary transcription of incoming ribonucleoproteins, separating a transcription defect from a downstream consequence of impaired replication. Knockdown of both human ANP32A and ANP32B addresses the possibility that mammalian ANP32A contributes to transcription of mammalian-adapted polymerases, which the avian rescue experiments cannot address. Immunoprecipitation of a catalytically inactive polymerase and single-molecule FRET with labeled promoters test whether the 627 position affects RNA binding. Short minigenome templates with and without nucleoprotein test whether the effect depends on nucleoprotein or on template length.\n\nFinally, to place the requirement on one of the two polymerases in the replicase, catalytically inactive or active polymerase is pre-expressed before infection under actinomycin D, so that replication depends entirely on the pre-expressed components and the encapsidation function can be examined with the replication function disabled.", "Key findings": "1. In human HEK-293T cells the avian-like WSN-K627E virus was significantly restricted in both replication and transcription relative to wild type, while in chicken DF-1 cells it showed slightly increased levels (Figure 2A, Figure 2B). Transient expression of chicken ANP32A restored WSN-K627E growth in human cells to near wild-type levels (Figure 2C, Figure 2D), and restored viral RNA accumulation, whereas human ANP32A did not, with neither affecting wild-type virus (Figure 2E).\n\n2. Reconstituting the minimal replication machinery in cells reproduced the restriction at the level of the polymerase, with PB2-627E ribonucleoproteins showing reduced production of all three viral RNA species and restoration by chicken ANP32A (Figure 2F), which localizes the effect to the polymerase rather than to other viral factors.\n\n3. Under cycloheximide, which permits primary transcription but blocks genome replication, wild-type and K627E viruses showed no difference in messenger RNA synthesis (Figure 2G). The observation is equivalence in primary transcription. The interpretation is that the reduced messenger RNA seen otherwise is a downstream consequence of impaired replication and reduced template.\n\n4. Knocking down both human ANP32A and ANP32B in A549 cells left primary transcription unchanged early in infection while strongly reducing genome replication (Figure 2H, Figure 2I), extending the transcription-independence conclusion to mammalian-adapted polymerase and to the mammalian proteins.\n\n5. Deleting the avian exon duplication from chicken ANP32A abolished rescue of the 627E polymerase, while a C-terminal or internal deletion of 31 amino acids in the low-complexity acidic region remained compatible with activity and longer deletions of that region abolished it (Figure 3A, Figure 3B, Figure 3C). The authors conclude that the low-complexity acidic region, and specifically its N-terminal portion, is pivotal.\n\n6. Immunoprecipitation of a TAP-tagged, catalytically inactive polymerase showed that genomic RNA binding by a 627E polymerase was not impaired relative to 627K, and that chicken ANP32A had no effect on binding (Figure 4A).\n\n7. Single-molecule FRET with fluorescently labeled promoters and recombinant catalytically inactive polymerases found comparable bound populations for genomic promoter binding whether the polymerase carried 627K, 627E, or lacked the entire 627-domain (Figure 4D), and comparable shifts from the unbound state for the complementary promoter (Figure 4E). The conclusion drawn is that the 627-domain plays no role in promoter binding, which contradicts earlier reports proposing weaker promoter binding by avian polymerases. The authors note that small amounts of endogenous human ANP32A and ANP32B may have co-purified with the recombinant polymerase, and argue this does not affect the outcome because the substitution had no significant effect on binding.\n\n8. In minigenome assays using a 76 nucleotide segment 5 template that does not require nucleoprotein, the K627E substitution still caused significant loss of activity and chicken ANP32A still restored it, with nucleoprotein presence or absence making no difference (Figure 5A). The same held on 47 nucleotide and 30 nucleotide templates (Figure 5B, Figure 5C), so the restriction and the ANP32A requirement are independent of nucleoprotein and of template length. The authors contrast this with an earlier report that restriction was diminished or abolished on short templates.\n\n9. The 3A8U genomic promoter mutation greatly enhanced replication overall, but the 627E polymerase remained significantly, if less markedly, restricted, and chicken ANP32A fully restored it (Figure 6B). The interpretation is that improved promoter strength alone does not overcome the restriction.\n\n10. With the G5U mutation limiting the template to primary complementary RNA synthesis, the 627E polymerase was unable to synthesize complementary RNA efficiently, and chicken ANP32A restored it, on both wild-type and 3A8U backgrounds (Figure 6C, Figure 6D, Figure 6E). This is the finding that contradicts the proposal that ANP32A acts only on the second step.\n\n11. With the 2C9G complementary promoter mutation preventing further complementary RNA synthesis, no genomic RNA synthesis was observed with the 627E polymerase, and chicken ANP32A significantly increased activity (Figure 6F, Figure 6G). Together with the preceding point, both directions of replication require ANP32A.\n\n12. Pre-expressing polymerase, nucleoprotein and chicken ANP32A before infection under actinomycin D showed that no complementary RNA was stabilized without polymerase, that 627K and 627E polymerases stabilized nascent complementary RNA equally well when their own catalytic activity was disabled, and that when active replication was permitted the 627E polymerase produced reduced genomic RNA, which chicken ANP32A overcame (Figure 6H). The observation is that the encapsidation function is unaffected by the 627 identity while the replication function is. The conclusion drawn is that ANP32A is needed for the actively replicating polymerase and not for the encapsidating one.", "Mechanistic model": "The model the data support is that ANP32A is required at both stages of genome replication and acts on the polymerase performing synthesis rather than on the polymerase capturing the nascent product. Species-specific differences in ANP32A therefore restrict avian-adapted polymerases at both steps in mammalian cells, and the PB2 627 substitution or supply of avian ANP32A restores the interaction.\n\nSeveral elements are directly demonstrated. The requirement for ANP32A at each step separately is demonstrated by the promoter-uncoupling minigenome experiments, which is the core new result. The independence of the requirement from nucleoprotein, from template length, and from promoter binding is demonstrated by the nucleoprotein-free templates, the short templates, the co-immunoprecipitation and the single-molecule FRET measurements. The dissociation between an intact encapsidation function and an impaired replication function is demonstrated by the pre-expression experiment with a catalytically inactive polymerase.\n\nThe bridging mechanism itself is not established here. The idea that ANP32A mediates assembly of a replicase complex by bridging an RNA-bound and an RNA-free polymerase comes from published cryo-electron microscopy of an influenza C virus polymerase dimer, and the authors present their data as consistent with that structure rather than as a demonstration of it. No structure, no dimer measurement and no direct assembly assay is presented in this study.\n\nThe evolutionary account in the discussion is explicitly marked as speculation by the authors, who write that it is tempting to speculate that influenza A viruses evolved this dependency in avian species and that the shorter mammalian ANP32A cannot efficiently support replicase assembly at both stages, thereby requiring adaptive mutations in avian strains.", "Conceptual or technical advance": "The methodological contribution is the use of single-step promoter mutations to separate two steps of genome replication that are obligately coupled during infection. The G5U and 2C9G mutations restrict a minigenome to one direction of synthesis, which makes it possible to ask where a host factor is required rather than only whether it is required. That approach is reusable for other factors and other steps in the replication cycle.\n\nConceptually, the result removes a model. The proposal that ANP32A is required only for genomic RNA synthesis from the complementary intermediate is inconsistent with a requirement demonstrated in both directions, and the further placement of the requirement on the replicating rather than the encapsidating polymerase constrains how the bridging role seen in structural work must operate functionally. The promoter binding measurements also weigh against the competing explanation that avian polymerase restriction reflects weaker template binding.", "Relationship to the broader research program": "This paper is led by the te Velthuis laboratory at Princeton, with the first author based in the tenOever laboratory at New York University, and the tenOever contribution is one component of a collaborative study rather than the direction of the work. The mechanistic questions addressed, host factor requirements and promoter behavior in influenza polymerase function, sit alongside the tenOever laboratory's own long-running interest in the influenza polymerase and the transcription-to-replication switch, including the earlier work on small viral RNAs as enhancers of polymerase activity. Both lines converge on the question of what converts the polymerase between its activities and what supplies the missing component in each mode, but relating them formally is category 3 synthesis and depends on the corpus records for those papers rather than on anything asserted here.\n\nThe host adaptation framing, in which a single host protein difference sets the species barrier for an avian virus in mammalian cells, also connects to the broader recurring theme of species-specific constraints on influenza replication.", "Related publications": "- Perez and colleagues 2012, Journal of Virology, a small-RNA enhancer of viral polymerase activity. Relationship conceptual extension, from the tenOever laboratory. Not cited in this paper, and the connection is at the level of shared subject matter, namely control of the influenza polymerase transition between transcription and replication, rather than a direct lineage.\n- Carrique and colleagues, cryo-electron microscopy of an influenza C virus polymerase dimer bridged by ANP32A. Relationship predecessor from another laboratory. This is the structural result the present functional data are presented as consistent with, cited as reference 7.\n- Long and colleagues, on avian ANP32A and its exon duplication restoring avian polymerase activity in mammalian cells. Relationship predecessor from another laboratory, cited as reference 20, and the source of the framing that the study sets out to test systematically.\n- Sugiyama and colleagues, on human ANP32A as an enhancer of genomic RNA synthesis with purified protein. Relationship predecessor from another laboratory, cited as reference 27, and the source of the single-step model that this study contradicts.\n- te Velthuis and colleagues, previous work from the senior author's group on the PB2 627-domain and basic polymerase function, cited as reference 37, and on promoter mutants and minigenome constructs. Relationship methodological foundation.", "Limitations and boundaries": "Avian polymerase is modeled by a single PB2 K627E substitution in the mammalian-adapted A/WSN/33 background rather than by an authentic avian isolate, so the conclusions apply to that polymorphism and not necessarily to every determinant of avian polymerase restriction. Likewise, avian ANP32A is represented by chicken ANP32A supplied by transient transfection, which is an overexpression condition and not endogenous avian expression.\n\nMuch of the mechanistic work uses minigenome assays with short internally truncated templates of 76, 47 and 30 nucleotides. These are deliberately chosen to remove nucleoprotein and template length as variables, but they do not reproduce a full-length ribonucleoprotein, and the paper itself notes that the promoter-uncoupling question can only be addressed in minigenomes because both steps necessarily occur during infection.\n\nThe single-molecule FRET experiments use recombinant catalytically inactive polymerases, and the authors acknowledge that small amounts of endogenous human ANP32A and ANP32B may have co-purified. Their argument that this does not matter rests on the absence of a 627-dependent difference rather than on demonstrating that the preparations were free of these proteins.\n\nThe knockdown experiment removes both ANP32A and ANP32B by small interfering RNA, so residual protein and the contributions of the two family members are not separated, and the transcription conclusion rests on an early time point at high multiplicity.\n\nThe encapsidation conclusion comes from one experimental configuration, pre-expression of catalytically inactive polymerase under actinomycin D, and rests on complementary RNA stabilization as the readout for encapsidation rather than on a direct measurement of complex composition.\n\nNo structural or biophysical evidence for replicase assembly is generated in this study, so the bridging model remains imported from published structural work. No animal experiments are included, and no claim is made about transmission or pathogenesis. The cell systems are human, chicken and canine lines, with no primary human airway tissue.", "Audience summaries": "### 25 words\n\nInfluenza copies its genome in two steps. Blocking each step separately with promoter mutations shows the host protein ANP32A is needed for both, not just one.\n\n### 75 words\n\nInfluenza A virus replicates its genome by first making a complementary copy and then copying that back into genomes, and the host protein ANP32A is essential for this and sets a barrier to avian viruses in mammalian cells. It had been proposed that ANP32A acts only at the second step. Using viral promoter mutations that permit one step at a time, this study shows both steps require ANP32A, acting on the actively synthesizing polymerase.\n\n### 150 words\n\nInfluenza A virus genome replication proceeds through a complementary RNA intermediate, and each nascent product is encapsidated by an additional polymerase, so more than one polymerase participates. The host factor ANP32A is essential for replication, bridges an RNA-bound and an RNA-free polymerase in published structures, and differs between birds and mammals in ways that restrict avian polymerases. Work with purified protein had suggested ANP32A is needed only for synthesis of genomic RNA from the intermediate. Using mutations in the viral promoters that block initiation on the resulting product, this study restricts minigenomes to one replication step at a time and finds that an avian-like PB2 627E polymerase is impaired at both steps and rescued by chicken ANP32A at both. Single-molecule FRET shows the 627 position does not affect promoter binding, and pre-expression experiments place the requirement on the replicating rather than the encapsidating polymerase." }, "discoveries": [], "relationships": [ { "from": "2022-nilsson-payant-the-host-factor-anp32a-is-required", "to": "2012-perez-a-small-rna-enhancer-of-viral-poly", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2022-nilsson-payant-the-host-factor-anp32a-is-required" } ], "canonical_url": "https://tenoeverlab.us/publications/2022-nilsson-payant-the-host-factor-anp32a-is-required/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus" ], "technologies": [ "plaque-assay", "reverse-genetics", "sirna-knockdown", "site-directed-mutagenesis", "minigenome-assay", "primer-extension", "rna-immunoprecipitation", "single-molecule-fret", "metabolic-inhibitor-blocks" ] } }, { "id": "2022-oishi-a-diminished-immune-response-under", "slug": "2022-oishi-a-diminished-immune-response-under", "url": "/publications/2022-oishi-a-diminished-immune-response-under/", "title": "A diminished immune response underlies age-related SARS-CoV-2 pathologies", "authors": [ "Kohei Oishi", "Shu Horiuchi", "Justin Frere", "Robert E. Schwartz", "Benjamin R. tenOever" ], "author_count": 5, "first_author": "Kohei Oishi", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 5, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2022, "journal": "Cell Reports", "volume": "39", "issue": "13", "pages": "111002", "doi": "10.1016/j.celrep.2022.111002", "doi_url": "https://doi.org/10.1016/j.celrep.2022.111002", "pmid": "35714615", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/35714615/", "pmcid": "PMC9181267", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9181267/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9181267/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "immunity-age-and-reinfection" ], "pathogens": [ "SARS-CoV-2" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "golden hamster", "human" ], "technologies": [ "intranasal infection of hamsters", "plaque assay", "RNA sequencing", "gene set enrichment analysis", "flow cytometry immune profiling", "antigen-specific B cell staining", "immunohistochemistry", "ELISA", "plaque reduction neutralization test" ], "biological_systems": [ "golden hamster", "hamster lung", "hamster spleen", "mediastinal lymph node", "Vero E6 cells", "human COVID-19 cadaver lung tissue" ], "key_concepts": [ "immunosenescence", "innate immune response kinetics", "interferon-stimulated genes", "NF-kappaB signalling", "tissue repair", "regulatory T cells", "TGF-beta signalling", "IL-17 and neutrophil recruitment", "germinal centre B cells", "affinity maturation", "neutralizing antibody potency" ], "keywords": [ "SARS-CoV-2", "aging", "golden hamster", "regulatory T cells", "IL-17", "neutrophils", "germinal center B cells", "neutralizing antibodies", "COVID-19", "innate immunity" ], "one_sentence_contribution": "Comparison of young and older golden hamsters infected with SARS-CoV-2 shows that age reduces the magnitude and duration of the innate response and of tissue repair, expands suppressor T cells and IL-17-driven neutrophil recruitment, and lowers germinal centre B cell frequency and neutralizing antibody potency without raising lung virus titres.", "summary_25": "Older hamsters infected with SARS-CoV-2 do not carry more virus. They mount a weaker, shorter immune response, repair their lungs more slowly, and make less potent antibodies.", "summary_75": "Golden hamsters aged forty weeks or more were compared with young animals after SARS-CoV-2 infection. Lung virus was not higher in older animals, yet their chemokine and interferon responses were lower and shorter-lived, lung repair markers were reduced, T cell expansion failed, and suppressor T cells, IL-17 and neutrophils increased. Spike-specific B cells and antibody titres were normal, but germinal centre B cells were scarce and neutralizing potency fell by more than sixty percent.", "summary_150": "Oishi and colleagues compared golden hamsters of six to nine weeks with animals of forty weeks or more after intranasal SARS-CoV-2 infection. Older animals had lower lung titres at one and three days and matched titres thereafter, so the aged phenotype does not follow from greater replication. Uninfected older lungs already showed reduced TGF-beta and NF-kappaB signalling and reduced proliferation signatures. After infection, chemokine induction was delayed and blunted, interferon-stimulated genes were not sustained past three days, and Ki67 staining indicated impaired repair. Flow cytometry showed failed T cell and Th1 expansion alongside expanded FoxP3 positive regulatory and CD8 T cells, elevated CCR6 positive CD8 cells, increased IL-17 in lymphocytes and epithelium, and more CD11b positive neutrophils. Spike-specific B cell frequency and anti-receptor-binding-domain IgG were unchanged, yet germinal centre B cells were markedly reduced and neutralizing titres fell from 1,549 to 514. Human post-mortem lung showed IL-17 correlating with age.", "citation": "Oishi K, Horiuchi S, Frere J, Schwartz RE, tenOever BR. A diminished immune response underlies age-related SARS-CoV-2 pathologies. Cell Reports. 2022. Volume 39, issue 13, article 111002. DOI 10.1016/j.celrep.2022.111002. PMID 35714615. PMCID PMC9181267. Oishi and Horiuchi contributed equally. tenOever is the lead contact.", "sections": { "Citation": "Oishi K, Horiuchi S, Frere J, Schwartz RE, tenOever BR. A diminished immune response underlies age-related SARS-CoV-2 pathologies. Cell Reports. 2022. Volume 39, issue 13, article 111002.\n\nDOI 10.1016/j.celrep.2022.111002. PMID 35714615. PMCID PMC9181267.\n\nOishi and Horiuchi contributed equally. tenOever is the lead contact.", "One-sentence contribution": "Comparison of young and older golden hamsters infected with SARS-CoV-2 shows that age reduces the magnitude and duration of the innate response and of tissue repair, expands suppressor T cells and IL-17-driven neutrophil recruitment, and lowers germinal centre B cell frequency and neutralizing antibody potency without raising lung virus titres.", "Executive summary": "Morbidity and mortality from SARS-CoV-2 rise sharply with age, and the biology behind that pattern was incompletely defined. The authors used the golden hamster, which reproduces many features of COVID-19, to compare animals of six to nine weeks with animals of forty weeks or more after intranasal infection. Lung virus titres were higher in young animals at one and three days and comparable between cohorts at five and seven days, so the aged phenotype is not explained by greater viral replication. Transcriptional profiling of uninfected lung showed that TGF-beta signalling, NF-kappaB signalling and cell division signatures were already lower in older animals at baseline. After infection, chemokine induction was diminished and delayed in older animals, and although the interferon-stimulated gene profile was comparable through three days it fell away rapidly thereafter despite equivalent virus. Ki67 staining showed reduced proliferation in the lungs of older animals, indicating slower repair. Flow cytometry showed that older animals failed to expand CD3 positive T cells or Th1 cells, carried higher frequencies of FoxP3 positive regulatory and CD8 T cells, showed elevated CCR6 positive CD8 T cells and IL-17 staining in both lymphocytes and epithelium, and recruited more CD11b positive neutrophils. Spike-specific B cell numbers and anti-receptor-binding-domain IgG titres were unchanged, but germinal centre B cells were markedly reduced in lung, spleen and draining lymph node, and serum neutralizing potency fell by more than sixty percent. Human COVID-19 cadaver lung transcriptomes showed IL-17 induction correlating with age.", "Scientific context": "Viruses that evade host defences generally cause most disease in the very young and the very old, but SARS-CoV-2 was noted early on to fall disproportionately on the aged. Ageing was known to alter immune function in several relevant ways described by other groups. Regulatory T cells, which secrete TGF-beta and suppress B and T cell function, accumulate with age. IL-17, the signature cytokine of T helper 17 cells, also increases with age and can activate neutrophils. Neutrophils contribute to early clearance but also to tissue injury, and neutrophil counts are used clinically as a marker related to acute respiratory distress syndrome in COVID-19. Previous hamster studies from other laboratories had established the model as reproducing COVID-19 biology and had reported that greater disease severity in older animals corresponded with lower rather than higher virus replication together with significant tissue damage. What had not been done was a molecular characterisation of the virus-host interaction and of both immune arms across age in that model, which is the gap this study addresses. The authors state that they corroborate the earlier severity and replication observations and extend them.", "Central question": "What changes in the innate and adaptive immune response to SARS-CoV-2 with age, and can those changes rather than differences in virus replication account for the greater morbidity seen in older hosts?", "Experimental strategy": "The design holds the virus constant and varies host age, using two golden hamster cohorts defined by published criteria for the species, six to nine weeks against forty weeks or more, both given the same intranasal dose. Because earlier work had already indicated that older animals do not carry more virus, the strategy is built to look past titre. Lung transcriptomes are taken from uninfected animals of both ages first, so any post-infection difference can be read against a documented baseline difference, and then across a time course of one, three, five and seven days, with the days five and seven comparisons singled out because virus levels are equivalent there and any host difference therefore cannot be attributed to viral load. Transcriptional inference is then checked against protein and cell-level measurement, with Ki67 immunohistochemistry validating the proliferation signature and flow cytometry resolving the immune populations that bulk RNA can only imply. The adaptive arm is approached through methods the group had previously developed for the hamster, which matters because commercial reagents for this species are limited. Antibody quantity and antibody quality are deliberately separated, with ELISA against the receptor binding domain measuring the former and a plaque reduction neutralization test measuring the latter, so that a defect in affinity maturation can be distinguished from a defect in antibody production. Finally, the IL-17 finding is tested for relevance beyond the model by examining RNA sequencing of human COVID-19 cadaver lung tissue stratified by age.", "Key findings": "1. Lung virus titres were significantly higher in young hamsters at one and three days post-infection and comparable between cohorts at five and seven days (Figure 1B). Greater disease in older animals is therefore not accompanied by greater lung replication.\n\n2. In uninfected animals, gene set enrichment showed TGF-beta signalling, NF-kappaB signalling and proliferation signatures significantly diminished in older lungs (Figure 1C, Table S1). The authors note that NF-kappaB signalling has been reported as required for SARS-CoV-2 replication and suggest this may contribute to the lower early virus in older animals, which is interpretation drawn from prior work rather than tested here.\n\n3. Chemokine induction after infection was diminished in older animals, with lower Ccl2, Ccl4, Ccl8, Ccl28 and Il1b. Older animals showed a rebound at three days but never reached the peak levels seen in young animals at five and seven days (Figure 1D).\n\n4. Interferon-stimulated gene profiles were comparable between cohorts through the first three days but declined rapidly in older animals thereafter despite equivalent virus at five days (Figure 1E). The authors state that the reason for this decline remains unclear and later attribute it, as an interpretation, to diminished immune cell recruitment.\n\n5. At five days, older animals showed a marked increase in neutrophil-associated transcripts S100a8 and S100a9 with log2 fold change above five, and significant downregulation of macrophage and dendritic cell markers Cd163, Clec4a, Mafb and Slamf8 (Figure S1C, Table S4). At seven days, histone-modification-associated transcripts including Tet2, Tet3, Ogt and Suz12 and the activated T cell marker Nfat5 were lost in older animals (Table S5).\n\n6. Ki67 transcript was reduced roughly fourfold in older animals, and immunohistochemistry confirmed markedly fewer Ki67 positive cells in older lungs at five and seven days (Figure 1F, Table S3). The authors read this as a defect in the kinetics or the biology of lung repair.\n\n7. Total immune-enriched cell recruitment to the lung was comparable between cohorts, but older animals had significantly higher CD45 positive cells at baseline and this reversed by seven days (Figures 2A and 2B). Young animals expanded CD3 positive T cells while older animals did not (Figure 2C), CD4 positive T cells plateaued in older animals from an already high baseline (Figure 2D), and Th1 cells defined as CXCR3 positive CD4 cells rose in young and fell in older animals (Figure 2E). CXCR3 positive CD8 cells expanded significantly in both cohorts (Figure 2F).\n\n8. Older animals showed a pronounced increase in FoxP3 positive CD4 regulatory T cells and FoxP3 positive CD8 T cells at seven days (Figure 3A), accompanied by a fivefold rise in Tgfb transcript. RNA sequencing showed loss of TGF-beta receptor subunit expression approaching an order of magnitude (Figure S4A). The authors interpret this combination as elevated TGF-beta production in an environment depleted of its receptor, invoking published negative feedback on receptor expression, and note that it sits awkwardly with the reduced repair signalled by Ki67.\n\n9. CCR6 positive CD8 T cells were elevated in older animals while CCR6 positive CD4 cells were not (Figure 3B), and immunohistochemistry showed markedly more IL-17 positive cells in older lung parenchyma, in epithelium as well as in lymphocytes (Figure 3C). CD11b positive presumed neutrophils were significantly elevated in older animals after infection despite a lower baseline (Figure 3D), corroborated by CD11b and myeloperoxidase staining (Figure 3E, Figure S3).\n\n10. In human COVID-19 cadaver lung RNA sequencing, IL-17 induction correlated statistically with age, with IL-17 target genes including Il6, Cxcl2 and Cxcl1 trending in the same direction (Figures S4B and S4C).\n\n11. Total B cell frequency, class switching, and spike-specific B cell frequency in lung and spleen showed no significant age-related difference (Figure 4A, Figures S5A and S5B), and anti-receptor-binding-domain IgG and IgG2 titres were also unchanged (Figure 4C). Spike-specific germinal centre B cells defined as Bcl6 positive were markedly reduced in older animals in lung, spleen and mediastinal lymph node (Figure 4B).\n\n12. Serum from older animals neutralized SARS-CoV-2 less effectively, with 50 percent plaque reduction titres of 1,549 for young and 514 for older animals, a reduction of more than sixty percent (Figure 4D). Because antibody quantity was unchanged, the authors read this as a deficit in affinity maturation rather than in production.", "Mechanistic model": "The study does not establish a definitive causal mechanism, and the authors describe the adaptive phenotype as most certainly multifactorial. The account the data support is correlational and layered. Older animals begin with lower baseline NF-kappaB and TGF-beta signalling and lower proliferative capacity in the lung. On infection they mount chemokine and interferon-stimulated gene responses that are delayed, lower in magnitude, and not sustained, which the authors attribute to reduced immune cell recruitment rather than to differences in virus. In parallel the suppressor compartment expands, with more regulatory T cells and FoxP3 positive CD8 T cells producing more TGF-beta, which the authors propose creates an environment favouring IL-17-producing cells, whose product then recruits neutrophils through NF-kappaB-driven chemokine induction. Neutrophil influx is offered as the link to tissue damage, citing prior work on neutrophils in COVID-19 pathology. On the humoral side, the proposed chain runs from expanded regulatory T cells to suppression of germinal centre formation to impaired affinity maturation and therefore to weaker neutralizing antibody at unchanged antibody titre. Each link in these chains is supported by an association measured in the same animals rather than by intervention. No depletion, blockade, or transfer experiment is performed, so causality between regulatory T cell expansion and germinal centre loss, or between IL-17 and neutrophil recruitment, is proposed rather than demonstrated. The authors also flag an internal tension they do not resolve, namely that TGF-beta is associated with tissue repair yet repair markers are reduced, and offer loss of receptor expression as a candidate reconciliation. The suggestion that IL-17 antagonists might be therapeutically useful is stated explicitly as requiring further testing and validation.", "Conceptual or technical advance": "The study separates three things that age-related severity might otherwise be assumed to share a cause. Virus burden is not elevated in older animals, antibody quantity is not reduced, and immune cell recruitment in bulk is not reduced, yet the quality and duration of the response are all diminished. That decomposition is what makes the phenotype tractable, because it points at affinity maturation, at response duration, and at the suppressor and IL-17 axis rather than at replication control. The work also applies flow cytometry and antigen-specific B cell methods developed by the group for the hamster, which extends what can be measured in a model that had mostly been characterised transcriptionally and histologically, and it supplies matched innate and adaptive profiling across age in one experiment. The IL-17 correlation with age in human post-mortem lung provides a point of contact between the model and patients.", "Relationship to the broader research program": "The paper depends directly on hamster immune profiling methods the laboratory published previously, and it follows the group's earlier work establishing the imbalanced host response to SARS-CoV-2 and the use of the interferon system as a line of defence against SARS-CoV-2 pathogenicity. It shares first and second authors with the coinfection study published by the same group in the same year, and both draw on the same hamster infrastructure and the same framing that the host response rather than viral replication sets the outcome. The observation that NF-kappaB signalling supports SARS-CoV-2 replication is taken from the group's own work with Nilsson-Payant. Marked as category 3 synthesis, the recurring commitment across these papers is that the interesting variable is the shape of the host response over time rather than the peak virus titre, and that the same model can be interrogated by varying the host, whether that host difference is age here or a prior infection in the coinfection study.", "Related publications": "- Horiuchi et al. 2021 (Science Immunology), methodological foundation. Same laboratory, shares an author, and the source of the hamster flow cytometry and adaptive immune profiling methods used throughout, alongside its own finding on immune memory from SARS-CoV-2 infection in hamsters.\n- Blanco-Melo et al. 2020 (Cell), predecessor. Same laboratory. Established the imbalanced host response to SARS-CoV-2 and is cited here for neutrophil recruitment as a hallmark of COVID-19.\n- Hoagland et al. 2021 (Immunity), predecessor. Same laboratory, shares authors, and cited for neutrophil recruitment in the hamster model and for leveraging the type I interferon system against SARS-CoV-2 pathogenicity.\n- Nilsson-Payant et al. 2021, conceptual extension. Same laboratory. Cited as showing NF-kappaB signalling to be required for SARS-CoV-2 replication, which the authors use to interpret the lower early virus titres in older animals.\n- Oishi et al. 2022 (Journal of Virology), companion. Same laboratory, same first two authors, same hamster model and SARS-CoV-2 strain, addressing coinfection rather than age.", "Limitations and boundaries": "The authors provide their own limitations section, and it is substantial. Commercial anti-hamster reagents are limited, so B cells were defined by MHC class II positive and CD3 negative staining rather than by CD19 or CD20, and neutrophils could not be identified with anti-Ly6G, leaving CD11b and myeloperoxidase as markers that also cover monocytes, macrophages and dendritic cells. Because older hamsters grow large and must be housed individually, and biosafety level 3 caging is limited, cohort sizes were only three to five animals, with four per time point for the virus and transcriptome course. Beyond the authors' own list, the study rests on one virus isolate at one dose delivered intranasally, on lung as the principal tissue, and on a time course ending at seven days, so nothing is established about later convalescence, reinfection, or transmission, and the authors say explicitly that how age affects protection from secondary infection requires further analysis. The two age cohorts are compared as blocks with no intermediate ages, so no dose relationship with age is established. Every mechanistic link is correlational, with no depletion, blockade, adoptive transfer, or receptor knockout, so the causal role of regulatory T cells, TGF-beta, IL-17, or neutrophils in the observed pathology is proposed rather than shown. The human data are a transcriptional correlation in post-mortem lung, which supports relevance but not mechanism. The therapeutic suggestion regarding IL-17 antagonists is explicitly extrapolation.", "Audience summaries": "### 25 words\n\nOlder hamsters infected with SARS-CoV-2 do not carry more virus. They mount a weaker, shorter immune response, repair their lungs more slowly, and make less potent antibodies.\n\n### 75 words\n\nGolden hamsters aged forty weeks or more were compared with young animals after SARS-CoV-2 infection. Lung virus was not higher in older animals, yet their chemokine and interferon responses were lower and shorter-lived, lung repair markers were reduced, T cell expansion failed, and suppressor T cells, IL-17 and neutrophils increased. Spike-specific B cells and antibody titres were normal, but germinal centre B cells were scarce and neutralizing potency fell by more than sixty percent.\n\n### 150 words\n\nOishi and colleagues compared golden hamsters of six to nine weeks with animals of forty weeks or more after intranasal SARS-CoV-2 infection. Older animals had lower lung titres at one and three days and matched titres thereafter, so the aged phenotype does not follow from greater replication. Uninfected older lungs already showed reduced TGF-beta and NF-kappaB signalling and reduced proliferation signatures. After infection, chemokine induction was delayed and blunted, interferon-stimulated genes were not sustained past three days, and Ki67 staining indicated impaired repair. Flow cytometry showed failed T cell and Th1 expansion alongside expanded FoxP3 positive regulatory and CD8 T cells, elevated CCR6 positive CD8 cells, increased IL-17 in lymphocytes and epithelium, and more CD11b positive neutrophils. Spike-specific B cell frequency and anti-receptor-binding-domain IgG were unchanged, yet germinal centre B cells were markedly reduced and neutralizing titres fell from 1,549 to 514. Human post-mortem lung showed IL-17 correlating with age." }, "discoveries": [], "relationships": [ { "from": "2022-oishi-a-diminished-immune-response-under", "to": "2021-horiuchi-immune-memory-from-sars-cov-2-infe", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2022-oishi-a-diminished-immune-response-under" }, { "from": "2022-oishi-a-diminished-immune-response-under", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2022-oishi-a-diminished-immune-response-under" }, { "from": "2022-oishi-a-diminished-immune-response-under", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2022-oishi-a-diminished-immune-response-under" } ], "canonical_url": "https://tenoeverlab.us/publications/2022-oishi-a-diminished-immune-response-under/", "controlled_vocabulary": { "pathogens": [ "sars-cov-2" ], "technologies": [ "bulk-rna-seq", "plaque-assay", "flow-cytometry", "pathway-enrichment-analysis", "in-vivo-infection-route", "elisa", "immunohistochemistry", "antigen-specific-b-cell-detection", "neutralization-assay" ] } }, { "id": "2022-oishi-the-host-response-to-influenza-a-v", "slug": "2022-oishi-the-host-response-to-influenza-a-v", "url": "/publications/2022-oishi-the-host-response-to-influenza-a-v/", "title": "The Host Response to Influenza A Virus Interferes with SARS-CoV-2 Replication during Coinfection", "authors": [ "Kohei Oishi", "Shu Horiuchi", "Judith M. Minkoff", "Benjamin R. tenOever" ], "author_count": 4, "first_author": "Kohei Oishi", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 4, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2022, "journal": "Journal of Virology", "volume": "96", "issue": "15", "pages": "e00765-22", "doi": "10.1128/jvi.00765-22", "doi_url": "https://doi.org/10.1128/jvi.00765-22", "pmid": "35862681", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/35862681/", "pmcid": "PMC9364782", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9364782/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9364782/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "immunity-age-and-reinfection" ], "pathogens": [ "SARS-CoV-2", "influenza A virus" ], "viral_families": [ "Coronaviridae", "Orthomyxoviridae" ], "host_species": [ "golden hamster", "human" ], "technologies": [ "plaque assay", "in vitro competition assay", "intranasal infection of hamsters", "messenger RNA sequencing", "differential gene expression analysis", "quantitative RT-PCR", "haematoxylin and eosin histology" ], "biological_systems": [ "Vero E6 cells", "A549-ACE2 cells", "golden hamster", "MDCK cells" ], "key_concepts": [ "viral interference", "coinfection", "type I and type III interferon", "immune priming", "interferon-stimulated genes", "replication kinetics", "sequential infection", "airway host response" ], "keywords": [ "SARS-CoV-2", "influenza A virus", "coinfection", "viral interference", "golden hamster", "interferon", "ISG15", "IRF7", "H1N1", "respiratory virus" ], "one_sentence_contribution": "In golden hamsters, influenza A virus infection reduces SARS-CoV-2 replication during coinfection, after preinfection, and even one to two weeks after influenza has been cleared, while SARS-CoV-2 leaves influenza replication in vivo unchanged.", "summary_25": "In hamsters, influenza infection holds back SARS-CoV-2 replication during coinfection and for up to two weeks afterwards, while SARS-CoV-2 does nothing to influenza in the animal.", "summary_75": "Golden hamsters were given SARS-CoV-2, influenza A virus, or both, either together or three, seven or fourteen days apart. Influenza consistently reduced SARS-CoV-2 replication and hastened its clearance, including when given a week or two before, when influenza itself was gone but interferon-stimulated genes remained elevated. SARS-CoV-2 never changed influenza titres in the animal, although it did suppress influenza in interferon-competent cells. Coinfected animals looked like SARS-CoV-2 infection alone by weight and histology.", "summary_150": "Oishi and colleagues compared SARS-CoV-2 and influenza A virus H1N1 coinfection in interferon-deficient Vero E6 cells, in interferon-competent A549-ACE2 cells, and in golden hamsters. In culture, influenza delayed SARS-CoV-2 in both cell lines, while SARS-CoV-2 suppressed influenza only in the interferon-competent line. In hamsters, simultaneous coinfection lowered SARS-CoV-2 titres at three days and cleared the virus by five days without altering influenza at any point. Influenza given three days earlier reduced subsequent SARS-CoV-2 replication, and the reverse arrangement had no effect on influenza. The suppression persisted when SARS-CoV-2 was given seven or fourteen days after influenza, a point at which only twenty-five lung genes were differentially expressed although ISG15 and IRF7 remained elevated. The authors attribute the effect to interferon-mediated immune priming rather than direct competition, state that they cannot distinguish direct from bystander effects, and conclude that cocirculation is unlikely to raise disease severity.", "citation": "Oishi K, Horiuchi S, Minkoff JM, tenOever BR. The host response to influenza A virus interferes with SARS-CoV-2 replication during coinfection. Journal of Virology. 2022. Volume 96, issue 15, article e00765-22. DOI 10.1128/jvi.00765-22. PMID 35862681. PMCID PMC9364782.", "sections": { "Citation": "Oishi K, Horiuchi S, Minkoff JM, tenOever BR. The host response to influenza A virus interferes with SARS-CoV-2 replication during coinfection. Journal of Virology. 2022. Volume 96, issue 15, article e00765-22.\n\nDOI 10.1128/jvi.00765-22. PMID 35862681. PMCID PMC9364782.", "One-sentence contribution": "In golden hamsters, influenza A virus infection reduces SARS-CoV-2 replication during coinfection, after preinfection, and even one to two weeks after influenza has been cleared, while SARS-CoV-2 leaves influenza replication in vivo unchanged.", "Executive summary": "SARS-CoV-2 and influenza A virus both spread by the airborne route, both infect the human airway, and both were documented as coinfections early in the COVID-19 pandemic. Whether coinfection makes disease worse was unsettled, with published animal studies reaching different conclusions depending on model, strain and sex. The authors asked how the two viruses interact when a single host carries both. In Vero E6 cells, which lack the type I and type III interferon genes, SARS-CoV-2 was modestly delayed by influenza at twenty-four hours and influenza was unaffected. In interferon-competent A549 cells expressing ACE2, SARS-CoV-2 was again delayed while influenza titres fell substantially at every time point. In golden hamsters infected intranasally with either virus alone or with both simultaneously, influenza replication was unchanged by SARS-CoV-2 at every time point, while SARS-CoV-2 titres were lower at three days and cleared from the lung by five days in coinfected animals rather than persisting to five and seven days as in single infection. Weight and lung histology tracked with SARS-CoV-2 rather than with coinfection as such. Preinfection with influenza three days ahead reduced subsequent SARS-CoV-2 replication, whereas preinfection with SARS-CoV-2 did not change influenza. The suppression persisted when SARS-CoV-2 was given seven or fourteen days after influenza, at which point only twenty-five genes remained differentially expressed although ISG15 and IRF7 remained elevated. The authors read this as immune priming rather than direct competition.", "Scientific context": "Coinfection with SARS-CoV-2 and other respiratory viruses was reported repeatedly early in the pandemic, with a meta-analysis of the first four months finding viral coinfection in about three percent of hospitalised COVID-19 patients and influenza A virus among the most common partners. More than half of the roughly thirty SARS-CoV-2 proteins had been reported by others to interfere with interferon induction or signalling, which raised the concern that a SARS-CoV-2-infected airway might be unusually permissive to a second pathogen. The two viruses use different entry receptors, ACE2 for SARS-CoV-2 spike and sialic acid alpha 2,6-galactose for human influenza haemagglutinin, but both are reported to infect alveolar type II cells. Prior experimental work by other groups had produced divergent results. In K18-hACE2 mice, coinfection increased disease severity and both preinfection and coinfection with influenza raised SARS-CoV-2 loads with delayed clearance. In golden hamsters, one study found increased severity with lower SARS-CoV-2 lung loads but prolonged shedding, and another using an H3N2 strain in female hamsters found robust SARS-CoV-2 replication alongside low influenza titres, with Mx1 implicated in inhibition of influenza. The present study enters this disagreement with a single model, matched strains, and both simultaneous and sequential designs.", "Central question": "When one host carries both SARS-CoV-2 and influenza A virus, does either virus interfere with the replication of the other, and does the direction and timing of that interference depend on the host interferon response rather than on direct competition for cells?", "Experimental strategy": "The design isolates the contribution of the interferon system twice over, once in cell culture and once in time. In culture, the same coinfection is run in Vero E6 cells, which lack the type I and type III interferon genes, and in A549 cells engineered to express ACE2, which are interferon competent and support both viruses, so a phenotype present only in the latter can be attributed to the host response rather than to receptor competition or cell death. In vivo, the golden hamster is used because it supports robust replication of both viruses in the respiratory tract and reproduces pathological and immunological features of human disease. Three temporal arrangements are then compared. Simultaneous coinfection asks whether the two viruses interfere while both are replicating. Preinfection at three days in each direction asks whether an established infection changes what follows. Challenge at seven and fourteen days after influenza, when no infectious influenza is recoverable, separates any effect of the ongoing infection from a residual change in the airway. Readouts combine plaque titration of each virus on its own indicator cell line, body weight, lung histology, messenger RNA sequencing of lung at five days after coinfection and at seven days after influenza alone, and quantitative RT-PCR for ISG15 and IRF7 as markers of a persisting interferon-stimulated state.", "Key findings": "1. In Vero E6 cells at low multiplicity, SARS-CoV-2 titres were lower in the presence of influenza at twenty-four hours but comparable at forty-eight and seventy-two hours, while influenza titres were unchanged at all time points (Figures 1A and 1B).\n\n2. In A549-ACE2 cells, SARS-CoV-2 again showed reduced titre at twenty-four hours and recovery by forty-eight hours, while influenza titres were substantially lower in the presence of SARS-CoV-2 at every time point (Figures 1C and 1D). Because the two cell lines differ in interferon competence, the authors read the influenza phenotype as host-response dependent.\n\n3. In simultaneously coinfected hamsters, SARS-CoV-2 lung titres matched single infection at one day, were lower at three days, and were cleared by five days, whereas infectious SARS-CoV-2 was recovered from all singly infected animals at five days and from a quarter at seven days (Figure 2B). Influenza titres were unaffected by coinfection at every time point (Figure 2C).\n\n4. Weight change and lung histology followed SARS-CoV-2 rather than coinfection. Coinfected animals and those given SARS-CoV-2 alone showed a delayed lack of weight gain relative to control and influenza-only animals, and both showed extensive mononuclear infiltration on haematoxylin and eosin staining at three days (Figures 2D and 2E).\n\n5. Messenger RNA sequencing of lung at five days showed that coinfection produced a more pronounced inflammatory response than influenza alone, and that the genes induced by SARS-CoV-2 or by coinfection included those induced by influenza plus additional ones, with the SARS-CoV-2-associated set enriched for NF-kappaB-dependent chemokines including CCL2, CCL5, CCL7 and CXCL10 (Figures 2F and 2G, Table 1).\n\n6. Preinfection with influenza three days before SARS-CoV-2 reduced SARS-CoV-2 titres at every time point tested, while influenza titres were unchanged by SARS-CoV-2 given during active influenza replication (Figures 3B and 3C).\n\n7. Preinfection with SARS-CoV-2 three days before influenza did not affect influenza replication, and SARS-CoV-2 titres were also unchanged by the later influenza challenge (Figures 3E and 3F). No delay in clearance of either virus was observed in either sequential arrangement.\n\n8. Challenging influenza-recovered hamsters with SARS-CoV-2 seven days after influenza, when infectious influenza was no longer detectable, gave lower SARS-CoV-2 titres at one and three days, with titres converging at five days (Figures 4A and 4B). At fourteen days after influenza, SARS-CoV-2 titres were still lower at one day (Figures 4C and 4D).\n\n9. Sequencing of lung at seven days after influenza alone found only twenty-five differentially expressed genes relative to control, which the authors take as indicating a largely restored baseline. Among the modestly enriched transcripts they note markers the paper associates with immune priming and with increased macrophage presence, including C1QA, C1QB and C1QC (Table 2).\n\n10. By quantitative RT-PCR, ISG15 and IRF7 were elevated in lung at seven days after influenza, with ISG15 still elevated at fourteen days (Figures 4E and 4F). The authors read this as a residual interferon-stimulated state, or alternatively as increased resident immune cells in the airway, and explicitly present these as two possibilities rather than one conclusion.", "Mechanistic model": "The study does not establish a definitive mechanism, and the authors state directly that it remains unclear whether the inhibition of SARS-CoV-2 reflects direct effects within coinfected cells or bystander effects on neighbouring cells, and that resolving this would require single-cell approaches not attempted here. What the data constrain is the direction, the durability, and the plausible source of the effect. Because suppression of early SARS-CoV-2 replication persists a week or two after influenza has been cleared, when almost no transcriptional difference remains detectable in bulk lung, direct competition for target cells is not a sufficient explanation, and the authors describe the phenotype as likely a product of immune priming with type I and type III interferon. They note two candidate substrates for that priming, namely residual low-level interferon-stimulated gene expression, supported by the elevated ISG15 and IRF7, and an increased population of resident immune cells, supported by the complement component enrichment. They do not distinguish these. They also invoke kinetics, citing earlier work from the group in the same model showing that interferon-stimulated gene and chemokine peaks occur around three days for influenza and later and more persistently, from five to seven days, for SARS-CoV-2, and propose that this offset contributes to asymmetric interference. The failure of SARS-CoV-2 to suppress influenza in hamsters despite Mx1 induction is interpreted as influenza being able to replicate in an interferon-primed lung given enough susceptible cells, and the authors note that their own earlier work found Mx1 induction by SARS-CoV-2 to be delayed in the airway and more evident in tissues that are not productively infected. The in vitro suppression of influenza by SARS-CoV-2 in interferon-competent cells but not in hamsters is attributed to the lung offering an expansive and heterogeneous cellular landscape not reproduced in culture, which is interpretation rather than demonstration.", "Conceptual or technical advance": "The work supplies a controlled comparison in one model of all three timings that matter epidemiologically, namely simultaneous infection and each virus preceding the other, and adds the recovered-host arm that separates an active infection from its aftermath. That last arm is what makes the argument, because it shows an effect on SARS-CoV-2 replication persisting into a window where the airway looks transcriptionally almost normal. Practically, the finding that coinfected animals show a host response comparable to SARS-CoV-2 alone, with no prolonged shedding and no delayed clearance, gives a reason to doubt that cocirculation of the two viruses would by itself increase disease severity, a conclusion the authors state while also noting that whether the interference affects severity remains unclear. The study also documents an asymmetry that makes the interferon-competence of the experimental system a variable rather than a detail, since the suppression of influenza by SARS-CoV-2 appears in interferon-competent cells, not in interferon-deficient cells, and not in the animal.", "Relationship to the broader research program": "The paper sits within the laboratory's use of the golden hamster as a model for comparing host responses to respiratory viruses, and it relies directly on earlier work from the group in the same model with the same virus strains for the kinetics of interferon-stimulated gene and chemokine induction. It also builds on the group's earlier finding that SARS-CoV-2 induces a more pronounced inflammatory signature than influenza in hamsters, an observation reported as consistent with patient data and with the ferret model. Marked as category 3 synthesis, a recurring thread across the corpus is the treatment of the host response, rather than the virus, as the variable that determines outcome, whether that is defective genome production driving interferon in the nucleoprotein work or interferon priming setting the ceiling on a second infection here. A second thread is the pairing of interferon-deficient and interferon-competent systems as a routine control, used in this paper as Vero E6 against A549-ACE2.", "Related publications": "- Horiuchi et al., predecessor. Same laboratory, cited for the hamster kinetics of interferon-stimulated gene and chemokine induction in response to the same SARS-CoV-2 and influenza strains, and shares an author with the present paper.\n- Blanco-Melo et al. 2020 and related work on the SARS-CoV-2 host response, predecessor. Same laboratory, cited for the finding that SARS-CoV-2 induces a stronger inflammatory signature than influenza and for Mx1 induction patterns across tissues.\n- Published golden hamster and K18-hACE2 mouse coinfection studies from other groups, companion. Cited and contrasted throughout the discussion as producing partly conflicting results attributed to model, strain and sex, and explicitly attributed by the authors to those other laboratories.", "Limitations and boundaries": "The animal work uses male Syrian golden hamsters only, at six to eight weeks of age, which matters because the authors themselves note that another group's divergent result came from female hamsters and that sex, age and strain may influence the outcome. One SARS-CoV-2 isolate and one H1N1 strain were used, at fixed doses delivered intranasally, so dose and strain dependence is untested. Group sizes are small, with three or four animals per time point for titration and eight per condition for weight. Titres are from lung homogenate, with no assessment of the upper respiratory tract, no transmission or shedding measurements, and no barcoded viruses to resolve whether interference occurs within the same cells, an experiment the authors name as needed. The mechanistic claim rests on an association between residual ISG15 and IRF7 expression and reduced early SARS-CoV-2 replication, with no interferon blockade, receptor knockout, or cell depletion to test causation, and the authors offer immune priming and increased resident immune cells as alternatives they cannot separate. Cell marker antibodies for hamster are limited, which the authors give as the reason cellular resolution was not attempted. Transcriptomics is bulk lung at two time points. The in vitro and in vivo results disagree on whether SARS-CoV-2 suppresses influenza, and the explanation offered for that disagreement is interpretive. Finally, the conclusion that cocirculation is unlikely to increase disease severity is drawn from an animal model and does not rest on clinical data.", "Audience summaries": "### 25 words\n\nIn hamsters, influenza infection holds back SARS-CoV-2 replication during coinfection and for up to two weeks afterwards, while SARS-CoV-2 does nothing to influenza in the animal.\n\n### 75 words\n\nGolden hamsters were given SARS-CoV-2, influenza A virus, or both, either together or three, seven or fourteen days apart. Influenza consistently reduced SARS-CoV-2 replication and hastened its clearance, including when given a week or two before, when influenza itself was gone but interferon-stimulated genes remained elevated. SARS-CoV-2 never changed influenza titres in the animal, although it did suppress influenza in interferon-competent cells. Coinfected animals looked like SARS-CoV-2 infection alone by weight and histology.\n\n### 150 words\n\nOishi and colleagues compared SARS-CoV-2 and influenza A virus H1N1 coinfection in interferon-deficient Vero E6 cells, in interferon-competent A549-ACE2 cells, and in golden hamsters. In culture, influenza delayed SARS-CoV-2 in both cell lines, while SARS-CoV-2 suppressed influenza only in the interferon-competent line. In hamsters, simultaneous coinfection lowered SARS-CoV-2 titres at three days and cleared the virus by five days without altering influenza at any point. Influenza given three days earlier reduced subsequent SARS-CoV-2 replication, and the reverse arrangement had no effect on influenza. The suppression persisted when SARS-CoV-2 was given seven or fourteen days after influenza, a point at which only twenty-five lung genes were differentially expressed although ISG15 and IRF7 remained elevated. The authors attribute the effect to interferon-mediated immune priming rather than direct competition, state that they cannot distinguish direct from bystander effects, and conclude that cocirculation is unlikely to raise disease severity." }, "discoveries": [], "relationships": [ { "from": "2022-oishi-the-host-response-to-influenza-a-v", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2022-oishi-the-host-response-to-influenza-a-v" } ], "canonical_url": "https://tenoeverlab.us/publications/2022-oishi-the-host-response-to-influenza-a-v/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sars-cov-2" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "in-vivo-infection-route", "histopathology", "differential-expression-analysis", "competition-assay" ] } }, { "id": "2022-yaron-host-protein-kinases-required-for-", "slug": "2022-yaron-host-protein-kinases-required-for-", "url": "/publications/2022-yaron-host-protein-kinases-required-for-/", "title": "Host protein kinases required for SARS-CoV-2 nucleocapsid phosphorylation and viral replication", "authors": [ "Tomer M. Yaron", "Brook E. Heaton", "Tyler M. Levy", "Jared L. Johnson", "Tristan X. Jordan", "Benjamin M. Cohen", "Alexander Kerelsky", "Ting-Yu Lin", "Katarina M. Liberatore", "Danielle K. Bulaon", "Samantha J. Van Nest", "Nikos Koundouros", "Edward R. Kastenhuber", "Marisa N. Mercadante", "Kripa Shobana-Ganesh", "Long He", "Robert E. Schwartz", "Shuibing Chen", "Harel Weinstein", "Olivier Elemento", "Elena Piskounova", "Benjamin E. Nilsson-Payant", "Gina Lee", "Joseph D. Trimarco", "Kaitlyn N. Burke", "Cait E. Hamele", "Ryan R. Chaparian", "Alfred T. Harding", "Aleksandra Tata", "Xinyu Zhu", "Purushothama Rao Tata", "Clare M. Smith", "Anthony P. Possemato", "Sasha L. Tkachev", "Peter V. Hornbeck", "Sean A. Beausoleil", "Shankara K. Anand", "François Aguet", "Gad Getz", "Andrew D. Davidson", "Kate Heesom", "Maia Kavanagh-Williamson", "David A. Matthews", "Benjamin R. tenOever", "Lewis C. Cantley", "John Blenis", "Nicholas S. Heaton" ], "author_count": 47, "first_author": "Tomer M. Yaron", "senior_authors": [ "Benjamin R. tenOever", "Lewis C. Cantley", "John Blenis", "Nicholas S. Heaton" ], "corresponding_authors": [ "Nicholas S. Heaton", "John Blenis", "Lewis C. Cantley", "Benjamin R. tenOever" ], "tenoever_position": 44, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2022, "journal": "Science Signaling", "volume": "15", "issue": "757", "pages": "eabm0808", "doi": "10.1126/scisignal.abm0808", "doi_url": "https://doi.org/10.1126/scisignal.abm0808", "pmid": "36282911", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/36282911/", "pmcid": "PMC9830954", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9830954/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9830954/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "host-factors-and-druggable-signaling" ], "pathogens": [ "SARS-CoV-2", "human coronavirus 229E" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "human", "African green monkey" ], "technologies": [ "phosphoproteomics by liquid chromatography mass spectrometry", "combinatorial peptide substrate specificity profiling", "in vitro kinase assays", "Phos-tag gel electrophoresis", "radiolabeled ATP incorporation", "site-directed mutagenesis", "small interfering RNA knockdown", "small molecule kinase inhibition", "plaque assay", "quantitative RT-PCR", "immunofluorescence", "comparative sequence conservation analysis" ], "biological_systems": [ "ACE2-expressing A549 cells", "Vero E6 cells", "Calu-3 cells", "Huh7 cells", "primary human type II pneumocytes", "recombinant protein in vitro kinase reactions" ], "key_concepts": [ "nucleocapsid SR-rich domain", "phospho-priming", "SRPK1 and SRPK2", "GSK-3", "casein kinase 1", "sequential multisite phosphorylation", "host-directed antiviral strategy", "kinase substrate motif prediction", "alectinib repurposing", "conservation across coronaviruses" ], "keywords": [ "SARS-CoV-2", "nucleocapsid protein", "phosphorylation", "SRPK1", "GSK-3", "casein kinase 1", "alectinib", "host-directed antiviral", "phosphoproteomics" ], "one_sentence_contribution": "Kinase substrate specificity profiling assigns the phosphorylation cluster in the SARS-CoV-2 nucleocapsid SR-rich domain to a cascade initiated by SRPK1 and SRPK2 and extended by GSK-3 and casein kinase 1, whose inhibition suppresses coronavirus replication.", "summary_25": "Three host kinase families phosphorylate the SARS-CoV-2 nucleocapsid in a set order, and blocking the first of them, including with the approved drug alectinib, suppresses coronavirus replication.", "summary_75": "The SARS-CoV-2 nucleocapsid protein carries a cluster of phosphorylation sites in a region conserved across coronaviruses. By matching each site to the kinases whose sequence preferences fit it, this work reconstructed an ordered cascade in which SRPK enzymes act first and GSK-3 and casein kinase 1 extend the modification. Removing or inhibiting the initiating kinases reduced viral replication in several human cell systems, and an already approved cancer drug that hits them worked too.", "summary_150": "Host-directed antivirals need well-defined host dependencies. This study focuses on the serine and arginine rich domain of the SARS-CoV-2 nucleocapsid protein, which phosphoproteomics identified as the most heavily modified region of any viral protein and which is unusually conserved across 82 coronaviruses. Using measured substrate specificity matrices rather than candidate screening, the authors predicted that SRPK enzymes phosphorylate serine 206 and serine 188 first, that GSK-3 then propagates along chains of residues spaced every four positions toward the amino terminus, and that casein kinase 1 covers threonine 205 after serine 202 is modified. In vitro reactions with recombinant protein supported the order, and mutating both priming serines abolished the cascade. Knockdown of SRPK1 and treatment with SPHINX31, SRPIN340 or the approved inhibitor alectinib reduced SARS-CoV-2 replication in engineered A549 cells, Calu-3 cells and primary human pneumocytes, and alectinib also suppressed HCoV-229E. The functional role of the phosphorylation itself remains undefined.", "citation": "Yaron TM, Heaton BE, Levy TM, Johnson JL, Jordan TX, Cohen BM, Kerelsky A, Lin TY, Liberatore KM, Bulaon DK, Van Nest SJ, Koundouros N, Kastenhuber ER, Mercadante MN, Shobana-Ganesh K, He L, Schwartz RE, Chen S, Weinstein H, Elemento O, Piskounova E, Nilsson-Payant BE, Lee G, Trimarco JD, Burke KN, Hamele CE, Chaparian RR, Harding AT, Tata A, Zhu X, Tata PR, Smith CM, Possemato AP, Tkachev SL, Hornbeck PV, Beausoleil SA, Anand SK, Aguet F, Getz G, Davidson AD, Heesom K, Kavanagh-Williamson M, Matthews DA, tenOever BR, Cantley LC, Blenis J, Heaton NS. Host protein kinases required for SARS-CoV-", "sections": { "Citation": "Yaron TM, Heaton BE, Levy TM, Johnson JL, Jordan TX, Cohen BM, Kerelsky A, Lin TY, Liberatore KM, Bulaon DK, Van Nest SJ, Koundouros N, Kastenhuber ER, Mercadante MN, Shobana-Ganesh K, He L, Schwartz RE, Chen S, Weinstein H, Elemento O, Piskounova E, Nilsson-Payant BE, Lee G, Trimarco JD, Burke KN, Hamele CE, Chaparian RR, Harding AT, Tata A, Zhu X, Tata PR, Smith CM, Possemato AP, Tkachev SL, Hornbeck PV, Beausoleil SA, Anand SK, Aguet F, Getz G, Davidson AD, Heesom K, Kavanagh-Williamson M, Matthews DA, tenOever BR, Cantley LC, Blenis J, Heaton NS. Host protein kinases required for SARS-CoV-2 nucleocapsid phosphorylation and viral replication. Science Signaling. 2022. 15(757), eabm0808.\n\nDOI 10.1126/scisignal.abm0808. PMID 36282911. PMCID PMC9830954.\n\nTomer M. Yaron, Brook E. Heaton, Tyler M. Levy, Jared L. Johnson and Tristan X. Jordan are marked as having contributed equally. Correspondence is addressed to Nicholas S. Heaton, John Blenis, Lewis C. Cantley and Benjamin R. tenOever.", "One-sentence contribution": "Kinase substrate specificity profiling assigns the phosphorylation cluster in the SARS-CoV-2 nucleocapsid SR-rich domain to a cascade initiated by SRPK1 and SRPK2 and extended by GSK-3 and casein kinase 1, whose inhibition suppresses coronavirus replication.", "Executive summary": "Host-directed antivirals are attractive because they can act across related viruses and are harder for a virus to escape by mutation, but they require knowing which host enzymes the virus actually depends on. This work targets the SARS-CoV-2 nucleocapsid protein, whose serine and arginine rich domain is heavily phosphorylated and unusually conserved across coronaviruses. Phosphoproteomics of infected ACE2-expressing A549 and Vero E6 cells found the nucleocapsid to be the most phosphorylated viral protein, with most sites in that domain and most shared with five previously published datasets. Conservation analysis across 82 coronaviruses showed the domain, its phosphoacceptor residues, and the motif residues around the proposed priming sites all constrained. Rather than screening kinases, the authors used measured substrate specificity motifs for the SRPK, GSK-3 and CK1 families to compute which kinase most plausibly acts at each site, and that analysis produced an ordered model in which SRPKs phosphorylate serine 206 and serine 188 first, GSK-3 walks toward the amino terminus in four-residue steps, and CK1 handles threonine 205 once serine 202 is phosphorylated. In vitro reactions with recombinant protein supported the order, since combining all three kinases gave more phosphate incorporation than the sum of individual reactions and mutating both priming serines abolished the effect. Knockdown of SRPK1 and treatment with SPHINX31, SRPIN340 or alectinib reduced viral RNA and infectious titer across several cell systems, and alectinib selectively reduced SR-rich domain phosphorylation.", "Scientific context": "Vaccines against SARS-CoV-2 had been deployed rapidly and effectively by the time of this work, but breakthrough infections and unvaccinated patients left a need for antivirals, and viral evolution places a limit on approaches directed at viral proteins. Targeting host factors offers breadth and a higher barrier to escape, and protein kinases are attractive because they are well characterized and widely druggable. On the viral side, the importance of nucleocapsid phosphorylation was already suggested by several lines of work. Studies of SARS-CoV had implicated GSK-3 and SRPK families in phosphorylating the SR-rich domain, SRPK1 had been shown to affect SARS-CoV nucleocapsid multimerization without a reported effect on viral growth, and GSK-3 inhibitors suppressed both SARS-CoV and mouse hepatitis virus. Other studies had linked general kinase families including CK2, CDKs and protein kinase C to SARS-CoV-2 replication without tying them to nucleocapsid phosphorylation. Separately, the SR-rich domain had been reported to drive gel to liquid phase transitions of nucleocapsid and RNA condensates. What was missing was an assignment of specific sites to specific kinases with an order of events, and a test of whether that cascade is required for replication.", "Central question": "Which host protein kinases phosphorylate the serine and arginine rich domain of the SARS-CoV-2 nucleocapsid protein, in what order do they act, and is that phosphorylation cascade required for coronavirus replication in human cells?", "Experimental strategy": "The strategy inverts the usual screening logic. Instead of testing candidate kinases against the substrate, the authors start from independently measured substrate specificity matrices for the relevant kinase families, obtained by combinatorial peptide library profiling, and use them to score every phosphoacceptor in the domain, which yields a prediction that is ordered in time because two of the three families require phospho-priming. GSK-3 prefers a phosphorylated residue four positions to the carboxyl side, CK1 prefers one three positions to the amino side, and the domain happens to contain three chains of serines and threonines spaced every fourth residue, so the geometry of the substrate and the geometry of the kinase preferences together dictate a direction of travel. Evolutionary conservation is used as independent evidence, comparing the phosphosites, the surrounding motif residues, and the remaining residues of each domain across 82 coronaviruses. The ordered model is then tested biochemically with purified components, using a Phos-tag gel for occupancy and radiolabeled ATP for total incorporation, with a double phospho-null mutant at the two proposed priming serines as the critical test. Requirement during infection is approached in three independent ways, knockdown, two research-grade SRPK inhibitors, and an approved drug with known off-target SRPK activity, each applied across an engineered line, a naturally infectable line, and primary human cells, with viability measured alongside so that antiviral effect is not confused with toxicity. Finally, phosphoproteomics after drug treatment closes the loop by asking whether the drug does to the nucleocapsid what the model says it should.", "Key findings": "1. Phosphoproteomics of infected cells identified phosphosites across several viral proteins, with nucleocapsid by far the most phosphorylated, 14 sites in ACE2-A549 cells of which 11 were in the SR-rich domain and 26 sites in Vero E6 cells of which 15 were in that domain (Fig. 1B and 1C). Most SR-rich domain sites were also present in five previously published datasets.\n2. Across 82 coronaviruses the SR-rich domain was significantly more conserved than the linker domain and as conserved as the amino-terminal and carboxyl-terminal functional domains, with serine, threonine and arginine the most conserved residues (Fig. 1C and 1D). The SR-rich domain was the only domain in which detected phosphosites were significantly more conserved than the other residues of that domain (Fig. 1E).\n3. Measured substrate specificity showed SRPK1, SRPK2 and SRPK3 selecting arginine at positions minus three and plus three, serine at minus two and plus two, and proline at plus one, GSK-3 alpha and beta selecting a phosphorylated residue at plus four, and CK1 isoforms selecting a phosphorylated residue at minus three with partial preference for serine at minus four (Fig. 2A).\n4. Scoring each site produced an ordered model. SRPKs favor serine 206 and serine 188 as priming sites, GSK-3 then extends along two chains toward the amino terminus, and CK1 is predicted for threonine 205 once serine 202 is phosphorylated (Fig. 2B and 2C). This assignment is a computational prediction from specificity matrices and is stated as such.\n5. The motif residues predicted to direct SRPK to the two priming sites are as conserved across coronaviruses as the phosphoacceptors themselves (Fig. 2D). The authors read this as evidence that the cascade matters to the virus family, which is an inference from conservation rather than a functional test.\n6. In vitro, SRPK1 alone shifted recombinant nucleocapsid on a Phos-tag gel while GSK-3 or CK1 alone had only modest effects, and all three together increased phosphate incorporation more than the sum of the individual reactions (Fig. 2E), consistent with priming.\n7. The double phospho-null mutant carrying serine 188 to alanine and serine 206 to alanine abolished the SRPK-driven Phos-tag shift and reduced radioactive incorporation by all three kinases (Fig. 2F). This is the strongest direct support for the priming step of the model.\n8. Small interfering RNA knockdown of SRPK1 reduced SRPK1 messenger RNA and protein, reduced viral RNA, and shifted the ratio of phosphorylated to unphosphorylated nucleocapsid toward the unphosphorylated form (Fig. 3A to 3D).\n9. The SRPK1 and SRPK2 inhibitors SPHINX31 and SRPIN340 reduced viral RNA and infectious titer in a dose-dependent manner at concentrations tolerated by the cells, in ACE2-A549 cells, in naturally infectable Calu-3 cells, and in primary human type II pneumocytes from four donors (Fig. 3E to 3N).\n10. Alectinib, an approved anaplastic lymphoma kinase inhibitor with known SRPK1 and SRPK2 activity, reduced viral RNA and infectious titer dose-dependently in ACE2-A549 cells, Calu-3 cells and primary pneumocytes (Fig. 4A to 4F), and reduced replication of the distantly related alphacoronavirus HCoV-229E in Huh7 cells by more than a thousandfold (Fig. 4G).\n11. Phosphoproteomics after alectinib treatment showed reduced phosphorylation at most SR-rich domain sites while sites outside that domain did not decrease (Fig. 4I), and phosphosites scoring above the ninetieth percentile for SRPK specificity were significantly overrepresented among sites reduced by the drug (Fig. 4J).\n12. Proteomic analysis also showed that some interferon-stimulated gene products increased in abundance after alectinib treatment. The authors state that their experiments do not discriminate between SRPKs being needed for normal antiviral gene expression and reduced nucleocapsid phosphorylation compromising viral immune suppression.", "Mechanistic model": "The model is a directional, priming-dependent cascade. SRPK1 and SRPK2 phosphorylate serine 206 and serine 188, each in a locally favorable arginine and serine context. Those marks license GSK-3, which requires a phosphorylated residue four positions to the carboxyl side and therefore propagates along the four-residue chains toward the amino terminus, covering serine 202 through serine 186 and serine 184 through serine 176. Casein kinase 1, which requires a phosphorylated residue three positions to the amino side, accounts for threonine 205 once serine 202 has been modified. Several parts of this are demonstrated and several are not. The site assignments are predictions from measured specificity matrices rather than direct mapping of each site to each kinase in cells. The requirement for the two priming events is shown biochemically by mutation, and the cooperativity among the three kinases is shown by comparing combined against individual reactions on purified protein. The requirement of SRPK activity for replication is shown genetically and pharmacologically, and the drug is shown to reduce phosphorylation specifically in the targeted domain. What is not established is why the phosphorylation is needed. The authors say directly that future work is required to establish the functional role of nucleocapsid phosphorylation in the replication cycle, and they note that the inhibitors may act on aspects of the life cycle unrelated to nucleocapsid, that other kinases may phosphorylate other domains and other viral proteins, and that effects on host protein phosphorylation cannot be ruled out as contributors to the antiviral phenotype.", "Conceptual or technical advance": "The work shows that a dense, ambiguous cluster of phosphorylation sites can be resolved into an ordered sequence using measured kinase substrate preferences rather than candidate testing, and that the ordering falls out of the combination of substrate geometry with the priming requirements of two kinase families. That is a general method, applicable wherever phosphosite spacing is regular and specificity matrices exist. Practically, it nominates SRPK1 and SRPK2 as host targets whose inhibition suppresses two coronaviruses from different genera, and it identifies an already approved drug that reaches them, which shortens the distance between a mechanistic finding and something testable in people. The conservation analysis extends the relevance beyond currently circulating viruses, since the same sites and surrounding motifs are conserved in bat coronaviruses.", "Relationship to the broader research program": "The tenOever contribution here is one component of a multi-laboratory study led from Duke and Weill Cornell, and it lies in the SARS-CoV-2 infection and phosphoproteomic work carried out at the New York sites, with several co-authors from that group. The broader corpus interest it connects to is the host determinants of coronavirus replication and the use of host-directed rather than virus-directed intervention, a theme that recurs in the laboratory's COVID-19 work. The observation that alectinib treatment coincided with increased abundance of interferon-stimulated gene products touches the laboratory's long-standing interest in how SARS-CoV-2 suppresses the interferon response, though the paper does not resolve the direction of that relationship. Any statement linking this study to the laboratory's other coronavirus work would be category 3 synthesis and should be assembled centrally.", "Related publications": "- Blanco-Melo et al. 2020 and associated tenOever laboratory work establishing the ACE2-expressing A549 infection system. Methodological foundation, the cell system used for the New York arm of this study is cited as previously described.\n- Prior reports from other groups on GSK-3 and SRPK phosphorylation of the SARS-CoV nucleocapsid, and on suppression of SARS-CoV and mouse hepatitis virus replication by GSK-3 inhibitors. Predecessors, the direct antecedents of the kinase families examined here.\n- Five previously published SARS-CoV-2 phosphoproteomic datasets from other groups. Companion, used here for cross-validation of detected sites.\n- Published work on gel to liquid phase transition of nucleocapsid and RNA condensates. Conceptual extension from other groups, cited as a candidate function for the phosphorylation described here but not tested in this study.", "Limitations and boundaries": "The kinase assignments to individual sites are computational predictions from substrate specificity matrices, validated in aggregate by in vitro reactions and by drug-induced changes in phosphorylation, not by direct site-by-site demonstration in infected cells, and the third priming site threonine 205 is assigned to CK1 largely by elimination. The biochemical reconstitution uses purified recombinant protein and single representative isoforms, SRPK1, GSK-3 alpha and CK1 epsilon, so isoform redundancy in cells is untested. The authors state that they cannot rule out effects of the inhibitors on aspects of the viral life cycle unrelated to nucleocapsid, that other kinases may contribute to phosphorylation of other domains and other viral proteins, and that altered phosphorylation of host proteins may contribute to the antiviral effect. The functional consequence of nucleocapsid phosphorylation for replication is not established here. All infection work is in cell culture, immortalized lines and primary pneumocytes, and the authors say explicitly that whether targeting SRPK1 and SRPK2 in vivo produces a similar magnitude of effect requires future study. The clinical observation offered in support, favorable COVID-19 outcomes in two lung cancer patients receiving alectinib, is described by the authors as not definitive and amounts to two case reports. Conservation across coronaviruses is sequence evidence for importance and not a functional demonstration in any virus other than the two tested.", "Audience summaries": "### 25 words\n\nThree host kinase families phosphorylate the SARS-CoV-2 nucleocapsid in a set order, and blocking the first of them, including with the approved drug alectinib, suppresses coronavirus replication.\n\n### 75 words\n\nThe SARS-CoV-2 nucleocapsid protein carries a cluster of phosphorylation sites in a region conserved across coronaviruses. By matching each site to the kinases whose sequence preferences fit it, this work reconstructed an ordered cascade in which SRPK enzymes act first and GSK-3 and casein kinase 1 extend the modification. Removing or inhibiting the initiating kinases reduced viral replication in several human cell systems, and an already approved cancer drug that hits them worked too.\n\n### 150 words\n\nHost-directed antivirals need well-defined host dependencies. This study focuses on the serine and arginine rich domain of the SARS-CoV-2 nucleocapsid protein, which phosphoproteomics identified as the most heavily modified region of any viral protein and which is unusually conserved across 82 coronaviruses. Using measured substrate specificity matrices rather than candidate screening, the authors predicted that SRPK enzymes phosphorylate serine 206 and serine 188 first, that GSK-3 then propagates along chains of residues spaced every four positions toward the amino terminus, and that casein kinase 1 covers threonine 205 after serine 202 is modified. In vitro reactions with recombinant protein supported the order, and mutating both priming serines abolished the cascade. Knockdown of SRPK1 and treatment with SPHINX31, SRPIN340 or the approved inhibitor alectinib reduced SARS-CoV-2 replication in engineered A549 cells, Calu-3 cells and primary human pneumocytes, and alectinib also suppressed HCoV-229E. The functional role of the phosphorylation itself remains undefined." }, "discoveries": [], "relationships": [ { "from": "2022-yaron-host-protein-kinases-required-for-", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2022-yaron-host-protein-kinases-required-for-" } ], "canonical_url": "https://tenoeverlab.us/publications/2022-yaron-host-protein-kinases-required-for-/", "controlled_vocabulary": { "pathogens": [ "sars-cov-2", "hcov-229e" ], "technologies": [ "rt-qpcr", "plaque-assay", "immunofluorescence-microscopy", "sirna-knockdown", "site-directed-mutagenesis", "small-molecule-inhibitor-profiling", "in-vitro-kinase-assay", "comparative-sequence-analysis", "phosphoproteomics", "kinase-substrate-profiling", "phos-tag-electrophoresis" ] } }, { "id": "2022-zazhytska-non-cell-autonomous-disruption-of-", "slug": "2022-zazhytska-non-cell-autonomous-disruption-of-", "url": "/publications/2022-zazhytska-non-cell-autonomous-disruption-of-/", "title": "Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia", "authors": [ "Marianna Zazhytska", "Albana Kodra", "Daisy A. Hoagland", "Justin Frere", "John F. Fullard", "Hani Shayya", "Natalie G. McArthur", "Rasmus Moeller", "Skyler Uhl", "Arina D. Omer", "Max E. Gottesman", "Stuart Firestein", "Qizhi Gong", "Peter D. Canoll", "James E. Goldman", "Panos Roussos", "Benjamin R. tenOever", "Jonathan B. Overdevest", "Stavros Lomvardas" ], "author_count": 19, "first_author": "Marianna Zazhytska", "senior_authors": [ "Benjamin R. tenOever", "Jonathan B. Overdevest", "Stavros Lomvardas" ], "corresponding_authors": [ "Benjamin R. tenOever", "Jonathan B. Overdevest", "Stavros Lomvardas" ], "tenoever_position": 17, "tenoever_role": "senior", "contribution_character": "co-led", "year": 2022, "journal": "Cell", "volume": "185", "issue": "6", "pages": "1052-1064.e12", "doi": "10.1016/j.cell.2022.01.024", "doi_url": "https://doi.org/10.1016/j.cell.2022.01.024", "pmid": "35180380", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/35180380/", "pmcid": "PMC8808699", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8808699/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8808699/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "post-acute-sequelae" ], "pathogens": [ "SARS-CoV-2", "human coronavirus OC43" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "golden hamster", "human" ], "technologies": [ "single-cell RNA sequencing", "bulk RNA sequencing", "in situ Hi-C", "fluorescence-activated nuclei sorting", "RNA in situ hybridization", "immunofluorescence microscopy", "hidden Markov model compartment analysis", "gene ontology and gene set enrichment analysis", "serum transfer with ultraviolet inactivation" ], "biological_systems": [ "golden hamster olfactory epithelium", "human olfactory epithelium autopsy tissue", "olfactory sensory neurons", "sustentacular cells", "FACS-sorted olfactory sensory neuron nuclei" ], "key_concepts": [ "non-cell-autonomous transcriptional effect", "olfactory receptor gene choice", "interchromosomal genomic compartments", "nuclear architecture disruption", "anosmia", "sustentacular cell tropism", "olfactory signal transduction genes", "Lhx2 and Ebf transcription factors", "nuclear memory", "circulating inflammatory signal" ], "keywords": [ "SARS-CoV-2", "COVID-19", "anosmia", "olfactory receptor", "olfactory sensory neuron", "nuclear architecture", "Hi-C", "golden hamster", "sustentacular cells", "Adcy3" ], "one_sentence_contribution": "SARS-CoV-2 infection of the olfactory epithelium reorganizes the nuclear architecture of uninfected olfactory sensory neurons, dissipating the interchromosomal compartments that hold olfactory receptor genes and suppressing receptor and signal transduction transcription in both hamsters and human autopsy tissue.", "summary_25": "A virus that does not enter smell neurons still silences them, unraveling the chromosome contacts that hold odorant receptor genes together and shutting down odor detection machinery.", "summary_75": "SARS-CoV-2 infects support cells in the nose but rarely the sensory neurons themselves, which made COVID-19 smell loss hard to explain. In hamsters and in human autopsy tissue, infection collapsed the unusual contacts that gather odorant receptor genes from many chromosomes into shared nuclear compartments, and receptor and signaling gene expression fell and stayed low after the virus was gone. Serum from infected hamsters, with virus inactivated, reproduced the nuclear change in naive animals.", "summary_150": "Olfactory sensory neurons express one receptor each, supported by interchromosomal compartments that gather receptor gene clusters. The authors asked how SARS-CoV-2 causes anosmia without infecting those neurons. Single-cell sequencing of infected hamster olfactory epithelium showed the virus in sustentacular cells, which were transiently depleted, while neuron numbers held steady. Those uninfected neurons nonetheless mounted an antiviral response and then lost olfactory receptor and signal transduction transcripts, a loss beginning at two days, peaking at four and persisting at ten days after viral clearance. In situ Hi-C showed receptor cluster contacts falling from one day and staying low, before genome-wide compartment changes and before Lhx2 and Ebf downregulation. Ultraviolet-inactivated serum from infected animals reproduced the contact loss in naive hamsters without transferring virus. Human autopsies showed matching receptor downregulation and reduced contacts in sorted neuronal nuclei. The circulating mediator and the receiving pathway were not identified.", "citation": "Zazhytska M, Kodra A, Hoagland DA, Frere J, Fullard JF, Shayya H, McArthur NG, Moeller R, Uhl S, Omer AD, Gottesman ME, Firestein S, Gong Q, Canoll PD, Goldman JE, Roussos P, tenOever BR, Overdevest JB, Lomvardas S. Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia. Cell. 2022. Volume 185, issue 6, pages 1052-1064.e12. DOI 10.1016/j.cell.2022.01.024. PMID 35180380. PMCID PMC8808699.", "sections": { "Citation": "Zazhytska M, Kodra A, Hoagland DA, Frere J, Fullard JF, Shayya H, McArthur NG, Moeller R, Uhl S, Omer AD, Gottesman ME, Firestein S, Gong Q, Canoll PD, Goldman JE, Roussos P, tenOever BR, Overdevest JB, Lomvardas S. Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia. Cell. 2022. Volume 185, issue 6, pages 1052-1064.e12.\n\nDOI 10.1016/j.cell.2022.01.024. PMID 35180380. PMCID PMC8808699.", "One-sentence contribution": "SARS-CoV-2 infection of the olfactory epithelium reorganizes the nuclear architecture of uninfected olfactory sensory neurons, dissipating the interchromosomal compartments that hold olfactory receptor genes and suppressing receptor and signal transduction transcription in both hamsters and human autopsy tissue.", "Executive summary": "Loss of smell is among the most common symptoms of COVID-19 and one of the hardest to explain, because olfactory sensory neurons do not express the entry factors SARS-CoV-2 requires and are infected only rarely. The authors ask how a virus that does not enter these neurons nonetheless silences their defining function. They combine an infected golden hamster time course with human olfactory epithelium autopsies, using single-cell and bulk RNA sequencing to describe the transcriptional response and in situ Hi-C to describe nuclear organization. In hamsters the virus infects sustentacular cells, which are transiently depleted and then restored, while neuron representation stays constant. Despite escaping infection the neurons mount an antiviral response and then, with a delay, lose expression of olfactory receptor genes and of the signal transduction genes that make odor detection possible, a loss still present at ten days when other markers have recovered and the virus has been cleared. Preceding that loss, the long-range cis and trans contacts among olfactory receptor gene clusters, which normally converge into specialized genomic compartments, are reduced from one day after infection and remain reduced at ten days. Ultraviolet-inactivated serum from infected hamsters, applied intranasally to naive animals for 12.5 hours, reproduces the loss of contacts without transferring virus. Human autopsies show the same receptor downregulation and the same loss of interchromosomal contacts in sorted neuronal nuclei. The circulating factor responsible was not identified.", "Scientific context": "Olfactory dysfunction emerged early as a common neurological symptom of COVID-19, usually resolving within about six weeks but persisting in roughly ten percent of patients. Unlike smell loss in other upper respiratory infections, it is not attributable to congestion blocking odorants from reaching the epithelium. That made the mechanism puzzling, because published work had established that olfactory sensory neurons do not express ACE2 and TMPRSS2 and are not appreciably infected by the virus, while sustentacular cells are. Separately, work largely from the senior authors' field had established that olfactory receptor choice depends on an unusual nuclear organization. Receptor gene clusters from many chromosomes converge into a small number of specialized genomic compartments, and this convergence supports stable expression of a single receptor per neuron. Transcription factors including Lhx2 and Ebf family members are required for receptor and signaling gene expression. Prior work by other groups had also linked innate immune signaling in the olfactory epithelium to reduced odorant receptor levels. What had not been examined was whether an infection confined to neighboring cells could reach into the neuron and disassemble that architecture.", "Central question": "How does SARS-CoV-2 cause loss of smell when it does not infect olfactory sensory neurons, and specifically does infection of the surrounding epithelium act on those neurons through a non-cell-autonomous route that alters the nuclear organization on which olfactory receptor expression depends?", "Experimental strategy": "The design pairs a controllable animal time course with human tissue that carries the actual clinical condition, and reads each at two levels, transcription and genome organization. Golden hamsters are used because their ACE2 resembles the human protein and because pathogenesis and immune response in this species have been characterized. Single-cell RNA sequencing across mock and infected animals at one, three and ten days establishes which cells are infected, whether any population is lost, and what each cell type does transcriptionally. Because the single-cell chemistry used a 5-prime approach that is well suited to detecting viral transcripts but poorly suited to olfactory receptor messages given hamster genome annotation, bulk RNA sequencing at one, two, four and ten days is added specifically to quantify receptor expression. In situ Hi-C on the epithelium at matched time points asks whether the receptor compartments are intact. The causal step separating virus from effect is a serum transfer, in which serum drawn at three days from infected or mock animals is ultraviolet-irradiated to inactivate virus and inoculated intranasally into naive hamsters, with sequencing used to confirm no viral genome was carried over. On the human side, a specific region at the roof of the nasal cavity enriched for olfactory neurons is identified and sampled from control and COVID-19 autopsies, assessed by histology and in situ hybridization, profiled by bulk RNA sequencing with batch correction and outlier removal, and subjected to in situ Hi-C after fluorescence-activated sorting of neuronal nuclei.", "Key findings": "1. The virus targets sustentacular cells and largely spares neurons. Across 68,951 hamster cells and thirteen annotated types, viral RNA was present in about 5 percent of cells at one and three days and gone by ten days. At one day roughly 47 percent of infected cells were sustentacular, about 40 percent of that population, while only about 6 percent of infected cells were neurons (Figure 1C-1E and Figure S1B). Spike protein colocalized with sustentacular and microglial markers, and colocalization with neuronal markers was rare and confined to regions of shedding and structural damage (Figures S1C and S1E).\n\n2. Sustentacular cells are transiently depleted and neurons are not. Sustentacular representation fell from 20.6 percent in mock animals to 6 percent at three days, with a concurrent rise in microglia and other immune cells, and both returned to preinfection levels by ten days. Neuronal representation was stable throughout (Figures 1A and 1B).\n\n3. Infected sustentacular cells show cell-autonomous changes. Splitting infected epithelia into virus-positive and virus-negative sustentacular cells revealed upregulated cytokines and chemokines and downregulated sustentacular markers in the positive fraction (Figure 2B).\n\n4. Uninfected neurons respond anyway. Olfactory sensory neurons activated antiviral gene expression at three days, declining by ten days as virus cleared, and genes required for smell, notably Adcy3, were significantly reduced at three days (Figures 2C, 2D and S2). Reduction of Adcy3 message and protein was confirmed by in situ hybridization and immunofluorescence at four days, including in regions with little detectable virus (Figures 2E and 2F), which the authors take as evidence that the effect does not require local infection.\n\n5. Receptor gene downregulation is the dominant transcriptional change and it persists. In bulk sequencing, viral load rose through four days and was eliminated by ten. Olfactory receptor downregulation began at two days, peaked at four and continued through ten days, when other neuronal markers had recovered and when the most variable genes in the epithelium had returned to baseline (Figures 3F, 3G and 3H). Adcy3, Gng13, Cnga2, Rtp1 and Gfy followed the same pattern (Figure 3I).\n\n6. Cell-type responses are staggered. Sustentacular and immediate neuronal precursor changes came early and resolved, with precursor transcription factors Lhx2, Ebf1 and Ebf2 reduced mainly at two days, while neuronal and globose basal cell changes were delayed, and globose basal markers peaked at ten days, which the authors read as progenitor activation toward replacement (Figures 3D and 3E).\n\n7. Receptor gene compartments come apart. In situ Hi-C showed the expected strong long-range cis contacts and widespread trans contacts among hamster receptor clusters in controls. Contacts were reduced from one day, most strongly at three days, and remained low at ten days (Figures 4A-4C). Hidden Markov model compartment scores showed widespread genome-wide compartment changes by three days, later than the receptor-specific disruption, and still disrupted at ten days after viral clearance (Figure 4D).\n\n8. Serum from infected animals reproduces the nuclear effect without virus. Ultraviolet-inactivated serum collected at three days and given intranasally to naive hamsters for 12.5 hours significantly reduced trans contacts among receptor clusters and changed genome-wide compartment scores, with sequencing confirming no viral genome in the recipients (Figures 5B-5E). At this early point receptor transcription had not yet changed significantly, though a downward trend across most receptor genes was present and more pronounced than at one day of actual infection (Figure S4A). Sustentacular markers did not respond to infected serum (Figure S4C), consistent with those changes being cell autonomous.\n\n9. Human autopsies show the same transcriptional signature. Across eighteen infected and a small number of control olfactory epithelium samples, viral RNA was detected in every infected specimen at variable levels while epithelial and respiratory marker representation did not change with viral load. Controls had higher receptor message levels, principal component analysis separated infected from control samples when restricted to receptor genes but not when using the whole transcriptome, and sensory perception of smell was the most significantly enriched gene ontology term among downregulated genes (Figures 6A, 6E-6H, 6J and 6K). One nominal control carried human coronavirus OC43 and clustered with the infected samples, and was excluded from the control pool.\n\n10. Human neurons lose the same contacts. In situ Hi-C on nuclei sorted from two control and four infected autopsies showed conserved receptor-specific long-range cis and trans contacts in humans and their reduction in infected samples, including in specimens where genome-wide compartmentalization was not comparably altered (Figures 7A-7E), and interchromosomal compartments containing Adcy3 and other olfaction genes also dissipated (Figure S6B).\n\n11. Compartment disruption can precede transcription factor loss. In hamsters the loss of receptor compartments preceded downregulation of Lhx2 and Ebf and persisted after their restoration, and in two infected human specimens receptor transcription and compartmentalization were disrupted while Lhx2 and Ebf were near control levels. The authors use this ordering to argue that compartment disruption is upstream rather than a consequence of transcription factor loss, which is an argument from temporal and correlative evidence rather than from perturbation.", "Mechanistic model": "The study does not establish a definitive mechanism, and the authors say so in their own limitations. What the data support is the following chain. SARS-CoV-2 infects sustentacular cells and elicits a local and systemic inflammatory response. Something in circulation, present in serum by three days and surviving ultraviolet inactivation, reaches olfactory sensory neurons that the virus itself does not enter. In those neurons the interchromosomal compartments that gather olfactory receptor gene clusters lose their contacts, and receptor and signal transduction transcription falls and stays low beyond viral clearance. Reduced expression of receptors, of the chaperones Rtp1 and Rtp2, of the signaling components Adcy3 and Gng13 and of the channel Cnga2 would be expected to impair odor detection, an inference the authors draw explicitly from the phenotypes of knockout mice rather than from measurement of smell in this study.\n\nSeveral elements are proposals. The identity of the circulating molecule or molecules is unknown and the neuronal signaling pathway that receives the signal was not determined. The nuclear memory idea, that compartments may form only during differentiation so that their disruption in mature neurons could be effectively irreversible and recovery would require neuronal replacement, is offered as a hypothesis with a stated reason rather than as a finding. The suggestion that restored contacts might reassemble differently and produce odor misrepresentation is a reasonable extrapolation the authors raise and do not test. The causal link from receptor downregulation to anosmia in patients is an inference, since olfactory function was not measured in the autopsy cohort beyond one self-reported case. The direction of the compartment to transcription factor relationship rests on timing and on two human specimens, not on manipulation.", "Conceptual or technical advance": "The work supplies a concrete cellular route by which an infection confined to a small fraction of cells can change the identity and function of cells it never enters, which reframes a general puzzle about SARS-CoV-2 pathology in terms of systemic signals acting on nuclear organization. It also brings three-dimensional genome organization into the interpretation of an acute infectious symptom, and it demonstrates that in situ Hi-C can be performed on neuronal nuclei sorted from human autopsy tissue, which makes nuclear architecture an accessible readout in post-mortem material generally. The serum transfer establishes an assay in which a systemic consequence of infection can be separated from the virus and delivered to a naive animal, and the observation that the receptor compartments are the earliest and most durable casualty, lost before genome-wide compartmentalization changes and before the relevant transcription factors fall, makes the fragility of these particular interchromosomal contacts a testable property.", "Relationship to the broader research program": "Within the tenOever laboratory's line of work the paper connects to the group's characterization of the host transcriptional response to SARS-CoV-2 and to the use of the golden hamster as a model for that response, both of which supplied the infection system and the antiviral gene framework used here. The recurring question the paper shares with that work is how much of the damage attributed to a virus is done by the host response rather than by infection of the damaged cells themselves, a question that also runs through the group's earlier interferon signaling studies from the opposite direction. Reading the antiviral program in uninfected neurons here alongside the group's work on interferon-driven transcriptional programs as a single thread about the costs of the response is category 3 synthesis, offered as such, and it is not a claim this paper makes.", "Related publications": "- Hoagland, Moeller, Uhl, Oishi, Frere, Golynker and colleagues including tenOever, 2021, Immunity, on leveraging the type I interferon system against SARS-CoV-2 pathogenicity. Methodological foundation and predecessor from the same laboratory, source of the hamster infection model and of the antiviral response framework referenced here.\n- Blanco-Melo, Nilsson-Payant, Liu, Uhl, Hoagland, Møller and colleagues, 2020, Cell, on the imbalanced host response to SARS-CoV-2. Predecessor from the same laboratory, cited in support of the observed antiviral gene induction.\n- Frere and colleagues, 2022, on lasting and systemic perturbations after SARS-CoV-2 infection. Companion, cited by the authors as a parallel account of sustained antiviral programs that could contribute to persistent neurological deficits.\n- Clowney and colleagues, 2012, and Monahan, Horta and Lomvardas, 2019, on nuclear aggregation of olfactory receptor genes and on trans interactions in receptor choice. Methodological foundation from the senior author's laboratory, the basis for interpreting the compartments measured here.\n- Khan and colleagues, 2021, on how SARS-CoV-2 attacks the respiratory and olfactory mucosae. Predecessor from another laboratory, providing the histological picture of infrequent neuronal infection that motivates this study and the spatial transcriptomic result the authors reconcile with their own.\n- Zazhytska and colleagues, 2021, preprint on disruption of nuclear architecture as a cause of COVID-19 induced anosmia. Predecessor, the preprint version of this work, also cited for its control single-cell analysis of an uninfected human autopsy.", "Limitations and boundaries": "The authors state their own limitations plainly and those are reproduced here. The circulating molecule or molecules responsible were not identified, nor was the neuronal signaling pathway that transduces the signal, so generalization to other neuronal populations is speculation they say they have not explored. They did not establish that receptor and signaling gene downregulation causes the anosmia, inferring this instead from knockout mouse phenotypes. In humans they can only deduce that infection caused the downregulation, because expression before infection cannot be measured, and although the hamster experiments support the interpretation they cannot exclude rodent-specific mechanisms. Beyond those, the human cohort is small and unbalanced, with eighteen infected samples against a handful of controls, one of which was removed as an outlier and one of which carried a different coronavirus, and olfactory status is known for only one patient, with the prevalence of deficits in the remainder estimated from the literature. Autopsy material carries variable post-mortem intervals, treatments and disease durations, and batch correction was applied. The human Hi-C comparison rests on two control and four infected autopsies. The hamster time course extends only to ten days, so persistence beyond that, and any recovery, is not observed, and the proposed irreversibility of compartment loss is untested. The serum transfer used a single 12.5 hour exposure to serum from a single time point and shows sufficiency of something in that serum, not necessity of any identified factor, and no smell testing was performed in hamsters or humans.", "Audience summaries": "### 25 words\n\nA virus that does not enter smell neurons still silences them, unraveling the chromosome contacts that hold odorant receptor genes together and shutting down odor detection machinery.\n\n### 75 words\n\nSARS-CoV-2 infects support cells in the nose but rarely the sensory neurons themselves, which made COVID-19 smell loss hard to explain. In hamsters and in human autopsy tissue, infection collapsed the unusual contacts that gather odorant receptor genes from many chromosomes into shared nuclear compartments, and receptor and signaling gene expression fell and stayed low after the virus was gone. Serum from infected hamsters, with virus inactivated, reproduced the nuclear change in naive animals.\n\n### 150 words\n\nOlfactory sensory neurons express one receptor each, supported by interchromosomal compartments that gather receptor gene clusters. The authors asked how SARS-CoV-2 causes anosmia without infecting those neurons. Single-cell sequencing of infected hamster olfactory epithelium showed the virus in sustentacular cells, which were transiently depleted, while neuron numbers held steady. Those uninfected neurons nonetheless mounted an antiviral response and then lost olfactory receptor and signal transduction transcripts, a loss beginning at two days, peaking at four and persisting at ten days after viral clearance. In situ Hi-C showed receptor cluster contacts falling from one day and staying low, before genome-wide compartment changes and before Lhx2 and Ebf downregulation. Ultraviolet-inactivated serum from infected animals reproduced the contact loss in naive hamsters without transferring virus. Human autopsies showed matching receptor downregulation and reduced contacts in sorted neuronal nuclei. The circulating mediator and the receiving pathway were not identified." }, "discoveries": [ "claim-14" ], "relationships": [ { "from": "2022-zazhytska-non-cell-autonomous-disruption-of-", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2022-zazhytska-non-cell-autonomous-disruption-of-" }, { "from": "2022-zazhytska-non-cell-autonomous-disruption-of-", "to": "2022-frere-sars-cov-2-infection-in-hamsters-a", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2022-zazhytska-non-cell-autonomous-disruption-of-" }, { "from": "2022-zazhytska-non-cell-autonomous-disruption-of-", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2022-zazhytska-non-cell-autonomous-disruption-of-" }, { "from": "2022-zazhytska-non-cell-autonomous-disruption-of-", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2022-zazhytska-non-cell-autonomous-disruption-of-" }, { "from": "2022-zazhytska-non-cell-autonomous-disruption-of-", "to": "2022-frere-sars-cov-2-infection-in-hamsters-a", "relationship": "companion", "evidence": "stated in the Related publications section of 2022-zazhytska-non-cell-autonomous-disruption-of-" } ], "canonical_url": "https://tenoeverlab.us/publications/2022-zazhytska-non-cell-autonomous-disruption-of-/", "controlled_vocabulary": { "pathogens": [ "sars-cov-2", "hcov-oc43" ], "technologies": [ "bulk-rna-seq", "immunofluorescence-microscopy", "pathway-enrichment-analysis", "rna-in-situ-hybridization", "single-cell-rna-seq", "cell-sorting", "in-situ-hi-c", "serum-transfer" ] } }, { "id": "2023-carrau-delayed-engagement-of-host-defense", "slug": "2023-carrau-delayed-engagement-of-host-defense", "url": "/publications/2023-carrau-delayed-engagement-of-host-defense/", "title": "Delayed engagement of host defenses enables SARS-CoV-2 viremia and productive infection of distal organs in the hamster model of COVID-19", "authors": [ "Lucia Carrau", "Justin J. Frere", "Ilona Golynker", "Alvaro Fajardo", "Cristobal F. Rivera", "Shu Horiuchi", "Tyler Roonprapunt", "Judith M. Minkoff", "Daniel Blanco-Melo", "Benjamin TenOever" ], "author_count": 10, "first_author": "Lucia Carrau", "senior_authors": [ "Benjamin TenOever" ], "corresponding_authors": [ "Benjamin TenOever" ], "tenoever_position": 10, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2023, "journal": "Science Signaling", "volume": "16", "issue": "789", "pages": "eadg5470", "doi": "10.1126/scisignal.adg5470", "doi_url": "https://doi.org/10.1126/scisignal.adg5470", "pmid": "37311033", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/37311033/", "pmcid": null, "pmc_url": null, "pmc_pdf_url": null, "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling", "pandemic-host-response" ], "themes": [ "calibration-of-interferon-in-vivo", "interferon-as-intervention" ], "pathogens": [ "SARS-CoV-2", "SARS-CoV-2 USA-WA1/2020" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "golden hamster", "hamster cells", "African green monkey cells" ], "technologies": [ "bulk RNA sequencing", "real-time quantitative RT-PCR", "plaque assay", "virus amplification on permissive cells", "interferon bioassay", "immunohistochemistry", "immunofluorescence microscopy", "flow cytometry", "intravenous infection", "dexamethasone immunosuppression" ], "biological_systems": [ "golden hamster", "hamster lung", "kidney", "liver", "spleen", "brain", "olfactory bulb", "heart", "gastrointestinal tract", "pancreas", "whole blood", "BHK-21 cells", "Vero E6 cells" ], "key_concepts": [ "type I and type III interferon", "systemic antiviral priming", "interferon-stimulated genes", "viremia", "viral tropism", "extrapulmonary manifestations", "delayed innate immune engagement", "immunosuppression", "route of inoculation", "COVID-19 heterogeneity" ], "keywords": [ "SARS-CoV-2", "golden hamster", "interferon priming", "viremia", "distal organ infection", "dexamethasone", "intravenous infection", "kidney", "COVID-19 extrapulmonary disease", "innate immunity" ], "one_sentence_contribution": "In golden hamsters, productive SARS-CoV-2 replication in the airways generates circulating type I and III interferon that primes every organ against infection, and blunting or bypassing that airway response permits viremia and productive infection of liver, kidney, spleen and brain.", "summary_25": "Airway infection with SARS-CoV-2 sends interferon into the bloodstream that protects distant organs, and hamsters whose airway response is blunted or bypassed develop infection in those organs.", "summary_75": "SARS-CoV-2 provokes an antiviral response in organs it barely reaches. In hamsters, that response comes from interferon produced in the infected lung and carried in the blood, not from local replication. Suppressing the airway response with a steroid, or injecting virus directly into the bloodstream to skip the lung, allowed productive infection of liver, kidney, spleen and brain. Infecting through the airway first protected those organs against a subsequent bloodstream challenge.", "summary_150": "Golden hamsters infected intranasally with SARS-CoV-2 showed an interferon signature in all nine organs profiled, while infectious virus was largely confined to lung and olfactory bulb. Whole blood carried an interferon stimulated gene signature without interferon transcripts of its own, and a fibroblast bioassay detected circulating interferon by one day after infection. Dexamethasone delayed airway interferon stimulated gene induction and phagocyte infiltration without changing early lung titers, and under that treatment virus appeared in liver, spleen, olfactory bulb and gastrointestinal tract, with low level viremia detectable only after amplification on permissive cells. Intravenous inoculation bypassed the airway and produced productive infection of kidney, liver, spleen, heart and gastrointestinal tract, confirmed in kidney by nucleocapsid staining of parenchymal and endothelial cells. Circulating interferon was present only in animals with lung titers, and prior airway infection reduced distal viral loads after an intravenous challenge, indicating that airway derived interferon restricts tropism.", "citation": "Carrau L, Frere JJ, Golynker I, Fajardo A, Rivera CF, Horiuchi S, Roonprapunt T, Minkoff JM, Blanco-Melo D, TenOever B. Delayed engagement of host defenses enables SARS-CoV-2 viremia and productive infection of distal organs in the hamster model of COVID-19. Science Signaling. 2023. Volume 16, issue 789, article eadg5470. DOI 10.1126/scisignal.adg5470. PMID 37311033.", "sections": { "Citation": "Carrau L, Frere JJ, Golynker I, Fajardo A, Rivera CF, Horiuchi S, Roonprapunt T, Minkoff JM, Blanco-Melo D, TenOever B. Delayed engagement of host defenses enables SARS-CoV-2 viremia and productive infection of distal organs in the hamster model of COVID-19. Science Signaling. 2023. Volume 16, issue 789, article eadg5470.\n\nDOI 10.1126/scisignal.adg5470. PMID 37311033.", "One-sentence contribution": "In golden hamsters, productive SARS-CoV-2 replication in the airways generates circulating type I and III interferon that primes every organ against infection, and blunting or bypassing that airway response permits viremia and productive infection of liver, kidney, spleen and brain.", "Executive summary": "COVID-19 presents very differently between individuals, and complications outside the lung are common in severe cases, which has raised the question of whether extrapulmonary replication contributes to severity. Working in the golden hamster, which reproduces many features of human infection, the authors asked where the virus actually replicates early after airway exposure, where the host antiviral response appears, and what connects the two. Bulk RNA sequencing of nine organs three days after intranasal challenge showed an interferon signature in every organ examined, while infectious virus by plaque assay was largely confined to lung and olfactory bulb, with less consistent recovery from heart and gastrointestinal tract. Whole blood carried an interferon stimulated gene signature without interferon transcripts of its own, and a bioassay using hamster fibroblasts detected roughly sixty units per millilitre of circulating interferon at one day after infection. Dexamethasone treatment delayed airway interferon stimulated gene induction and reduced lung phagocyte infiltration without changing early lung titers, and under that treatment infectious virus appeared in liver, spleen, olfactory bulb and gastrointestinal tract, with low level viremia detectable after amplification. Delivering virus intravenously bypassed the airway entirely and produced productive infection of kidney, liver, spleen, heart and gastrointestinal tract. Priming animals by intranasal infection before an intravenous challenge reduced distal viral loads. Circulating interferon was detected only in animals with lung titers.", "Scientific context": "SARS-CoV-2 delays the host interferon response by compartmentalizing double-stranded RNA in double membrane vesicles and by degrading host mRNA, while NF kappa B driven chemokine and cytokine production proceeds, producing an unbalanced response in the lungs characterized by high chemokines with delayed type I and III interferon and heavy neutrophil recruitment. The interferon response that does appear is thought to arise once infected cells die and their contents, including viral double-stranded RNA and host DNA, are sampled by phagocytes and other cells bearing Toll-like receptors. Clinically, COVID-19 shows substantial heterogeneity, with severe disease concentrated among older and comorbid individuals and frequently accompanied by cardiac, cognitive and gastrointestinal complications, which has led several groups to propose that distal replication contributes to severity. Deficits in innate immune signaling, including inborn errors and autoantibodies against type I interferon, are associated with critical outcomes, and genetic ablation of type I but not type III interferon signaling in hamsters had been shown to raise viral RNA. The golden hamster had been established by this laboratory and by others as a model reproducing transmission, clinical presentation and comorbidity effects. What had not been resolved was whether the interferon response seen in organs away from the airway reflects local replication or a signal arriving from elsewhere, and whether that response does anything.", "Central question": "Does the antiviral state observed throughout the body after airway SARS-CoV-2 infection arise from productive replication in those organs or from interferon generated in the lung and carried in the circulation, and does that systemic priming restrict where the virus can establish infection?", "Experimental strategy": "The design separates the question of where virus is from the question of where the response is, and then removes the response to see what changes. First, the two are measured in parallel in the same animals, with bulk RNA sequencing across nine organs set against plaque assay and viral RNA quantification in the same tissues at one and three days, plus immunohistochemistry for the interferon stimulated protein Mx alongside viral nucleocapsid in lung and kidney. Second, the blood is examined as the candidate conduit, using RNA sequencing of whole blood to show an interferon stimulated gene signature and using the absence of Ifnb and Ifnl reads in blood to argue that those cells are responding to interferon rather than sensing virus, since interferon transcripts are induced only on direct detection of viral double-stranded RNA. Because no hamster interferon reagents were available, circulating interferon was quantified indirectly with a bioassay in which serum is applied to BHK-21 hamster fibroblasts, which cannot themselves make type I or III interferon, and Mx induction is read against a universal interferon standard curve. Third, the priming is removed in two independent ways that fail differently. Dexamethasone suppresses de novo transcription and therefore delays the response while leaving the airway infection in place, and intravenous inoculation places virus in the circulation without an airway infection to generate the signal. Fourth, priming is restored deliberately by infecting intranasally and then challenging intravenously three days later, with control groups receiving buffer by one route or the other, and organs harvested at four days so that the readout precedes an adaptive response. Detection of very low viremia used an amplification step on permissive Vero E6 cells before plaque assay.", "Key findings": "1. Three days after intranasal challenge, transcriptional profiling of olfactory bulb, brain, liver, kidney, gastrointestinal tract, pancreas, spleen, heart and lung showed organ specific clustering and shared responses, with type I and III interferon signatures the most prominent enriched annotation in every organ, corroborated by Isg15 quantitative RT-PCR (Figure 1A and Figure S1).\n\n2. Infectious virus was not present in most of those organs. Plaque assay recovered high titers in lung, consistent recovery from olfactory bulb, less consistent recovery from heart, and a single gastrointestinal tract sample the authors attribute to possible ingestion of inoculum (Figure 1B). Viral RNA was elevated only in lung and gastrointestinal tract, and the same confinement was seen at one day after infection (Figure 1C and Figure S1D). Mx protein was detected in kidney as well as lung by immunohistochemistry (Figure 1D).\n\n3. Whole blood showed an interferon stimulated gene signature at both one and three days, including Ifit3, Mx, Isg15, Oasl and Irf7, while no replication competent virus was recovered from blood and viral N RNA was detected only at one day at low level (Figures 2A and 2B, Figures S2A and S2B).\n\n4. No Ifnb or Ifnl reads were found in blood, whereas Ifnl appeared in lung at one day and rose by three days and Ifnb appeared in lung at three days (Figures 2C and 2D). The authors read the absence of interferon transcripts in blood as indicating that blood cells are responding to interferon rather than directly sensing virus. Ifna transcripts were not detected in any sample, which the authors attribute to incomplete or incorrect annotation of that gene family in the hamster genome.\n\n5. The serum bioassay detected roughly sixty units per millilitre of interferon equivalents at one day after infection, stable at three days (Figure 2E). Taken with the preceding points, the authors support a model in which lung derived interferon enters the circulation and primes distal organs.\n\n6. Dexamethasone delayed rather than abolished the airway response. Lung titers were unchanged over the first three days, but treated animals failed to control replication thereafter, with titers rising after three days, replication competent virus present at seven days and clearance only by nine days (Figure 3A). Mx positive cell counts were reduced at three days (Figure 3B), lung interferon stimulated gene induction was delayed by RNA sequencing without other aspects of host biology being affected (Figure 3C), and IBA1 positive phagocyte infiltration was reduced (Figure 3D). Flow cytometry showed reduced B cell populations and reduced T cell frequency after challenge (Figures S3B and S3C).\n\n7. Dexamethasone reduced distal Isg15 induction at one day across kidney, liver, gastrointestinal tract, brain and heart, with levels similar to vehicle by three days (Figure 3E).\n\n8. Under dexamethasone, infectious virus appeared in liver, spleen, olfactory bulb and gastrointestinal tract in addition to lung, although not with complete penetrance (Figures 3F and 3G). Direct plaque assay of blood and sera was negative, but amplification on Vero E6 cells revealed low level viremia in roughly fifty to seventy five percent of dexamethasone treated animals at one day only (Figure 3H). Animals with no detectable blood virus at one day were also negative in kidney, liver, spleen, heart and gastrointestinal tract.\n\n9. Intravenous administration bypassed airway control and produced infection of lung, kidney, liver, spleen, heart and gastrointestinal tract by three days, with kidney titers near ten to the fourth plaque forming units per millilitre at both one and three days (Figures 4A and 4B). Kidney infection was confirmed as productive by nucleocapsid staining in parenchymal cells and in endothelial cells of the peritubular capillary network and renal medulla, with elevated Mx in surrounding tissue (Figures 4C to 4F).\n\n10. Intranasal infection generated more circulating interferon than intravenous infection at one day, with comparable levels by three days (Figures 4G and 4H). Across all animals, circulating interferon was detected only in those with positive lung titers regardless of inoculation route, while kidney viral load did not affect circulating interferon (Figures 4I and 4J). The same pattern held using an RNA sequencing derived interferon score (Figures 4K and 4L). A single intravenously infected animal with lung infectious virus at one day also had the highest circulating interferon.\n\n11. Kidney interferon stimulated gene profiles were similar whether infection was intranasal or intravenous for the most highly expressed canonical genes, which the authors interpret as the airway response producing distal protection comparable to what a locally infected organ generates (Figure S4C).\n\n12. Prior airway infection protected distal organs against an intravenous challenge. Animals infected intranasally and then challenged intravenously three days later had significantly less virus in blood and sera, kidney, liver and gastrointestinal tract at four days than animals given buffer intranasally before the same intravenous challenge (Figures 4M and 4N), while Isg15 induction was comparable across groups (Figure S4E).", "Mechanistic model": "The study does not establish a definitive molecular mechanism, and the central claim is a causal relationship established by intervention rather than a pathway. What the data support is that productive replication in the airway is the source of type I and type III interferon that enters the circulation, that this circulating interferon rather than local replication accounts for the antiviral transcriptional state in organs where no virus is recovered, and that this state restricts where the virus can subsequently establish infection. The strongest support comes from three independent manipulations pointing the same way, namely that dexamethasone delays the airway response and permits distal infection with transient viremia, that intravenous delivery bypasses the airway response and permits productive distal infection, and that deliberately priming by airway infection before intravenous challenge reduces distal viral loads. The inference that blood cells respond to interferon rather than sense virus rests on the absence of Ifnb and Ifnl reads in blood together with their presence in lung, which is indirect. The circulating interferon measurement is a bioassay reading Mx induction in hamster fibroblasts rather than a direct measurement of interferon protein, since the authors state that hamster reagents were unavailable. The data do not identify which cells produce the circulating interferon, do not distinguish the relative contributions of type I and type III interferon, and do not establish the compartment through which virus disseminates, since infectious material was recoverable from blood only after an amplification step. The authors also do not exclude that dexamethasone acts on the distal organs directly in addition to its effect on the airway. The extension of this model to severe human COVID-19 or to Long Covid is offered explicitly as speculation requiring further study.", "Conceptual or technical advance": "Separating where a respiratory virus replicates from where the host responds, and then showing by intervention that the response in the second place depends on replication in the first, reframes extrapulmonary antiviral signaling as a protective output of the lung rather than as evidence of distal infection. It follows that the heterogeneity of COVID-19 presentation can be considered as a function of the speed and strength of early airway immune engagement, and that individuals whose interferon response is blunted by age, immunosuppression, inborn error or autoantibody may permit the transient viremia that seeds distal organs. Practically, the work supplies a serum bioassay that makes circulating interferon quantifiable in hamsters despite the absence of species specific reagents, and it provides two complementary ways to remove airway priming, one pharmacological and one by route of inoculation, that can be applied to other respiratory pathogens.", "Relationship to the broader research program": "This extends the laboratory's prior hamster work on SARS-CoV-2, which described a wave of inflammation reaching tissues with little productive replication and proposed that disseminated viral material might account for it. The present study tests the alternative explanation and finds for circulating interferon, and then shows that the systemic response is protective rather than merely present. The recurring position across these studies is that the host response, measured transcriptionally and across whole animals, is the primary object of study and that the model must be chosen so that response can be seen. Category 3 synthesis, visible only when several corpus papers are read together, is that the laboratory repeatedly treats the site of replication and the site of response as separable variables and designs interventions that decouple them. That statement rests on more than this paper alone.", "Related publications": "- Hoagland et al. 2021, leveraging the antiviral type I interferon system as a first line of defense against SARS-CoV-2 pathogenicity, predecessor. The same laboratory's hamster study describing systemic inflammation despite minimal distal replication, which frames the question addressed here.\n- Blanco-Melo et al. 2020, imbalanced host response to SARS-CoV-2 drives development of COVID-19, predecessor. Source of the low interferon and high chemokine framing used in the introduction, and Blanco-Melo is a co-author here.\n- Boudewijns and colleagues, cited by the authors as showing that ablation of STAT2, and of type I but not type III interferon signaling, increases viral RNA and dissemination in the hamster model, companion. Provides the genetic counterpart to the pharmacological suppression used here.", "Limitations and boundaries": "All in vivo work is in young male golden hamsters at five to seven weeks, so age and sex effects are not addressed even though advanced age is the comorbidity the authors invoke in interpreting their results. Group sizes are small, generally three or four animals per condition. The authors themselves note inconsistency in recovering infectious material from nonpulmonary tissue and attribute it to genetic diversity in outbred hamsters together with stochasticity, which limits how firmly penetrance can be stated for the dexamethasone results. Dexamethasone is a broad suppressor of de novo transcription and is not specific to interferon signaling, so effects beyond delayed priming cannot be excluded, including direct effects on distal organs. Intravenous administration uses a thousandfold higher dose than the intranasal route, so route and dose are not independent in that comparison. Circulating interferon is measured by bioassay rather than directly, and the assay does not distinguish type I from type III interferon. Ifna could not be assessed at all because of hamster genome annotation. Viremia was detectable only after amplification, so its magnitude and duration are not quantified, and the route of dissemination is not established. The priming experiment was read at four days to precede adaptive immunity, so it speaks to innate priming only, and one animal in the control group was excluded for failing to become infected. Finally, the extension to severe human COVID-19 and to Long Covid is presented by the authors as speculation and is not tested here.", "Audience summaries": "### 25 words\n\nAirway infection with SARS-CoV-2 sends interferon into the bloodstream that protects distant organs, and hamsters whose airway response is blunted or bypassed develop infection in those organs.\n\n### 75 words\n\nSARS-CoV-2 provokes an antiviral response in organs it barely reaches. In hamsters, that response comes from interferon produced in the infected lung and carried in the blood, not from local replication. Suppressing the airway response with a steroid, or injecting virus directly into the bloodstream to skip the lung, allowed productive infection of liver, kidney, spleen and brain. Infecting through the airway first protected those organs against a subsequent bloodstream challenge.\n\n### 150 words\n\nGolden hamsters infected intranasally with SARS-CoV-2 showed an interferon signature in all nine organs profiled, while infectious virus was largely confined to lung and olfactory bulb. Whole blood carried an interferon stimulated gene signature without interferon transcripts of its own, and a fibroblast bioassay detected circulating interferon by one day after infection. Dexamethasone delayed airway interferon stimulated gene induction and phagocyte infiltration without changing early lung titers, and under that treatment virus appeared in liver, spleen, olfactory bulb and gastrointestinal tract, with low level viremia detectable only after amplification on permissive cells. Intravenous inoculation bypassed the airway and produced productive infection of kidney, liver, spleen, heart and gastrointestinal tract, confirmed in kidney by nucleocapsid staining of parenchymal and endothelial cells. Circulating interferon was present only in animals with lung titers, and prior airway infection reduced distal viral loads after an intravenous challenge, indicating that airway derived interferon restricts tropism." }, "discoveries": [ "claim-13" ], "relationships": [ { "from": "2023-carrau-delayed-engagement-of-host-defense", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2023-carrau-delayed-engagement-of-host-defense" }, { "from": "2023-carrau-delayed-engagement-of-host-defense", "to": "2020-blanco-melo-imbalanced-host-response-to-sars-c", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2023-carrau-delayed-engagement-of-host-defense" } ], "canonical_url": "https://tenoeverlab.us/publications/2023-carrau-delayed-engagement-of-host-defense/", "controlled_vocabulary": { "pathogens": [ "sars-cov-2" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "immunofluorescence-microscopy", "flow-cytometry", "in-vivo-infection-route", "immunohistochemistry", "virus-amplification-assay", "interferon-bioassay", "immunosuppression-treatment" ] } }, { "id": "2023-oishi-archaeal-kink-turn-binding-protein", "slug": "2023-oishi-archaeal-kink-turn-binding-protein", "url": "/publications/2023-oishi-archaeal-kink-turn-binding-protein/", "title": "Archaeal Kink-Turn Binding Protein Mediates Inhibition of Orthomyxovirus Splicing Biology", "authors": [ "Kohei Oishi", "Daniel Blanco-Melo", "Andrew P. Kurland", "Jeffrey R. Johnson", "Benjamin R. tenOever" ], "author_count": 5, "first_author": "Kohei Oishi", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 5, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2023, "journal": "Journal of Virology", "volume": "97", "issue": "4", "pages": "e01813-22", "doi": "10.1128/jvi.01813-22", "doi_url": "https://doi.org/10.1128/jvi.01813-22", "pmid": "36943134", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/36943134/", "pmcid": "PMC10134859", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10134859/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10134859/pdf/", "publication_type": "primary research", "declared_conflicts": "Benjamin tenOever is a co-founder of Archean Biologics and is named as an author on patent WO2022061216A1 covering commercialization of L7Ae, as declared in the paper.", "research_areas": [ "influenza-genome-regulation", "small-rna-antiviral-defense" ], "themes": [ "evolution-of-antiviral-defense", "splicing-and-temporal-control" ], "pathogens": [ "influenza A virus", "influenza B virus", "infectious salmon anemia virus", "vesicular stomatitis virus" ], "viral_families": [ "Orthomyxoviridae", "Rhabdoviridae" ], "host_species": [ "human", "dog", "African green monkey" ], "technologies": [ "expression screening of codon-optimized proteins", "doxycycline-inducible lentiviral expression", "influenza reverse genetics", "plaque assay", "quantitative RT-PCR", "RNA sequencing", "mass spectrometry proteomics", "UV crosslinking immunoprecipitation sequencing", "serial passage selection", "chimeric minigene reporters", "alanine scanning mutagenesis" ], "biological_systems": [ "HEK293T cells", "A549 cells", "MDCK cells", "Vero E6 cells" ], "key_concepts": [ "L7Ae", "kink-turn RNA structure", "orthomyxovirus splicing", "M2 and NS2 splice products", "noncanonical splicing", "splicing-independent virus", "escape mutant fitness cost", "L30 protein family", "3 prime splice acceptor site", "molecular timer of infection" ], "keywords": [ "L7Ae", "kink turn", "influenza virus", "splicing", "M2", "NS2", "Orthomyxoviridae", "isavirus", "archaea", "antiviral" ], "one_sentence_contribution": "Archaeal members of the L30 protein family, but not their orthologues from any other domain of life, block production of the spliced influenza A virus, influenza B virus and isavirus transcripts without measurably disturbing host splicing, identifying a shared and apparently noncanonical feature of orthomyxovirus splicing.", "summary_25": "A protein borrowed from archaea blocks the splicing step that influenza and related viruses depend on, without disturbing the same process in human cells.", "summary_75": "Influenza viruses make two of their proteins by splicing, and the pace of that splicing schedules their replication. Screening archaeal RNA binding proteins identified L7Ae, which eliminated both spliced products of influenza A virus while leaving the unspliced ones intact. Orthologues from every other domain of life failed to do this. L7Ae also suppressed influenza B virus and a salmon orthomyxovirus, and virus passaged under selection could not escape without a severe fitness penalty.", "summary_150": "Orthomyxoviruses splice segment 7 and segment 8 transcripts to produce M2 and NS2, and the slow accumulation of these products separates early from late infection. A screen of codon-optimized archaeal RNA binding proteins identified the kink-turn binding protein L7Ae as eliminating M2 and NS2 while M1 and NS1 accumulated. The effect reproduced without infection or viral polymerase, required nuclear L7Ae, and disappeared against a virus engineered to make both products through 2A peptides rather than splicing. Only archaeal orthologues had this activity, with the archaea-defining residues contributing most, while substitutions that abolish canonical box C and D binding did not. Host transcriptome, proteome and an intron-containing reporter were largely unaffected. Chimeric minigenes localized sensitivity to the 3 prime splice acceptor region and a short adjoining coding stretch in both segments, but crosslinking yielded no footprint. Twenty passages under selection produced only a variant that raises splicing efficiency at substantial fitness cost.", "citation": "Oishi K, Blanco-Melo D, Kurland AP, Johnson JR, tenOever BR. Archaeal Kink-Turn Binding Protein Mediates Inhibition of Orthomyxovirus Splicing Biology. Journal of Virology. 2023. Volume 97, issue 4, article e01813-22. DOI 10.1128/jvi.01813-22. PMID 36943134. PMCID PMC10134859.", "sections": { "Citation": "Oishi K, Blanco-Melo D, Kurland AP, Johnson JR, tenOever BR. Archaeal Kink-Turn Binding Protein Mediates Inhibition of Orthomyxovirus Splicing Biology. Journal of Virology. 2023. Volume 97, issue 4, article e01813-22. DOI 10.1128/jvi.01813-22. PMID 36943134. PMCID PMC10134859.", "One-sentence contribution": "Archaeal members of the L30 protein family, but not their orthologues from any other domain of life, block production of the spliced influenza A virus, influenza B virus and isavirus transcripts without measurably disturbing host splicing, identifying a shared and apparently noncanonical feature of orthomyxovirus splicing.", "Executive summary": "Orthomyxoviruses replicate in the nucleus and expand their coding capacity by splicing segment 7 and segment 8 transcripts, producing M2 and NS2 respectively. The slow accumulation of these spliced products acts as a timer that separates early and late events of infection. How this splicing is executed has remained unclear, and it has never been reconstituted in vitro, which has led to proposals that it is noncanonical.\n\nThe authors screened a synthesized library of codon-optimized archaeal RNA binding proteins for effects on influenza A virus protein accumulation. One member, the kink-turn binding protein L7Ae, selectively eliminated M2 and NS2 while M1 and NS1 accumulated. The effect reproduced without infection and without the viral polymerase, required nuclear localization of L7Ae, and was lost against a recombinant virus in which M2 and NS2 are made from 2A peptide constructs rather than by splicing. In inducible canine kidney cells, L7Ae reduced titers of several influenza A virus subtypes by two to three logs, influenza B virus by one to two logs, and blocked splice product formation in infectious salmon anemia virus, while leaving vesicular stomatitis virus untouched.\n\nSpecificity was assessed against an intron-containing reporter, host RNA sequencing and proteomics, all of which showed little change. Mapping localized the sensitive element to the 3 prime splice acceptor region and a short stretch of adjoining open reading frame, but crosslinking immunoprecipitation did not resolve a footprint. Twenty passages under selection produced no true escape, only a segment 8 variant that raises splicing efficiency at a severe fitness cost.", "Scientific context": "Orthomyxoviruses carry segmented negative sense genomes and complete replication in the nucleus, using splicing and ribosomal frameshifting to expand coding capacity. M2, from segment 7, is the ion channel required for uncoating. NS2, also called the nuclear export protein, exports viral ribonucleoproteins, modulates polymerase activity through enhanced complementary RNA production, and contributes to small viral RNA generation. Slow accumulation of the spliced products has been described as a molecular timer coordinating the infection cycle, a concept developed in part in earlier work from this laboratory. Splicing is ubiquitous in eukaryotes, absent in bacteria, and present in unusual forms in archaea, and influenza virus splicing has been argued to be noncanonical and possibly post-transcriptional, having never been reconstituted in vitro. The paper's stated entry point is that screening archaeal RNA binding proteins might reveal something about that mechanism.", "Central question": "Do any archaeal RNA binding proteins interfere with the splicing of influenza A virus transcripts, and if so, what does the specificity of that interference reveal about how orthomyxoviruses execute splicing and whether the feature is shared across the family?", "Experimental strategy": "The screen is the conceptual core. Rather than perturbing host factors, the authors introduce proteins from a domain of life whose RNA biology diverges from the host, on the reasoning that an archaeal protein might recognize a structural feature of viral RNA that the host machinery treats differently. Reading out four viral proteins at once, the unspliced M1 and NS1 alongside the spliced M2 and NS2, makes a splicing-specific hit distinguishable from a general replication defect from the outset.\n\nThe hit was then decomposed. Reconstitution with polymerase and viral RNA, and then with polymerase II driven plasmids, removes infection and then the viral polymerase from the requirement. Intronless M2 and NS2 constructs separate an RNA processing effect from protein instability. A recombinant virus in which the same products are made through a 2A peptide provides the decisive genetic test, since a splicing-directed inhibitor should have no effect on it. Orthologue panels across bacteria, protists, archaea, plants, arthropods and vertebrates convert the finding into a statement about lineage specificity, and truncation and alanine substitution map the requirement onto the protein.\n\nSpecificity for the virus was tested at three levels, an intron-containing fluorescent reporter, host transcriptome by RNA sequencing, and host proteome by mass spectrometry. Mapping on the RNA side used scanning 30 nucleotide scrambles across segment 7, chimeras between the L7Ae-sensitive viral minigene and the L7Ae-insensitive beta-globin reporter, and crosslinking immunoprecipitation sequencing. Serial passage under selection tests whether the virus can evolve around the inhibitor and at what cost.", "Key findings": "1. In a screen of codon-optimized archaeal RNA binding proteins expressed in fibroblasts and challenged with A/Puerto Rico/8/34, RBP14 and RBP16 of the RNase III family reduced viral proteins generally, while RBP11 reduced M2 and abolished NS2 despite accumulation of M1 and NS1 (Figure 1A). RBP11 is the L30 family member known in archaea as L7Ae.\n\n2. Reconstitution with the polymerase components, nucleoprotein and individual viral RNA segments showed that L7Ae from Archaeoglobus fulgidus blocked M2 and NS2 with little effect on neuraminidase, and raised M1 and NS1 correspondingly (Figure 1B). Polymerase II driven expression gave the same result, and intronless M2 and NS2 constructs were unaffected, which excludes protein instability as the explanation (Supplementary Figure 1A and 1B).\n\n3. Among L30 orthologues cloned from bacteria, protists, archaea, plants, arthropods and vertebrates, only the archaeal members inhibited M2 and NS2, and five additional archaeal L7Ae proteins all did so (Figure 1C and 1D, Supplementary Figure 1C to 1E).\n\n4. In doxycycline-inducible canine kidney cells, L7Ae induction caused specific loss of M2 and NS2 during H1N1 infection, and reduced wild-type virus titers by approximately three logs, while a recombinant virus expressing M2 and NS2 through 2A peptides was unaffected in both protein expression and titer (Figure 2A to 2D). This is the central genetic demonstration that the target is splicing.\n\n5. L7Ae reduced titers of H1N1 A/California/04/2009, H1N1 A/Texas/36/91, H3N2 A/Panama/99/2007 and live-attenuated H5N1 A/Vietnam/1203/2004 by two to three logs, influenza B virus by one to two logs, and suppressed splice product formation by infectious salmon anemia virus, while having no effect on vesicular stomatitis virus (Figure 2E to 2J and Supplementary Figure 2D).\n\n6. L7Ae did not affect expression from an intron-containing DsRed reporter, changed only 18 host genes with a Pearson correlation of 0.985 across normalized read counts, and produced no significant proteome changes by mass spectrometry (Figure 3B to 3D). Among the few enriched transcripts were small Cajal body-specific RNAs carrying box C and D elements, which the authors suggest were stabilized by L7Ae binding. That suggestion is an interpretation.\n\n7. During infection, M2 and NS2 messenger RNA measured across the spliced junction fell by about 50 percent with L7Ae, while M1 and NS1 rose modestly at 6 hours (Figure 3E). Inhibition required nuclear L7Ae, since a nuclear export sequence tagged version lost activity (Figure 3F and 3G).\n\n8. Truncation showed the flexible alpha 1 and alpha 6 terminal helices are dispensable. Substitutions at lysines 37 and 79, which are known to be required for binding box C and D elements, did not abolish inhibition, while the archaea-defining residues isoleucine 88, glutamate 89 and valine 90 contributed most (Figure 4A to 4E). The authors read the retention of activity by the K37A and K79A mutant as evidence that the viral substrate is not a canonical kink turn.\n\n9. Scanning 30 nucleotide scrambles across segment 7 identified four constructs that abolished M2 on their own by destroying splice sites, the branch point or the pyrimidine tract, while all other scrambles remained L7Ae-sensitive (Figure 5A and 5B). Chimeras with the beta-globin reporter restored splicing in the presence of L7Ae only when the 3 prime splice site of segment 7 was replaced, not the 5 prime site, and deletion series narrowed the requirement to roughly 20 nucleotides of intron upstream of the 3 prime splice site plus about 9 nucleotides of the M2 open reading frame, with a similar result for segment 8 (Figure 5C to 5I).\n\n10. Crosslinking immunoprecipitation sequencing enriched viral RNA relative to control but produced no clear footprint, although the 3 prime splice sites of segments 7 and 8 were captured (Supplementary Figure 4). This is a negative result and the authors attribute it to a dynamic or transient substrate.\n\n11. Twenty passages under L7Ae selection yielded two segment 8 variants and no true escape. The C508A and G576A changes truncated NS1 prematurely. The G60A variant permitted low-level NS2 by increasing splicing efficiency rather than by losing L7Ae sensitivity (Figure 6A). An engineered virus with an optimal vertebrate 5 prime donor site in segment 8 also remained sensitive, and L7Ae still raised NS1 from that construct, which the authors take as further evidence that the 3 prime acceptor region is the point of engagement (Supplementary Figure 5A).\n\n12. In competition, the G60A variant was outcompeted by wild-type virus by passage 2 without L7Ae and dominated with L7Ae, and its titers did not exceed 100,000 plaque forming units per milliliter under either condition (Figure 6B and 6C). Resistance therefore carries a substantial fitness cost.", "Mechanistic model": "The study does not establish the molecular mechanism, and the authors state their account as a postulate. What the data establish is that an archaeal L30 family protein, acting in the nucleus, prevents maturation of the spliced orthomyxovirus transcripts without requiring the viral polymerase, without acting on the proteins themselves, and without detectable effect on host splicing, and that sensitivity maps to the 3 prime splice acceptor region plus a short adjoining coding stretch in both segment 7 and segment 8.\n\nThe proposed model is that orthomyxoviruses form a kink-turn-like structure during lariat formation, used to recruit host splicing components, and that archaeal L7Ae binds that structure and occludes it. Several observations complicate a simple canonical kink turn account, and the authors say so. No canonical kink turn was found in the viral genomes. Mutations that abolish canonical box C and D binding leave the inhibition intact, which points to a substrate that deviates from the canonical fold. Crosslinking did not yield a footprint, and published SHAPE-seq work has also failed to detect the M2 or NS2 lariat, so the authors argue the relevant structure is transient and forms only during splicing. The residues that matter most are the ones that distinguish archaeal L7Ae from its orthologues, which is why the specificity exists, but the RNA counterpart of those contacts is unidentified. The mapping data are consistent with direct engagement near the 3 prime acceptor site, and also with an indirect effect in which L7Ae occupancy elsewhere prevents assembly of the splicing complex at that site. The data do not distinguish these.", "Conceptual or technical advance": "A single heterologous protein now separates orthomyxovirus splicing from host splicing experimentally, which makes the long-standing claim that influenza virus splicing is noncanonical testable rather than inferential. The same tool works across three genera of the family, including a fish pathogen, so it also converts a feature described for influenza A virus into a family-level property. The finding that the virus cannot escape without either truncating NS1 or driving splicing efficiency up at a large fitness cost is informative about how tightly the timer is constrained. The authors propose therapeutic and transgenic animal applications. Those are prospects, and the reader should note the declared conflict of interest, since tenOever is a co-founder of Archean Biologics and an author of a patent covering commercialization of L7Ae.", "Relationship to the broader research program": "The paper builds on the laboratory's earlier work on segment 7 and segment 8 processing, in particular the finding that influenza A virus uses suboptimal splice sites to coordinate the timing of infection, published by Chua and colleagues in 2013, and on the splicing-independent recombinant virus constructed in that line of work, which supplies the decisive control here. The related question of what structural element in the NS segment governs this processing is pursued further in ongoing work in the laboratory on a conserved hairpin in segment 8. Category 3 synthesis, visible when this paper is set beside the earlier splicing work, is a sustained argument that viral splice site suboptimality is not incidental but is the mechanism by which the virus schedules its own replication cycle.", "Related publications": "- Chua and colleagues 2013, Cell Reports, on suboptimal splicing and the timing of influenza A virus infection. Predecessor and methodological foundation. Source of the timer concept and of the splicing-independent 2A recombinant virus used here as the key control.\n- Oishi and colleagues on the segment 8 conserved hairpin. Follow-up. Continues the search for the RNA element responsible for the processing behavior probed here.", "Limitations and boundaries": "Everything is done in cultured cells, predominantly HEK293T, A549 and MDCK lines, with no animal infection, so in vivo efficacy and tolerability of L7Ae expression are untested. L7Ae is delivered by transfection or by doxycycline-induced lentiviral expression, which is a strong and non-physiological expression regime rather than a therapeutic one. Host impact was assessed as transcriptome and proteome under induction in one cell line and a single intron-containing reporter, which does not exclude effects on specific splicing events, on small nucleolar and small Cajal body RNA function, or in other cell types, and the authors note it is unclear how the observed enrichment of those RNAs might affect host biology. The RNA element is localized to a region rather than identified, and the key crosslinking experiment returned no footprint, so direct binding to the proposed structure is not demonstrated. The proposed kink-turn-like structure is inferred from the identity of the protein and from mutational data, not observed. Escape selection was 20 passages from a single starting strain in one cell line, which bounds but does not exclude the emergence of resistance under other conditions. The potency measurements cover a small panel of laboratory strains, and the influenza B virus effect is smaller than the influenza A virus effect. The isavirus experiment reports loss of splice products and not titers. Finally, the authors declare a competing interest tied to commercialization of the protein studied here.", "Audience summaries": "### 25 words\n\nA protein borrowed from archaea blocks the splicing step that influenza and related viruses depend on, without disturbing the same process in human cells.\n\n### 75 words\n\nInfluenza viruses make two of their proteins by splicing, and the pace of that splicing schedules their replication. Screening archaeal RNA binding proteins identified L7Ae, which eliminated both spliced products of influenza A virus while leaving the unspliced ones intact. Orthologues from every other domain of life failed to do this. L7Ae also suppressed influenza B virus and a salmon orthomyxovirus, and virus passaged under selection could not escape without a severe fitness penalty.\n\n### 150 words\n\nOrthomyxoviruses splice segment 7 and segment 8 transcripts to produce M2 and NS2, and the slow accumulation of these products separates early from late infection. A screen of codon-optimized archaeal RNA binding proteins identified the kink-turn binding protein L7Ae as eliminating M2 and NS2 while M1 and NS1 accumulated. The effect reproduced without infection or viral polymerase, required nuclear L7Ae, and disappeared against a virus engineered to make both products through 2A peptides rather than splicing. Only archaeal orthologues had this activity, with the archaea-defining residues contributing most, while substitutions that abolish canonical box C and D binding did not. Host transcriptome, proteome and an intron-containing reporter were largely unaffected. Chimeric minigenes localized sensitivity to the 3 prime splice acceptor region and a short adjoining coding stretch in both segments, but crosslinking yielded no footprint. Twenty passages under selection produced only a variant that raises splicing efficiency at substantial fitness cost." }, "discoveries": [ "claim-02" ], "relationships": [ { "from": "2023-oishi-archaeal-kink-turn-binding-protein", "to": "2013-chua-influenza-a-virus-utilizes-subopti", "relationship": "methodological foundation", "evidence": "stated in the Related publications section of 2023-oishi-archaeal-kink-turn-binding-protein" } ], "canonical_url": "https://tenoeverlab.us/publications/2023-oishi-archaeal-kink-turn-binding-protein/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "vsv", "infectious-salmon-anemia-virus", "influenza-b-virus" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "reverse-genetics", "site-directed-mutagenesis", "mass-spectrometry-proteomics", "serial-passage", "inducible-expression", "splicing-reporter", "heterologous-protein-expression-screen", "clip-seq" ] } }, { "id": "2023-paget-stress-granules-are-shock-absorber", "slug": "2023-paget-stress-granules-are-shock-absorber", "url": "/publications/2023-paget-stress-granules-are-shock-absorber/", "title": "Stress granules are shock absorbers that prevent excessive innate immune responses to dsRNA", "authors": [ "Max Paget", "Cristhian Cadena", "Sadeem Ahmad", "Hai-Tao Wang", "Tristan X. Jordan", "Ehyun Kim", "Beechui Koo", "Shawn M. Lyons", "Pavel Ivanov", "Benjamin tenOever", "Xin Mu", "Sun Hur" ], "author_count": 12, "first_author": "Max Paget", "senior_authors": [ "Sun Hur" ], "corresponding_authors": [ "Sun Hur" ], "tenoever_position": 10, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2023, "journal": "Molecular Cell", "volume": "83", "issue": "7", "pages": "1180-1196.e8", "doi": "10.1016/j.molcel.2023.03.010", "doi_url": "https://doi.org/10.1016/j.molcel.2023.03.010", "pmid": "37028415", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/37028415/", "pmcid": "PMC10170497", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10170497/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10170497/pdf/", "publication_type": "primary research", "declared_conflicts": "Benjamin tenOever is a co-founder of Archean Biologics, as declared in the paper.", "research_areas": [ "innate-immune-signaling" ], "themes": [ "sensing-aberrant-rna" ], "pathogens": [ "Sendai virus", "influenza A virus", "vesicular stomatitis virus", "encephalomyocarditis virus" ], "viral_families": [ "Paramyxoviridae", "Orthomyxoviridae", "Rhabdoviridae", "Picornaviridae" ], "host_species": [ "human" ], "technologies": [ "CRISPR knockout cell lines", "immunofluorescence microscopy and colocalization analysis", "RNA sequencing", "RT-qPCR", "ELISA", "immunoblotting", "cell-free IRF3 dimerization assay", "Sytox and caspase activity cell death assays", "siRNA knockdown", "puromycin incorporation translation assay" ], "biological_systems": [ "U2OS cells", "A549 cells", "HeLa cells", "human bronchial epithelial cells", "G3BP1 and G3BP2 knockout cells", "UBAP2L knockout cells", "PKR knockout cells", "MAVS knockout cells", "RNase L knockout cells", "cell-free mitochondrial fraction assay" ], "key_concepts": [ "stress granules", "biomolecular condensates", "RIG-I-like receptors", "MAVS signaling", "immune-mediated apoptosis", "PKR", "OAS and RNase L", "ADAR1 deficiency", "self-derived double-stranded RNA", "negative feedback by apoptotic caspases" ], "keywords": [ "stress granules", "G3BP1", "UBAP2L", "dsRNA", "RIG-I", "MDA5", "MAVS", "apoptosis", "ADAR1", "innate immunity" ], "one_sentence_contribution": "Stress granules restrain rather than amplify double-stranded RNA sensing, and cells lacking the granule nucleators G3BP1 and G3BP2, UBAP2L or PKR respond to double-stranded RNA with excessive RIG-I-like receptor, PKR and OAS activation and MAVS-dependent apoptosis.", "summary_25": "Stress granules turn out to restrain double-stranded RNA sensing rather than promote it. Cells that cannot form them overreact and die by immune-triggered apoptosis.", "summary_75": "Cells detect double-stranded RNA through several sensors and also build stress granules, condensates once thought to serve as signaling platforms for those sensors. Using three separate genetic routes to granule deficiency, this study finds the opposite. Without granules, sensing is hyperactive, cells secrete excess tumor necrosis factor alpha and die by caspase-dependent apoptosis that requires MAVS but not interferon. The same protection applies to self-derived double-stranded RNA arising from ADAR1 deficiency.", "summary_150": "Stress granules had been proposed as platforms that promote RIG-I-like receptor signaling, though double-stranded RNA itself is excluded from them and granule-disrupting drugs do not impair signaling. Using cells lacking G3BP1 and G3BP2, UBAP2L or PKR, this study finds that granules instead restrain double-stranded RNA sensing. All three backgrounds showed stronger transcriptional and cytokine responses, higher IRF3 activation and greater MAVS signaling potential in a cell-free assay, along with hyperactive PKR and OAS-RNase L arms. Granule-deficient cells underwent caspase-dependent apoptosis that was largely rescued by deleting MAVS but not IRF3 and was relieved by blocking tumor necrosis factor alpha. Restoring granules in PKR-deficient cells with thapsigargin or starvation suppressed signaling, tying the effect to granules rather than to the route that makes them. During infection, granule loss both enhanced signaling and, independently of MAVS, raised viral protein per cell. The molecular mechanism is not established.", "citation": "Paget M, Cadena C, Ahmad S, Wang HT, Jordan TX, Kim E, Koo B, Lyons SM, Ivanov P, tenOever B, Mu X, Hur S. Stress granules are shock absorbers that prevent excessive innate immune responses to dsRNA. Molecular Cell. 2023. Volume 83, Issue 7, pages 1180-1196.e8. DOI 10.1016/j.molcel.2023.03.010. PMID 37028415. PMCID PMC10170497. Max Paget and Cristhian Cadena are designated as having contributed equally. Sun Hur is the lead contact and sole corresponding author.", "sections": { "Citation": "Paget M, Cadena C, Ahmad S, Wang HT, Jordan TX, Kim E, Koo B, Lyons SM, Ivanov P, tenOever B, Mu X, Hur S. Stress granules are shock absorbers that prevent excessive innate immune responses to dsRNA. Molecular Cell. 2023. Volume 83, Issue 7, pages 1180-1196.e8.\n\nDOI 10.1016/j.molcel.2023.03.010. PMID 37028415. PMCID PMC10170497.\n\nMax Paget and Cristhian Cadena are designated as having contributed equally. Sun Hur is the lead contact and sole corresponding author.", "One-sentence contribution": "Stress granules restrain rather than amplify double-stranded RNA sensing, and cells lacking the granule nucleators G3BP1 and G3BP2, UBAP2L or PKR respond to double-stranded RNA with excessive RIG-I-like receptor, PKR and OAS activation and MAVS-dependent apoptosis.", "Executive summary": "Double-stranded RNA is detected by RIG-I-like receptors, by protein kinase R and by the oligoadenylate synthetases, and it also triggers the assembly of stress granules, cytoplasmic condensates nucleated by G3BP1 and G3BP2 together with UBAP2L. Stress granules had been proposed to serve as signaling platforms for RIG-I-like receptors, based on the concentration of those receptors in granules together with viral RNA and on reduced interferon induction when G3BPs were knocked down. Other observations sat uneasily with that model, including the exclusion of double-stranded RNA from granules and the failure of granule-disrupting agents to impair signaling.\n\nThis study approaches the question with three independent genetic routes to granule deficiency, deleting G3BP1 and G3BP2, deleting UBAP2L, or deleting PKR, and with stimuli that decouple granule formation from double-stranded RNA sensing.\n\nAll three granule-deficient backgrounds showed stronger signaling in response to defined 162 base pair 5-prime triphosphate double-stranded RNA, measured by transcriptome, cytokine messenger RNA and protein, IRF3 phosphorylation and nuclear translocation, and MAVS activity in a cell-free assay. PKR and the OAS-RNase L arm were also hyperactive. Granule-deficient cells underwent pronounced caspase-dependent apoptosis, which was largely rescued by deleting MAVS but not by deleting IRF3, and which was relieved by blocking tumor necrosis factor alpha. The same protection extended to endogenous double-stranded RNA accumulating after ADAR1 knockdown. During infection, granule-deficient cells showed both stronger signaling and, independently of MAVS, higher viral protein per infected cell.", "Scientific context": "Double-stranded RNA was long regarded as a purely viral product, generated by RNA-dependent RNA polymerization or by convergent bidirectional transcription of DNA genomes, but the paper notes that dysregulated cellular processes can also produce it and that responses to double-stranded RNA underlie pathologies from autoimmunity to neurodegeneration and metabolic disease. RIG-I and MDA5 recognize double-stranded RNA, multimerize, and induce MAVS multimerization, leading to IRF3 and NF-kappaB activation and induction of type I interferons and other antiviral genes.\n\nDouble-stranded RNA also triggers stress granules, a conserved condensate response also induced by heat shock and oxidative stress. Kinases including PKR phosphorylate eIF2 alpha and suppress global translation, and stalled ribosome-messenger RNA complexes aggregate with cytoplasmic proteins including the nucleators G3BP1, G3BP2 and UBAP2L.\n\nThe paper states that the physiological functions of granule formation remain unclear. Granules were initially thought to be sites of translational suppression, a view recent work has argued against. In innate immunity they were proposed to be signaling scaffolds for RIG-I-like receptors, supported by receptor and viral RNA concentration in granules and by reduced interferon induction on G3BP knockdown, and consistent with the frequent targeting of granules by viruses. The paper then lists the contrary evidence. Double-stranded RNA is excluded from granules even though some viral RNAs are enriched. Granule-disrupting agents such as cycloheximide do not impair signaling. Stressors such as arsenite and heat shock produce granules with receptor colocalization but without receptor activation. And granules have been reported to suppress other innate pathways including the NLRP3 inflammasome and MAPK signaling.", "Central question": "Do stress granules serve as platforms that promote RIG-I-like receptor signaling in response to double-stranded RNA, or do they instead constrain the magnitude of that response, and what are the consequences for cell survival and for viral replication?", "Experimental strategy": "The design addresses two problems at once, the ambiguity of correlative colocalization data and the confounding of granule loss with loss of other functions of the nucleator proteins.\n\nThe first is handled by measuring signaling output at several levels rather than inferring it from localization, including whole transcriptome, cytokine messenger RNA and secreted protein, IRF3 phosphorylation and nuclear translocation, and MAVS activity assayed cell-free by mixing isolated mitochondrial fractions with a common pool of resting cytosolic extract and radiolabeled IRF3. That last assay is important because it reads the signaling potential of MAVS itself against a fixed downstream background.\n\nThe second is handled by using three genetically distinct routes to granule deficiency. G3BP1 and G3BP2 and UBAP2L are nucleators, PKR is not a nucleator but supplies the translational arrest that granule formation requires. The three have different additional functions, so a phenotype shared by all three is more plausibly attributable to granules. The paper is explicit that PKR alone would be a poor model, since it affects both translation and granule formation and is itself subject to granule-mediated feedback.\n\nA further decoupling exploits the fact that PKR-deficient cells fail to make granules in response to double-stranded RNA but still make them in response to thapsigargin, which acts through PERK, or to nutrient starvation. Adding those stressors to PKR-deficient cells restores granules without changing the double-stranded RNA stimulus, which tests whether granules as such suppress signaling rather than whether the specific route to them matters.\n\nThe stimulus itself is a defined in vitro transcribed 162 base pair double-stranded RNA bearing 5-prime triphosphate, chosen so that receptors are stimulated potently without the confounding effect of viral antagonists. Complementation of G3BPs back into knockout cells provides the gain-of-function control.\n\nDownstream, epistasis is used to identify what kills the cells. Knockouts of MAVS, IRF3, PKR and RNase L, plus inhibitors of TBK1, JAK, NF-kappaB and caspases and blocking antibody to tumor necrosis factor alpha, partition the death phenotype among the arms of the response.\n\nFinally, the question is asked in two more settings, during infection with four viruses selected to engage RIG-I or MDA5, and with endogenous double-stranded RNA produced by ADAR1 knockdown with interferon beta priming, which tests whether the protective role extends beyond exogenous stimulation.", "Key findings": "1. Transfected 162 base pair 5-prime triphosphate double-stranded RNA induced granules in U2OS cells that depended on G3BPs and were cycloheximide sensitive, and that were enriched for RIG-I, MAVS, TRAF proteins and TBK1, along with the mitochondrial proteins COXIV and NIX (Figure 1A, Figure S1B, Figure S1C). Cy5-labeled double-stranded RNA showed minimal granule localization whether delivered by lipid transfection or electroporation (Figure 1A). The observation is that signaling machinery concentrates in granules while the ligand does not, which the authors read as inconsistent with granules being sites of receptor activation.\n\n2. G3BP-deficient cells showed enhanced antiviral signaling at 6 hours by transcriptome (Figure 1B, Figure S2A), by IFNB, IL6 and RANTES messenger RNA (Figure 1C) and secreted protein (Figure 1D), across all doses tested (Figure 1E). Complementing G3BPs restored granules and suppressed signaling (Figure S2C).\n\n3. IRF3 phosphorylation and nuclear IRF3 were higher in G3BP-deficient cells at 6 hours but declined sharply by 24 hours, mirroring IFNB messenger RNA, while RANTES and IL6 remained elevated at both times (Figure 1C, Figure 1F, Figure 1G). The divergent time course of the IRF3 arm is what the authors later attribute to caspase feedback.\n\n4. In the cell-free assay, mitochondrial fractions from stimulated cells activated IRF3 dimerization, this required MAVS, and fractions from G3BP-deficient cells were more potent than wild type (Figure 1H, Figure 1I). This places the difference at the level of MAVS signaling potential rather than only at downstream readouts.\n\n5. The hyperactivation generalized to A549, HeLa and human bronchial epithelial cells, to both delivery methods, and to double-stranded RNAs of different lengths and sequences (Figure S3A to Figure S3G). Signaling driven by gain-of-function MDA5, gain-of-function RIG-I or STING, which occurs without granule formation, was unaffected by G3BP loss (Figure S3H, Figure S3I), which the authors read as indicating the effect is specific to granule-forming conditions. Basal transcriptomes showed no consistent pattern of baseline inflammation in G3BP-deficient cells (Figure S3E).\n\n6. PKR-deficient and UBAP2L-deficient cells formed G3BP1 foci, but these were smaller, less frequent and lacked MAVS or TIAR enrichment, distinguishing them from conventional granules (Figure 2A, Figure 2B). Both backgrounds nonetheless showed hyperactive signaling at 6 hours (Figure 2C, Figure 2D, Figure 2E). At 24 hours the PKR-deficient cells diverged from the other two, with IL6 and RANTES falling below wild type, which the authors attribute to PKR affecting translation as well as granules, noting that translation itself positively influences immune signaling (Figure 2F).\n\n7. In PKR-deficient cells, thapsigargin or nutrient starvation restored granules containing RIG-I, MAVS and TIAR and reduced signaling at 6 hours, while neither stressor suppressed signaling in wild-type cells where double-stranded RNA alone already makes granules (Figure 2G, Figure 2H, Figure 2I). The suppression therefore tracks with granule presence rather than with the route to granule formation.\n\n8. PKR, OAS3 and RNase L were enriched in granules (Figure 3B, Figure 3C). PKR phosphorylation and ATF4 were higher in G3BP-deficient cells (Figure 3D) and ribosomal RNA integrity was more degraded, indicating higher RNase L activity (Figure 3E). Deleting MAVS partly rescued RNA integrity, so part of the PKR and OAS hyperactivation is secondary to enhanced interferon-driven induction of those sensors. Granules therefore suppress all three arms.\n\n9. G3BP-deficient cells underwent pronounced cell death after double-stranded RNA at all doses, by detachment, Sytox uptake, caspase-3 and caspase-7 activity and live-dead staining (Figure 4A to Figure 4C, Figure S4A), while etoposide and staurosporine killed both genotypes equally (Figure 4D), so the hypersensitivity is stimulus specific. UBAP2L-deficient and PKR-deficient cells behaved similarly (Figure 4E), and the phenotype held in A549, HeLa and bronchial epithelial cells (Figure S4B to Figure S4H).\n\n10. Death showed apoptotic morphology, PARP and caspase-3 cleavage, and activation of caspase-8 and caspase-9 but not caspase-1 (Figure 4F to Figure 4H, Figure S4I). A pan-caspase inhibitor blocked death and PARP cleavage completely, while pyroptosis and necroptosis inhibitors did not (Figure 4I, Figure 4J, Figure S4J).\n\n11. The pan-caspase inhibitor restored IRF3 phosphorylation and IFNB messenger RNA at 24 hours without affecting them at 6 hours, in both U2OS and A549 G3BP-deficient cells, and had no effect in wild-type cells (Figure 4K to Figure 4M). Clear caspase-dependent cleavage of RIG-I, MAVS and IRF3 was not observed (Figure S4K). The interpretation offered is that caspase activation, rather than cleavage of any one identified substrate, produces the late decline in the IRF3 arm.\n\n12. Deleting MAVS largely rescued viability in G3BP-deficient U2OS and A549 cells and reduced caspase cleavage (Figure 5A, Figure 5B, Figure S5A), whereas deleting IRF3 did not rescue viability despite abolishing IFNB induction, and TBK1 or JAK inhibitors did not help (Figure 5A, Figure 5C, Figure S5B). Death is therefore MAVS dependent but IRF3 and interferon independent.\n\n13. Pro-apoptotic genes including TNF, FAS and TNFRSF10B were hyperinduced in a MAVS-dependent but IRF3-independent way (Figure 5C, Figure 5D), tumor necrosis factor alpha secretion was markedly elevated (Figure 5E), and blocking antibody to it significantly relieved death (Figure 5F). NF-kappaB inhibitors reduced both TNF expression and death (Figure S5C, Figure S5D). Tumor necrosis factor alpha alone did not kill cells (Figure S5E), so the authors conclude it cooperates with other factors rather than acting as a sufficient death signal.\n\n14. Deleting PKR or RNase L partially relieved death in G3BP-deficient U2OS cells, less completely than deleting MAVS (Figure 5G, Figure 5H). In A549 cells, deleting RNase L rescued significantly while deleting PKR did not (Figure S5F, Figure S5G), which the authors present as cell-type variation. They also note that deleting PKR increases death in a wild-type background while decreasing it in a G3BP-deficient background, and interpret this opposite behavior as reflecting granule loss in the first case and relief of translational inhibition in the second.\n\n15. Across Sendai virus, influenza A virus lacking NS1, the M51R variant of vesicular stomatitis virus and encephalomyocarditis virus, granule-deficient cells mounted stronger signaling and underwent more death, both predominantly MAVS dependent (Figure 6A to Figure 6G, Figure S6).\n\n16. Cell-to-cell spread of the three viruses with available antibodies was more restricted in G3BP-deficient cells, and this effect required MAVS, consistent with hyperactive signaling limiting spread. Unexpectedly, viral protein per infected cell was higher in G3BP-deficient cells and this was independent of MAVS (Figure 6H to Figure 6J, Figure S7A to Figure S7C), matching higher overall viral messenger RNA (Figure S7E to Figure S7H). The authors read this as two separable functions, receptor-dependent suppression of spread and receptor-independent restriction of replication.\n\n17. ADAR1 knockdown with interferon beta priming produced granules and signaling in wild-type cells, and stronger signaling in G3BP-deficient cells with or without priming (Figure 7A, Figure 7B). Death was greater in G3BP-deficient cells, predominantly MAVS dependent and partly PKR and RNase L dependent, and was relieved by blocking tumor necrosis factor alpha or by pan-caspase inhibition (Figure 7C, Figure 7D). UBAP2L-deficient cells were likewise hypersensitive (Figure 7E). The protective role therefore extends to self-derived double-stranded RNA.", "Mechanistic model": "The authors state in their own limitations section that it is currently unclear precisely how stress granules regulate either the immune response or viral replication, so the molecular mechanism is not established.\n\nWhat the data support is a functional model. Stress granules act as a buffer or shock absorber that slows the rate at which double-stranded RNA sensing ramps up and keeps its magnitude below a threshold at which the cell kills itself. Without granules, RIG-I-like receptors, PKR and the oligoadenylate synthetases are all more strongly activated, the MAVS arm drives NF-kappaB-dependent induction and secretion of tumor necrosis factor alpha along with other pro-apoptotic genes, and caspase-dependent apoptosis follows. The interferon arm through IRF3 does not contribute to that death and instead shows a spike followed by a sharp decline caused by caspase-dependent negative feedback.\n\nDirectly demonstrated are the hyperactivation of all three sensing arms in granule-deficient cells across three genetic backgrounds and several cell types, the increased MAVS signaling potential measured cell-free, the MAVS dependence and IRF3 independence of the death phenotype, the role of tumor necrosis factor alpha established by blocking antibody and by NF-kappaB inhibition, the caspase dependence of death established by inhibitor and cleavage markers, the caspase dependence of the late IRF3 decline, and the extension of all of this to infection and to ADAR1 deficiency.\n\nHow granules exert the suppression is proposed rather than shown. The authors offer that immune molecules appear to be recruited to granules independent of their activation state, which may exert a sequestration effect that retards activation, and they raise as an alternative that transient transit through granules may alter signaling activity through post-translational modification or association with inhibitory molecules. They also note that they do not know what fraction of signaling molecules is localized within granules at any time. The identification of tumor necrosis factor alpha as a contributor rather than a sufficient cause is explicitly stated, since the cytokine alone did not kill cells.\n\nThe reconciliation with earlier reports that granules amplify signaling is offered as a hypothesis. The authors propose that the IRF3 and interferon axis, commonly used as the single readout of receptor signaling, behaves non-monotonically in granule-deficient cells because of caspase feedback, and that this plus differences in how granules were disrupted may account for conflicting results in the literature.\n\nThe authors also flag, in their limitations, that other biological processes affected in common by all three genetic perturbations and all three chemical perturbations could in principle contribute to the observed effects.", "Conceptual or technical advance": "The study reverses the prevailing reading of a correlation. Concentration of sensing machinery inside a condensate had been taken as evidence of a signaling platform, and this work shows that the same colocalization is compatible with, and here accompanied by, suppression. The exclusion of the ligand from granules while the receptors are enriched makes that argument concrete.\n\nMethodologically, the combination of three genetic routes to granule deficiency with stressors that restore granules without changing the double-stranded RNA stimulus is a way to isolate the contribution of a condensate from the other functions of the proteins that build it. The PKR-deficient background in particular becomes an experimental instrument, because it separates the double-stranded RNA stimulus from granule formation in a way that pharmacological disruption cannot.\n\nThe finding also reframes what a condensate can be for. Rather than assigning granules an antiviral or proviral label, the study reports two effects that both preserve the cell, dampening a potentially lethal immune response and restricting viral replication through a receptor-independent route, and proposes homeostasis as the common denominator. The extension to ADAR1 deficiency connects the same mechanism to autoinflammatory pathology driven by self-derived double-stranded RNA.", "Relationship to the broader research program": "This study is led by the Hur laboratory at Harvard, and the author contributions statement records that the tenOever contribution, together with three other authors, was provision of reagents. It is a collaborative paper in which the tenOever laboratory supplied material rather than directing the work.\n\nIts subject matter nonetheless intersects several recurring interests in the tenOever corpus, including double-stranded RNA sensing during virus infection, the magnitude and control of the interferon response, and the use of engineered viruses such as influenza A virus lacking NS1 as tools that strip away viral antagonism. The question of what sets the ceiling on an antiviral response, as opposed to what initiates it, is a thread that appears elsewhere in the corpus, but drawing that connection formally is category 3 synthesis and would require the other records to support it.", "Related publications": "- Kedersha, Anderson, Ivanov and colleagues, on the molecular mechanisms of stress granule assembly and disassembly. Relationship methodological foundation from collaborating laboratories, cited as reference 14, with Ivanov and Lyons as co-authors here.\n- Onomoto and colleagues, and related work proposing stress granules as antiviral signaling platforms for RIG-I-like receptors. Relationship predecessor from other laboratories, cited as references 25 and 26, and the model this study argues against.\n- Yoo and colleagues, and related reports questioning whether granules are sites of receptor activation, including the exclusion of double-stranded RNA from granules and the failure of cycloheximide to impair signaling. Relationship predecessor from other laboratories, cited as references 30 and 31.\n- Work on ADAR1 deficiency leading to endogenous double-stranded RNA accumulation and aberrant MDA5, PKR and OAS activation. Relationship predecessor from other laboratories, cited as references 60 to 66, and the basis for the self-RNA experiments here.\n- Prior work from the Hur laboratory on cooperative MDA5 filament assembly and on MAVS multimerization. Relationship methodological foundation, cited as references 10 to 13, and the source of the cell-free IRF3 dimerization assay used here.", "Limitations and boundaries": "The authors state their own limitations plainly. It is unclear precisely how granules regulate either the immune response or viral replication, they do not know what fraction of signaling molecules resides in granules, and it remains possible that processes other than granules are commonly affected by all three genetic and all three chemical perturbations and contribute to the results.\n\nAll experiments are in human immortalized or primary cell lines, principally the osteosarcoma line U2OS with confirmation in A549, HeLa and bronchial epithelial cells. There is no animal work, so the proposed relevance to systemic inflammation, immunopathology and autoinflammatory disease is an extrapolation from cell-intrinsic observations.\n\nGranule deficiency is produced by complete genetic loss of nucleators or of PKR, which is a more absolute perturbation than any physiological modulation of granule assembly, and the knockout lines have been without these proteins before the stimulus is applied. The complementation experiment restores G3BPs and suppresses signaling, which supports the assignment, but the other backgrounds are not complemented.\n\nThe three granule-deficient backgrounds are not equivalent. PKR-deficient and UBAP2L-deficient cells still form G3BP1 foci, so the comparison is between granules of different composition and size rather than between presence and absence. The PKR background additionally alters translation and diverges from the others at 24 hours, and the authors caution against using it as the sole model.\n\nConclusions differ between cell types in ways the paper does not resolve. Deleting RNase L rescued viability in A549 cells while deleting PKR did not, the reverse of part of the U2OS result, and deleting PKR has opposite effects on death depending on whether G3BPs are present.\n\nThe apoptosis mechanism is only partly resolved. Tumor necrosis factor alpha contributes but is not sufficient, deleting MAVS rescues largely but not completely and reduces caspase cleavage but not to completion, and no caspase substrate accounting for the late IRF3 decline was identified, with cleavage of RIG-I, MAVS and IRF3 not observed.\n\nThe principal stimulus is a defined synthetic double-stranded RNA delivered by transfection or electroporation, which is chosen deliberately to avoid viral antagonists but does not reproduce the location, quantity or kinetics of double-stranded RNA generated during replication. The paper also notes that its poly(IC)-triggered granules behaved differently from published reports and attributes this to reported variability among commercial poly(IC) reagents, which bounds comparison with that literature.\n\nVirological readouts rely on immunofluorescence for spread and protein per cell using antibodies available for three of the four viruses, with no antibody for encephalomyocarditis virus, and infectious titer measurements are not the primary readout.\n\nThe ADAR1 experiments use transient small interfering RNA knockdown with interferon beta priming rather than genetic loss, so residual enzyme and the priming condition are part of the system.", "Audience summaries": "### 25 words\n\nStress granules turn out to restrain double-stranded RNA sensing rather than promote it. Cells that cannot form them overreact and die by immune-triggered apoptosis.\n\n### 75 words\n\nCells detect double-stranded RNA through several sensors and also build stress granules, condensates once thought to serve as signaling platforms for those sensors. Using three separate genetic routes to granule deficiency, this study finds the opposite. Without granules, sensing is hyperactive, cells secrete excess tumor necrosis factor alpha and die by caspase-dependent apoptosis that requires MAVS but not interferon. The same protection applies to self-derived double-stranded RNA arising from ADAR1 deficiency.\n\n### 150 words\n\nStress granules had been proposed as platforms that promote RIG-I-like receptor signaling, though double-stranded RNA itself is excluded from them and granule-disrupting drugs do not impair signaling. Using cells lacking G3BP1 and G3BP2, UBAP2L or PKR, this study finds that granules instead restrain double-stranded RNA sensing. All three backgrounds showed stronger transcriptional and cytokine responses, higher IRF3 activation and greater MAVS signaling potential in a cell-free assay, along with hyperactive PKR and OAS-RNase L arms. Granule-deficient cells underwent caspase-dependent apoptosis that was largely rescued by deleting MAVS but not IRF3 and was relieved by blocking tumor necrosis factor alpha. Restoring granules in PKR-deficient cells with thapsigargin or starvation suppressed signaling, tying the effect to granules rather than to the route that makes them. During infection, granule loss both enhanced signaling and, independently of MAVS, raised viral protein per cell. The molecular mechanism is not established." }, "discoveries": [ "claim-06" ], "relationships": [], "canonical_url": "https://tenoeverlab.us/publications/2023-paget-stress-granules-are-shock-absorber/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "vsv", "sendai-virus", "emcv" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "immunofluorescence-microscopy", "sirna-knockdown", "immunoblotting", "elisa", "in-vitro-reconstitution", "crispr-knockout", "cell-death-assays", "translation-measurement" ] } }, { "id": "2023-serafini-sars-cov-2-airway-infection-result", "slug": "2023-serafini-sars-cov-2-airway-infection-result", "url": "/publications/2023-serafini-sars-cov-2-airway-infection-result/", "title": "SARS-CoV-2 airway infection results in the development of somatosensory abnormalities in a hamster model", "authors": [ "Randal A. Serafini", "Justin J. Frere", "Jeffrey Zimering", "Ilinca M. Giosan", "Kerri D. Pryce", "Ilona Golynker", "Maryline Panis", "Anne Ruiz", "Benjamin R. tenOever", "Venetia Zachariou" ], "author_count": 10, "first_author": "Randal A. Serafini", "senior_authors": [ "Benjamin R. tenOever", "Venetia Zachariou" ], "corresponding_authors": [ "Benjamin R. tenOever", "Venetia Zachariou" ], "tenoever_position": 9, "tenoever_role": "senior", "contribution_character": "co-led", "year": 2023, "journal": "Science Signaling", "volume": "16", "issue": "784", "pages": "eade4984", "doi": "10.1126/scisignal.ade4984", "doi_url": "https://doi.org/10.1126/scisignal.ade4984", "pmid": "37159520", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/37159520/", "pmcid": "PMC10422867", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10422867/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10422867/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response" ], "themes": [ "post-acute-sequelae" ], "pathogens": [ "SARS-CoV-2", "influenza A virus" ], "viral_families": [ "Coronaviridae", "Orthomyxoviridae" ], "host_species": [ "golden hamster", "mouse" ], "technologies": [ "bulk RNA sequencing", "RNA sequencing deconvolution", "quantitative RT-PCR", "RNAscope in situ hybridization", "immunohistochemistry", "plaque assay", "von Frey monofilament testing", "Hargreaves thermal testing", "locomotor beam break assay", "Ingenuity Pathway Analysis", "cross-dataset meta-analysis" ], "biological_systems": [ "golden hamster dorsal root ganglia", "hamster spinal cord", "hamster lung", "mouse hindpaw inflammatory pain model", "mouse spared nerve injury comparison dataset", "mouse paw incision model", "Vero cells" ], "key_concepts": [ "post-acute sequelae of SARS-CoV-2 infection", "mechanical hypersensitivity", "dorsal root ganglia", "viral RNA dissemination without infectious virus", "type I interferon signalling in sensory tissue", "neuropathic transcriptome", "upstream regulator prediction", "ILF3", "neuroplasticity", "demyelination signature" ], "keywords": [ "SARS-CoV-2", "Long Covid", "golden hamster", "dorsal root ganglia", "mechanical hypersensitivity", "ILF3", "YM155", "influenza A virus", "interferon beta", "neuropathic pain", "RNA sequencing" ], "one_sentence_contribution": "Intranasal SARS-CoV-2 infection of golden hamsters deposits viral RNA without infectious virus in dorsal root ganglia and spinal cord, producing a milder but longer-lasting mechanical hypersensitivity than influenza A virus and a neuropathic dorsal root ganglion transcriptome at 31 days, from which ILF3 emerges as an analgesic target validated in mouse pain models.", "summary_25": "A nose-only SARS-CoV-2 infection in hamsters left viral RNA but no live virus in sensory nerve tissue, and produced touch sensitivity lasting a month after clearance.", "summary_75": "Many people report lingering pain and altered sensation after COVID-19. In hamsters infected through the nose, viral RNA but no infectious virus appeared in sensory nerve clusters within a day. Influenza caused sharp, brief touch sensitivity, while SARS-CoV-2 caused milder sensitivity that grew and then returned a month later, long after the virus was gone. Gene activity in those nerve clusters shifted toward a nerve-injury pattern, and a drug targeting one predicted regulator relieved pain in mice.", "summary_150": "Intranasal SARS-CoV-2 in golden hamsters raised viral nucleocapsid transcripts and Isg15 in cervical and thoracic dorsal root ganglia and spinal cord by one day, resolving by four to seven days, while plaque assay recovered infectious virus only from lung and nucleocapsid protein was undetectable in neural tissue. In situ hybridisation placed Spike RNA in both neuronal and satellite glial compartments. Influenza A virus produced strong mechanical hypersensitivity at one day that resolved by four, whereas SARS-CoV-2 produced milder hypersensitivity that reached significance at four days and reappeared at 28 days in both sexes. Acute dorsal root ganglion transcriptomes were neuronal in character for SARS-CoV-2 and interferon-dominated for influenza, and at 31 days SARS-CoV-2 produced 1065 differentially expressed genes with reduced myelin and synaptogenesis signatures and increased macrophage contribution by deconvolution. Upstream regulator analysis nominated ILF3, whose inhibitor reduced hypersensitivity in three mouse pain models, though not in infected animals.", "citation": "Serafini RA, Frere JJ, Zimering J, Giosan IM, Pryce KD, Golynker I, Panis M, Ruiz A, tenOever BR, Zachariou V. SARS-CoV-2 airway infection results in the development of somatosensory abnormalities in a hamster model. Science Signaling. 2023. Volume 16, issue 784, article eade4984. DOI 10.1126/scisignal.ade4984. PMID 37159520. PMCID PMC10422867.", "sections": { "Citation": "Serafini RA, Frere JJ, Zimering J, Giosan IM, Pryce KD, Golynker I, Panis M, Ruiz A, tenOever BR, Zachariou V. SARS-CoV-2 airway infection results in the development of somatosensory abnormalities in a hamster model. Science Signaling. 2023. Volume 16, issue 784, article eade4984.\n\nDOI 10.1126/scisignal.ade4984. PMID 37159520. PMCID PMC10422867.", "One-sentence contribution": "Intranasal SARS-CoV-2 infection of golden hamsters deposits viral RNA without infectious virus in dorsal root ganglia and spinal cord, producing a milder but longer-lasting mechanical hypersensitivity than influenza A virus and a neuropathic dorsal root ganglion transcriptome at 31 days, from which ILF3 emerges as an analgesic target validated in mouse pain models.", "Executive summary": "Sensory symptoms including pain, tingling and numbness occur both during acute COVID-19 and persistently in post-acute sequelae, yet the mechanism was unclear and it was not established whether SARS-CoV-2 reaches the peripheral sensory nervous system at all. Using the golden hamster model of respiratory infection, which reproduces COVID-19 without host or virus adaptation, the authors tracked viral material, host transcription and sensory behaviour in parallel, with influenza A virus as a comparator respiratory RNA virus. Viral nucleocapsid transcripts and Isg15 rose sharply in cervical and thoracic dorsal root ganglia and spinal cord within one day of infection and largely resolved by four to seven days, while plaque assay recovered infectious virus from lung and from no neural tissue. In situ hybridisation placed Spike RNA near nuclei in both neuronal and satellite glial compartments, and nucleocapsid protein was not detectable in these tissues. Behaviourally, influenza produced pronounced mechanical hypersensitivity at one day that resolved by four, whereas SARS-CoV-2 produced a slower, milder decline in withdrawal threshold that reached significance at four days and, unlike influenza, returned at 28 days in both sexes. Transcriptionally, acute SARS-CoV-2 dorsal root ganglia were dominated by neuronal signalling pathways and influenza by interferon pathways, and at 31 days SARS-CoV-2 produced 1065 differentially expressed genes with a neuropathic character. Upstream regulator analysis nominated ILF3, and its inhibitor YM155 reduced hypersensitivity in three mouse pain models.", "Scientific context": "Abnormal somatosensation accompanies both neuroinvasive and non-neuroinvasive viral infections, with mechanisms that differ by virus. Herpesviruses persist in dorsal root ganglia and drive abnormal sensory neuron activity on reactivation, while HIV induces sensory neuropathy through viral protein interaction with axons together with secondary inflammation. For coronaviruses the mechanism was much less understood. Whether SARS-CoV-2 crosses the blood-brain barrier and infects the central nervous system was unresolved, with ultrastructural studies of post-mortem tissue describing particle-like structures while other studies failed to recover replication-competent virus from brain. Hamster work, including from these groups, had found viral RNA in olfactory bulb, cortex, brainstem and cerebellum during active infection without evidence of infectious material, suggesting that airway replication disseminates viral RNA and inflammatory debris that provoke antiviral transcription in distal tissues.\n\nThe paper identifies the specific gap it addresses. Despite extensive study of central nervous system involvement, little clinical or preclinical work had examined penetration of SARS-CoV-2 into the peripheral nervous system and particularly the dorsal root ganglia, and reports on viral transcripts in cerebrospinal fluid conflicted. Earlier coronavirus literature in rodents, including hemagglutinating encephalomyelitis virus and mouse hepatitis virus, had described replication in dorsal root ganglia and spinal cord invasion, attributing neurological dysfunction either to direct viral damage or to collateral damage from antiviral immune responses.", "Central question": "Do somatosensory abnormalities after respiratory SARS-CoV-2 infection arise from exposure of dorsal root ganglion and spinal cord neurons to mature virus, or to circulating inflammatory material and viral RNA, and does the resulting transcriptional state differ from that produced by another respiratory RNA virus in a way that explains a different sensory time course.", "Experimental strategy": "The design rests on three comparisons layered onto one animal model.\n\nThe first is virus against virus. Influenza A virus is a respiratory RNA virus that also provokes a systemic inflammatory response and clinical myalgia, so running it in parallel separates whatever is specific to SARS-CoV-2 from what any acute respiratory infection produces. That comparison is what makes the neuronal versus interferon distinction in the transcriptomes interpretable, and it is what establishes that the persistent phenotype is not simply a consequence of having been acutely ill.\n\nThe second is material against material. Quantitative PCR for viral transcripts, plaque assay for infectious virus, in situ hybridisation for Spike RNA and immunohistochemistry for nucleocapsid protein are applied to the same tissues, so the question of whether virus replicates in sensory tissue or only deposits RNA there can be answered rather than assumed. Lung serves as the internal positive control for both the plaque assay and the protein stain.\n\nThe third is acute against chronic. Tissues and behaviour were sampled at 1 and 4 days, when viral RNA and interferon signalling are present, and again at 28 to 31 days, after clearance, which is the interval relevant to persistent symptoms. Both sexes were included at the late time point.\n\nTarget identification then proceeds by computational prediction followed by pharmacological test in a different species and different injury contexts. Upstream regulator analysis was filtered deliberately, retaining regulators predicted to be less active at time points where hypersensitivity was lower, on the reasoning that inhibiting such a regulator might be analgesic, and the selection of ILF3 among the candidates was driven by the availability of a systemically deliverable and clinically tested inhibitor. Testing that inhibitor in complete Freund's adjuvant inflammation, in interferon beta injection and in paw incision separates a general analgesic effect from one specific to interferon-driven sensitisation, and paired locomotor and weight measurements guard against mistaking sedation or illness for analgesia.", "Key findings": "1. Viral RNA and an interferon-stimulated transcript rise transiently in sensory tissue. Nucleocapsid transcripts and Isg15 increased at 1 day after infection in cervical dorsal root ganglia and spinal cord and in thoracic dorsal root ganglia and spinal cord, and were cleared or returned toward baseline in most samples by 4 to 7 days (Figure 1A to 1H).\n\n2. No infectious virus reaches sensory tissue. Plaque assay at 3 days recovered virus from lung homogenates of infected animals only, and from no dorsal root ganglion or spinal cord sample (Figure 1I).\n\n3. Spike RNA is present in dorsal root ganglia and spinal cord without detectable viral protein. In situ hybridisation at 1 day showed Spike puncta around DAPI-labelled nuclei, consistent in dorsal root ganglia with satellite glial cells, and in Rbfox3-labelled neuronal spaces, absent in mock animals (Figure 2A and 2B). Nucleocapsid protein was not notably detected in dorsal root ganglia while it was confirmed in lung (Figure 2C). These are representative images from two animals per group.\n\n4. The two viruses produce different acute sensory time courses. Influenza produced robust mechanical hypersensitivity at 1 day that had completely subsided by 4 days, while SARS-CoV-2 produced a gradual decline in withdrawal threshold reaching significance only at 4 days, and influenza hypersensitivity at 1 day was significantly greater than that of SARS-CoV-2 at the same time (Figure 3A).\n\n5. The two viruses produce different acute dorsal root ganglion transcriptomes. SARS-CoV-2 gave 344 differentially expressed genes at 1 day and 63 at 4 days, against 82 and 18 for influenza (Figure 3B). Pathway analysis placed axonal guidance signalling and synaptogenesis signalling as the top enriched pathways for SARS-CoV-2 at 1 day and neuroinflammation signalling among the top five at 4 days, while influenza pathways were consistently generic antiviral (Figure 3C). Shared upregulated genes between the two viruses were primarily antiviral (Figure 3D). Quantitative PCR confirmed bidirectional regulation of neuropathy-associated and pronociceptive genes including Sema3b, Vegfa and Rgs4 at 1 day and Mx1, Irf7, Slc6a4 and Rgs18 at 4 days (Figure 3E).\n\n6. Hypersensitivity returns after clearance and only for SARS-CoV-2. At 28 days, well after viral clearance, SARS-CoV-2 infected hamsters of both sexes showed substantial mechanical hypersensitivity while influenza and mock animals were normal (Figure 5A).\n\n7. A neuropathic transcriptome emerges late. At 31 days, thoracic dorsal root ganglia from SARS-CoV-2 infected animals showed 1065 differentially expressed genes, 170 up and 895 down (Figure 5B), with decreased synaptogenesis signalling and involvement of EIF2, mTOR, opioid and SNARE signalling pathways (Figure 5C). Disease association analysis linked these genes primarily to neuro-oncological and neurodegenerative conditions (Figure 5D), and affected transcripts included tubulin isoforms, myelin proteins, activity-related channels, extracellular matrix proteins and cytokine and interferon-related proteins (Figure 5E).\n\n8. Cell composition inference suggests a shift. Deconvolution of the 31-day data indicated reduced transcriptomic contribution from myelinating Schwann cells and increased contribution from macrophages (Figure 5F). The authors read this as suggesting a proinflammatory state with potentially impaired myelination, which is an inference from bulk deconvolution rather than a direct cell count.\n\n9. Peripheral and central pain-relevant tissues largely counter-regulate. Comparing 31-day dorsal root ganglion upstream regulators with striatum and thalamus data from the same model in prior work, most of the top 15 regulators showed similar predicted states in the two brain regions and generally opposite states in dorsal root ganglia, with PTPRR and MIR17HG commonly upregulated and FIRRE commonly downregulated between dorsal root ganglia and thalamus (Figure 5G).\n\n10. ILF3 was nominated and validated pharmacologically. Nine upstream regulators met the filtering criterion, and ILF3 was selected from among those not previously studied in pain because an inhibitor, YM155, exists. Ilf3 transcript itself did not change by whole-tissue quantitative PCR, which the authors read as indicating the change is at the protein or localisation level. YM155 at 5 milligrams per kilogram raised thermal and mechanical withdrawal thresholds in complete Freund's adjuvant treated mice both acutely and with sustained effect over days (Figure 4C to 4F), prevented interferon beta induced mechanical hypersensitivity when given from the time of injection and reversed it when started a day later at a lower dose (Figure 4I and 4J), and reduced hypersensitivity after paw incision when given prophylactically (Figure 4K), without changes in locomotion beyond 30 minutes or in weight (Figure 4B, 4G, 4L). Toxicity was lethal at 20 milligrams per kilogram and variable at 10, and male mice were more sensitive than female mice at 5.\n\n11. Comparison against published pain model datasets showed genes commonly upregulated between SARS-CoV-2 and complete Freund's adjuvant at 1 and 4 days and between SARS-CoV-2 and spared nerve injury at 1 day, a shared extracellular matrix remodelling set upregulated across all three acute conditions, and a strong counter-regulation between spared nerve injury and 31-day SARS-CoV-2 in genes related to nervous system development, myelination and synaptic transmission (Figure 6A to 6D).", "Mechanistic model": "The study does not establish a mechanism linking viral material in sensory tissue to persistent hypersensitivity, and the authors are explicit that the association is correlative. Their summary states that the presence of viral material and the resulting interferon response and gene expression changes correlated with the prolonged hypersensitivity signature.\n\nWhat the data constrain is the following. SARS-CoV-2 RNA reaches cervical and thoracic dorsal root ganglia and spinal cord within a day of intranasal infection, in both neuronal and satellite glial compartments, in the absence of recoverable infectious virus and without detectable nucleocapsid protein, so local replication is not supported and the material is best described as disseminated viral RNA. The acute transcriptional consequence in dorsal root ganglia is predominantly neuronal in character and differs from the predominantly interferon-driven response to influenza, even though both viruses provoke systemic inflammation, so the difference is not simply a matter of how much inflammation occurs. The late phenotype, both behavioural and transcriptional, appears after viral RNA has cleared and is specific to SARS-CoV-2 among the two viruses tested.\n\nThe interpretive hypothesis the authors advance, that certain acute SARS-CoV-2 transcriptional changes counteract interferon-induced sensitisation through a stronger neuronal adaptation signature and thereby produce the milder acute phenotype, is stated as a hypothesis that motivated the pathway analysis rather than as a result. Likewise the suggestion that the late phenotype reflects altered excitability, cytoskeletal architecture, extracellular remodelling and myelination is presented as what the transcriptome and deconvolution may indicate.\n\nFor ILF3 the causal claim is narrower and better supported but also displaced from the virus. YM155 reduces hypersensitivity in three mouse models including one driven purely by interferon beta, which supports ILF3 activity as a contributor to interferon-associated sensitisation. It was not tested in infected hamsters, so ILF3 inhibition is not shown to relieve SARS-CoV-2 associated hypersensitivity. The authors also note that YM155 is thought to act on ILF3 subcellular localisation rather than on its expression, and Ilf3 transcript was unchanged in the tissue.", "Conceptual or technical advance": "The work establishes that a purely airway infection can leave a durable transcriptional and behavioural mark on the peripheral sensory nervous system without any infectious virus reaching that tissue, which reframes post-viral sensory symptoms as a consequence of disseminated viral material and host response rather than requiring neuroinvasion. Comparing two respiratory RNA viruses in the same model and at the same time points is what allows a virus-specific claim to be made at all. The authors also propose the hamster respiratory SARS-CoV-2 model as a preclinical chronic pain model suitable for evaluating pharmacological treatments, and the ILF3 result illustrates the pipeline they intend, moving from an upstream regulator prediction in infected tissue to a validated analgesic effect in conventional injury models.", "Relationship to the broader research program": "The hamster model, the infection protocol and the longitudinal transcriptomic framing follow directly from the laboratory's prior work on systemic and brain consequences of SARS-CoV-2 in the same model, and the striatum and thalamus datasets used in the cross-tissue comparison come from that earlier study. The paper extends the same question, what a respiratory infection does to distal tissue in the absence of infectious virus there, from the central nervous system to the peripheral sensory nervous system. The tenOever contribution covers study design, the infection work, virology and supervision, with Frere as co-first author, while the sensory neuroscience, behavioural pharmacology and mouse pain models come from the Zachariou laboratory with Serafini as co-first author, and the two are joint corresponding authors and joint supervisors.\n\nCategory 3 synthesis. Read together with the laboratory's earlier hamster brain work and with its influenza work, this paper contributes to a recurring question in the corpus about how a localised respiratory infection produces effects in tissues the virus does not productively infect. That is a cross-paper theme and this study alone establishes it only for dorsal root ganglia and spinal cord.", "Related publications": "- Frere and colleagues, 2022, predecessor. The longitudinal hamster study of systemic and brain consequences of SARS-CoV-2 from the same laboratory, cited throughout as reference 34, source of the infection protocol and of the striatum and thalamus datasets reused in the cross-tissue upstream regulator comparison.\n- Barragán-Iglesias and colleagues, predecessor. Source of the interferon beta hindpaw hypersensitivity paradigm, replicated here before being used to test ILF3 inhibition.\n- Parisien and colleagues, methodological foundation. Source of the publicly available mouse dorsal root ganglion RNA sequencing datasets for complete Freund's adjuvant and spared nerve injury against which the hamster data were meta-analysed.", "Limitations and boundaries": "The infection model is the golden hamster, and all of the infection biology, transcriptomics and infection-associated behaviour come from that species, while every pharmacological test of ILF3 inhibition was performed in mice using non-infectious injury models, so the therapeutic claim is not demonstrated against SARS-CoV-2 associated hypersensitivity in any animal. Acute transcriptomic and imaging work used male hamsters only, with females included at the 31-day behavioural time point, and the 31-day sequencing was male only. Group sizes are small throughout, with n of 3 to 8 for quantitative PCR, n of 4 for acute sequencing, n of 3 for 31-day sequencing, n of 4 per group for the acute behaviour and n of 2 per group for the in situ hybridisation and immunohistochemistry images. Absence of infectious virus rests on plaque assay at a single time point, 3 days, and absence of viral protein on immunohistochemistry at 1 day, so neither excludes low-level or transient events below assay sensitivity. Sensory assessment is limited to mechanical withdrawal threshold in hamsters, with thermal testing performed only in the mouse models, and biosafety constraints required a linear perceived intensity scale rather than standard logarithmic force measurements for hamsters, which the authors state was done to accommodate a low number of animals at high variable forces. Pathway, upstream regulator and disease association results are predictions from bulk tissue transcriptomes, and the cell composition shift is a deconvolution inference rather than a measured change in cell numbers. Ilf3 transcript did not change, so the upstream regulator prediction was not confirmed at the level of the molecule itself in the infected tissue. YM155 is a single compound with documented toxicity at higher doses and sex-dependent sensitivity, and its selectivity for ILF3 in these assays was not independently established. The cross-species meta-analysis compares hamster infection data against mouse injury data generated elsewhere. Only one SARS-CoV-2 isolate and one influenza strain were used, and the latest time point examined is 31 days.", "Audience summaries": "### 25 words\n\nA nose-only SARS-CoV-2 infection in hamsters left viral RNA but no live virus in sensory nerve tissue, and produced touch sensitivity lasting a month after clearance.\n\n### 75 words\n\nMany people report lingering pain and altered sensation after COVID-19. In hamsters infected through the nose, viral RNA but no infectious virus appeared in sensory nerve clusters within a day. Influenza caused sharp, brief touch sensitivity, while SARS-CoV-2 caused milder sensitivity that grew and then returned a month later, long after the virus was gone. Gene activity in those nerve clusters shifted toward a nerve-injury pattern, and a drug targeting one predicted regulator relieved pain in mice.\n\n### 150 words\n\nIntranasal SARS-CoV-2 in golden hamsters raised viral nucleocapsid transcripts and Isg15 in cervical and thoracic dorsal root ganglia and spinal cord by one day, resolving by four to seven days, while plaque assay recovered infectious virus only from lung and nucleocapsid protein was undetectable in neural tissue. In situ hybridisation placed Spike RNA in both neuronal and satellite glial compartments. Influenza A virus produced strong mechanical hypersensitivity at one day that resolved by four, whereas SARS-CoV-2 produced milder hypersensitivity that reached significance at four days and reappeared at 28 days in both sexes. Acute dorsal root ganglion transcriptomes were neuronal in character for SARS-CoV-2 and interferon-dominated for influenza, and at 31 days SARS-CoV-2 produced 1065 differentially expressed genes with reduced myelin and synaptogenesis signatures and increased macrophage contribution by deconvolution. Upstream regulator analysis nominated ILF3, whose inhibitor reduced hypersensitivity in three mouse pain models, though not in infected animals." }, "discoveries": [ "claim-14" ], "relationships": [ { "from": "2023-serafini-sars-cov-2-airway-infection-result", "to": "2022-frere-sars-cov-2-infection-in-hamsters-a", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2023-serafini-sars-cov-2-airway-infection-result" } ], "canonical_url": "https://tenoeverlab.us/publications/2023-serafini-sars-cov-2-airway-infection-result/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sars-cov-2" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "plaque-assay", "pathway-enrichment-analysis", "immunohistochemistry", "rna-in-situ-hybridization", "behavioural-and-sensory-testing", "deconvolution", "meta-analysis" ] } }, { "id": "2023-uhl-adar1-biology-can-hinder-effective", "slug": "2023-uhl-adar1-biology-can-hinder-effective", "url": "/publications/2023-uhl-adar1-biology-can-hinder-effective/", "title": "ADAR1 Biology Can Hinder Effective Antiviral RNA Interference", "authors": [ "Skyler Uhl", "Chanyong Jang", "Justin J. Frere", "Tristan X. Jordan", "Anne E. Simon", "Benjamin R. tenOever" ], "author_count": 6, "first_author": "Skyler Uhl", "senior_authors": [ "Benjamin R. tenOever" ], "corresponding_authors": [ "Benjamin R. tenOever" ], "tenoever_position": 6, "tenoever_role": "senior", "contribution_character": "lab-led", "year": 2023, "journal": "Journal of Virology", "volume": "97", "issue": "4", "pages": "e00245-23", "doi": "10.1128/jvi.00245-23", "doi_url": "https://doi.org/10.1128/jvi.00245-23", "pmid": "37017521", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/37017521/", "pmcid": "PMC10134826", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10134826/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10134826/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "small-rna-antiviral-defense", "viral-populations-evolution" ], "themes": [ "reconstructing-antiviral-rnai", "recombination-and-escape" ], "pathogens": [ "Sendai virus", "influenza A virus" ], "viral_families": [ "Paramyxoviridae", "Orthomyxoviridae" ], "host_species": [ "human", "mouse", "canine", "hamster", "Nicotiana benthamiana" ], "technologies": [ "paramyxovirus reverse genetics", "microRNA target site engineering", "CRISPR knockout", "RNA sequencing", "amplicon sequencing", "high-content microscopy", "flow cytometry", "adenoviral reconstitution", "agroinfiltration", "phylogenetic analysis" ], "biological_systems": [ "A549 cells", "STAT1 knockout A549 cells", "ADAR1 knockout A549 cells", "MDCK cells", "mouse embryonic fibroblasts", "NoDice HEK293T cells", "BSRT7 cells", "Nicotiana benthamiana" ], "key_concepts": [ "antiviral RNA interference", "adenosine to inosine RNA editing", "ADAR1 p110 and p150 isoforms", "viral escape from silencing", "hypermutation", "negative-sense RNA virus constraints", "ribonucleoprotein protection of genomes", "viral suppressors of RNA silencing", "incompatibility of ADAR1 and RNAi" ], "keywords": [ "ADAR1", "RNA editing", "Sendai virus", "RNA interference", "microRNA targeting", "viral escape", "Nicotiana benthamiana", "interferon" ], "one_sentence_contribution": "Escape of a microRNA-targeted Sendai virus from engineered antiviral RNA interference comes not from the virus but from host ADAR1, whose adenosine to inosine editing destroys the target sites, and human ADAR1 also suppresses endogenous silencing in a plant.", "summary_25": "A virus put under engineered RNA silencing pressure escaped, but the change came from the host. ADAR1 edited the target sites away, rescuing the virus.", "summary_75": "Mammalian cells can be forced to silence a virus by inserting perfectly matched microRNA target sites into an essential viral gene. A Sendai virus under this pressure eventually escaped, not by mutating or deleting the cassette, but because the host enzyme ADAR1 edited adenosines within the target sites. Deleting ADAR1 abolished escape and restoring it brought escape back. Human ADAR1 expressed in a plant also suppressed that plant's own gene silencing.", "summary_150": "Negative-sense RNA viruses cannot recombine, and a previous study found they could not escape engineered microRNA-directed silencing, unlike positive-sense viruses. Holding infections without passage rather than serially passaging revealed escape of a five-target Sendai virus at six to eight days in about 8 percent of wells. Sequencing showed no deletion and no random mutation, but dense A to G transitions confined to the target sites. Knocking out ADAR1 in a STAT1 deficient background eliminated escape in all 96 wells, and adenoviral reconstitution restored it. Moving the cassette to the phosphoprotein gene, which attenuates without exposing the genome, also permitted escape, with editing on the antigenome rather than the genome. Whether ADAR1 is recruited by the silencing complex or acts on duplex viral RNA is unresolved. ADAR1 orthologs track with interferon-based defense across species, and human ADAR1 suppressed endogenous silencing in Nicotiana benthamiana.", "citation": "Uhl S, Jang C, Frere JJ, Jordan TX, Simon AE, tenOever BR. ADAR1 Biology Can Hinder Effective Antiviral RNA Interference. Journal of Virology. 2023. Volume 97, issue 4, article e00245-23. DOI 10.1128/jvi.00245-23. PMID 37017521. PMCID PMC10134826.", "sections": { "Citation": "Uhl S, Jang C, Frere JJ, Jordan TX, Simon AE, tenOever BR. ADAR1 Biology Can Hinder Effective Antiviral RNA Interference. Journal of Virology. 2023. Volume 97, issue 4, article e00245-23.\n\nDOI 10.1128/jvi.00245-23. PMID 37017521. PMCID PMC10134826.", "One-sentence contribution": "Escape of a microRNA-targeted Sendai virus from engineered antiviral RNA interference comes not from the virus but from host ADAR1, whose adenosine to inosine editing destroys the target sites, and human ADAR1 also suppresses endogenous silencing in a plant.", "Executive summary": "Mammalian cells can be made to mount something close to antiviral RNA interference by embedding perfectly complementary microRNA target sites in an essential viral gene, which converts endogenous microRNAs into cleaving guides through Argonaute 2. Earlier work from this laboratory had shown that positive-sense RNA viruses escape this pressure by homologous recombination while negative-sense viruses do not, apparently lacking a route out. Here a recombinant Sendai virus carrying five microRNA target sites in the 3' untranslated region of the nucleoprotein gene was held under sustained infection rather than serial passage, and escape appeared at six to eight days in roughly 8 percent of wells. Sequencing of escape variants showed neither deletion of the cassette nor random point mutation but a dense pattern of A to G changes confined to the target sites, the signature of adenosine deaminase acting on RNA. Knockout of ADAR1 in a STAT1 deficient background abolished escape in all 96 wells, and adenoviral reconstitution of ADAR1 restored it. Escape also occurred when the cassette was moved to the phosphoprotein gene, where editing appeared on the antigenome rather than the genome. Across a phylogenetic sampling, ADAR1 orthologs were found in species with interferon or interferon-like defenses and absent from those with well-characterized antiviral RNA interference, and expressing human ADAR1 in Nicotiana benthamiana suppressed silencing of a green fluorescent protein reporter.", "Scientific context": "Eukaryotic antiviral defenses against RNA fall broadly into two designs, an RNA-based one in which host nucleases are guided by small RNAs derived from the pathogen, found in plants, insects and nematodes, and a protein-based one in which pattern recognition receptors detect pathogen-associated molecular patterns and induce interferon and interferon-stimulated genes, used by vertebrates. Both are triggered by double-stranded RNA. Whether mammals also retain functional antiviral RNA interference has been reported by several groups and remains contested.\n\nTwo prior results from this laboratory set up the experiment. Benitez and colleagues had shown that inserting perfectly complementary microRNA target sites into a virus recreates something functionally equivalent to antiviral RNA interference in mammalian cells and can substitute for the interferon system. A later comparative study applied that same pressure across diverse RNA viruses and found that positive-sense viruses escape by homologous recombination, through polymerase template switching that removes the cassette, while negative-sense viruses showed no escape, consistent with their general inability to recombine. Because negative-sense RNA viruses nevertheless exist in hosts with functional antiviral RNA interference, the authors reasoned that some route to escape must exist, and asked whether more time or weaker pressure would reveal it.", "Central question": "Given that negative-sense RNA viruses cannot remove a microRNA targeting cassette by recombination, can they escape RNA interference pressure at all, and if so by what route.", "Experimental strategy": "The system imposes a defined, uniform silencing pressure whose escape can be read at single-well resolution. A green fluorescent protein expressing recombinant Sendai virus carries five distinct microRNA target sites in the 3' untranslated region of the nucleoprotein gene, so that endogenous microRNAs of epithelial cells direct Argonaute 2 cleavage of the positive-sense antigenome and nucleoprotein messenger RNA. Two controls define the axes. A construct with the same sequence in reverse complement orientation places the sites on the genome, which is inaccessible inside the ribonucleoprotein, and so reports the cost of the insert without the silencing. A single target site construct lowers the pressure, which separates escape that requires one change from escape that would require five.\n\nTwo design choices matter for what was found. Sustained infection replaces serial passage, so that rare escape events are not diluted away by transfer, and readout is by fluorescence in 96-well format, which turns escape frequency into a countable quantity. Genetics then tests causality rather than correlation. ADAR1 is knocked out in a STAT1 deficient background, necessary because losing ADAR1 in interferon-competent cells triggers interferon and cell death, and the phenotype is restored by adenoviral reconstitution so that clonal and off-target explanations are addressed.\n\nMoving the cassette from nucleoprotein to phosphoprotein separates two confounded variables, since silencing nucleoprotein exposes the genome and triggers innate sensing while silencing phosphoprotein attenuates comparably without that exposure. Finally, the question is taken outside mammals in two directions, by phylogenetic survey of the ADAR family against the defense system each species uses, and by expressing human ADAR1 in a plant that has endogenous RNA silencing and no ADARs, with a known viral suppressor as the positive control.", "Key findings": "1. Under serial passage, the five target virus was undetectable while the reverse control produced uniform fluorescence, reproducing prior work, and the single target virus produced scattered fluorescent cells (Figure S1A).\n2. Under sustained infection without passaging, the single target virus reached control levels of replication by six days, and the five target virus became fluorescent at six days and clearly positive by eight days, escaping in roughly 8 percent of individual wells. Flow cytometry and nucleoprotein immunoblotting agreed with the imaging (Figures 1B, 1C, 1D and S1C).\n3. Escape of the single target virus was by point mutation. Around 75 percent of reads at the miR-21 site carried an A to U change in the seed region, with three shared U to C transitions at 5 to 10 percent, two of them in the seed. No elevated mutation frequency was seen in the surrounding region (Figures 2B and S2A).\n4. Escape of the five target virus involved neither deletion of the cassette nor scattered point mutation, which is what recombination-based escape would have produced. Instead there were dense U to C transitions in the positive sense, corresponding to A to G changes in the genome, within each target site (Figures 2C and S2B).\n5. Amplicon sequencing over time detected low-frequency edits by two days that became dominant by four and eight days, indicating that edited genomes outcompete unedited ones rather than arising late (Figures 2D and S2B).\n6. Escape persisted in STAT1 knockout cells with the same editing signature, was observed in none of 96 wells when ADAR1 was additionally knocked out, and was restored when ADAR1 was reconstituted by adenovector, which expressed both the p110 and p150 isoforms (Figures 3A to 3D and S3C).\n7. Moving the cassette to the phosphoprotein gene gave comparable attenuation, more than a hundredfold reduction in viral RNA relative to control, but without the strong interferon-stimulated gene response seen with nucleoprotein targeting, consistent with the genome remaining protected (Figures 4B and 4C).\n8. The phosphoprotein-targeted virus also escaped, from four days, at lower frequency than the nucleoprotein-targeted virus, and with the same A to G signature. The editing was on the antigenome rather than the genome, and the pattern was more heterogeneous, not always disrupting all five sites, which the authors read as indicating that low levels of phosphoprotein may suffice for limited replication (Figures 4D, 4E, S4A to S4C).\n9. Escape with the same editing signature occurred in canine MDCK cells and in mouse embryonic fibroblasts, so the phenotype is not specific to human ADAR1. Escape mutants from these cells carried fewer edits across the cassette, which the authors attribute to the absence of some cognate microRNAs, noting that MDCK cells lack miR-192 and miR-31 (Figures 5B to 5D and S5).\n10. Across the species sampled, adenosine deaminases acting on transfer RNA were universal, while the ADAR1 ortholog was present in species with a known interferon or interferon-like system and absent from those with well-characterized antiviral RNA interference. The authors describe this analysis as limited in scope and note it agrees with more extensive published analyses from other groups (Figure 5A).\n11. Agroinfiltration of human ADAR1 into Nicotiana benthamiana alongside a green fluorescent protein plasmid increased reporter expression, with p110 more effective than p150, against the tomato bushy stunt virus P19 suppressor as positive control. The authors argue that the differential effect among P19, p110 and p150 rules out simple competition for translational machinery (Figure 5E and S5E).", "Mechanistic model": "The causal chain from ADAR1 to escape is established by genetics, since knockout abolishes escape and reconstitution restores it, and by the editing signature itself. What is not established, and the authors say so directly, is how ADAR1 comes to edit these particular sequences. They state that the dynamics of how this editing occurs remain somewhat uncertain and that distinguishing a direct from an indirect role for microRNAs will be difficult, because the escape frequency is too low for biochemistry and there are few other ways to impose this degree of attenuation while also generating double-stranded RNA.\n\nTwo models are laid out. In the indirect model, ADAR1 acts on double-stranded RNA formed between genome and antigenome, or on cleaved viral messenger RNA, or on a highly complementary species such as a defective viral genome, with editing more active in the nucleoprotein-targeted virus because silencing nucleoprotein exposes the genome. In the direct model, the RISC recruits ADAR1, for which there is published evidence of an ADAR1 interaction with Dicer from another group, and editing then occurs at or opposite the bound sites.\n\nThe data cut both ways and the authors present them that way. Editing of the nucleoprotein-targeted virus is on the genome, which microRNAs do not bind, which argues against direct recruitment, while editing of the phosphoprotein-targeted virus is on the antigenome, which does conform to the direct model. The observation that escape mutants from MDCK cells, which lack two of the five cognate microRNAs, carry edits at fewer sites is offered as tempting to speculate in favor of a direct role, since genome and antigenome duplex formation should produce editing across the whole cassette regardless of which microRNAs are present. The authors propose as one possibility that RISC engagement of the nascent messenger RNA recruits ADAR1 to the aligned genomic template, which would reconcile the two observations, but this is put forward as a possible mechanism and not demonstrated.\n\nThe evolutionary claim is correlative. The distribution of ADAR1 across species with interferon-like versus RNA interference-based defense, together with the plant experiment, is presented as evidence that ADAR1 is disruptive to RNA silencing and as a possible explanation for its absence where that defense is essential. The authors note that many explanations exist for gene loss and that if RNA interference does function antivirally in some mammalian cells then how ADAR1 operates there is unclear, raising the possibility that it negatively regulates both systems in a context-dependent way as reported for Caenorhabditis elegans ADARs by another group.", "Conceptual or technical advance": "The result reframes what escape from antiviral RNA interference can mean. Escape here is not a viral adaptation at all. The virus acquires no antagonist and performs no recombination, and the selective pressure is relieved by a host enzyme acting on the virus in a way that happens to destroy the guide binding sites. That makes a host editing enzyme, rather than a virally encoded suppressor, the relevant actor, and it explains why negative-sense viruses that cannot recombine are nonetheless not trapped.\n\nIt also supplies a functional argument that connects two literatures. If ADAR1 activity degrades the sequence fidelity that perfect complementarity requires, then ADAR1 and effective antiviral RNA interference are in tension, which offers a reason for the observed phylogenetic anticorrelation between them and connects to the broader question of why vertebrates use interferon rather than small RNA defense. The plant experiment is the most direct test of that tension available here, since it introduces the mammalian enzyme into a functioning endogenous silencing system.\n\nPractically, the finding matters for any attempt to build microRNA-based antiviral control or microRNA-based attenuation into a vector, since it identifies a host-driven route by which such designs erode over time.", "Relationship to the broader research program": "This paper continues a line running through the laboratory's work on whether mammals can be made to use RNA-based antiviral defense and what happens when that pressure is applied. Benitez and colleagues established that engineered microRNA targeting can substitute for interferon, and the immediately preceding comparative study established the difference between positive-sense and negative-sense viruses under that pressure. The recombinant Sendai virus constructs and the five target cassette come from that prior work.\n\nCategory 3 synthesis. Read with the laboratory's 2016 Perspective, which argued that chordates lost RNA interference because systemic small RNA defense is incompatible with interferon biology, this study supplies a second and different incompatibility, at the level of a single interferon-associated enzyme rather than at the level of the pathway. The two arguments are independent and are not claimed together in either paper, but together they make the loss of antiviral RNA interference in vertebrates look overdetermined. The interest in ribonucleoprotein protection of negative-strand genomes from small RNA targeting also connects back to the laboratory's early observation that influenza genomic RNA is not accessible to RISC.", "Related publications": "- Benitez and colleagues, 2015, Engineered Mammalian RNAi Can Elicit Antiviral Protection that Negates the Requirement for the Interferon Response, cited as reference 37 from the same laboratory. Methodological foundation and predecessor.\n- The immediately preceding comparative escape study from this laboratory, cited as reference 38, which established recombination-mediated escape in positive-sense viruses and its absence in negative-sense viruses. Predecessor, and the study this work directly extends.\n- The prior study supplying the recombinant Sendai virus genomes and the N5T and N5R stocks, cited as references 43 and 78. Methodological foundation.\n- Varble and colleagues, 2010, Engineered RNA viral synthesis of microRNAs. Predecessor from the same laboratory, reporting that negative-sense genomic RNA within the ribonucleoprotein is not accessible to microRNA-directed silencing.\n- tenOever, 2016, The Evolution of Antiviral Defense Systems. Review or synthesis from the same author, supplying the evolutionary framing of why vertebrates use interferon rather than RNA interference.\n- Work from another group on ADAR1 association with the RISC through Dicer, cited as reference 57, which motivates the direct recruitment model. Conceptual foundation.\n- Work from another group on Caenorhabditis elegans ADARs preventing Dicer processing of host transcripts, cited as reference 67. Conceptual foundation for the closing argument.", "Limitations and boundaries": "All virological work is in cultured cells, with no animal infection and no test of whether ADAR1-mediated escape occurs in vivo or affects pathogenesis. The central genetics is performed in a STAT1 knockout background, required because ADAR1 loss in interferon-competent cells causes interferon induction and death, so escape and its abolition were established in cells that cannot mount a STAT1-dependent response. The authors verified that escape and editing still occur in STAT1 knockout cells, but the ADAR1 requirement itself was not demonstrated in a fully interferon-competent setting.\n\nThe system is an engineered mimic of antiviral RNA interference, and the authors state its two departures from endogenous RNA interference. Endogenous small interfering RNAs require early viral replication to generate substrate, which gives a virus time to antagonize the pathway, and they are biased to the ends of the genome, whereas the microRNA targets used here are internal and act immediately. The authors argue the plant result mitigates concern about where targeting occurs, but the mismatch remains.\n\nEscape is rare, at roughly 8 percent of wells for the nucleoprotein-targeted virus and lower for the phosphoprotein-targeted one, and that rarity is itself a limit, since the authors note it puts biochemical characterization of the editing event out of reach. The mechanism of ADAR1 recruitment is explicitly unresolved, with two models that the data support unevenly, and the inference favoring direct microRNA involvement rests on fewer edits in MDCK cells, which is attributed to a published microRNA profile rather than measured here, with the microRNA profile of mouse embryonic fibroblasts stated to be unknown.\n\nThe result does not generalize across RNA viruses. The authors report that they again attempted to generate an escape mutant of a five target recombinant influenza A virus and observed neither editing nor escape, and offer replication site and genome packaging as possible reasons without resolving them. The phylogenetic analysis is described by the authors as limited in scope and is correlative, so it cannot establish that ADAR1 presence and antiviral RNA interference are mutually exclusive by causation. The plant experiment uses a transgene reporter and agroinfiltration rather than virus infection, shows suppression by both isoforms with p110 stronger, which differs from a published report in Drosophila where interference was attributed to p150, and that discrepancy is noted but not explained.", "Audience summaries": "### 25 words\n\nA virus put under engineered RNA silencing pressure escaped, but the change came from the host. ADAR1 edited the target sites away, rescuing the virus.\n\n### 75 words\n\nMammalian cells can be forced to silence a virus by inserting perfectly matched microRNA target sites into an essential viral gene. A Sendai virus under this pressure eventually escaped, not by mutating or deleting the cassette, but because the host enzyme ADAR1 edited adenosines within the target sites. Deleting ADAR1 abolished escape and restoring it brought escape back. Human ADAR1 expressed in a plant also suppressed that plant's own gene silencing.\n\n### 150 words\n\nNegative-sense RNA viruses cannot recombine, and a previous study found they could not escape engineered microRNA-directed silencing, unlike positive-sense viruses. Holding infections without passage rather than serially passaging revealed escape of a five-target Sendai virus at six to eight days in about 8 percent of wells. Sequencing showed no deletion and no random mutation, but dense A to G transitions confined to the target sites. Knocking out ADAR1 in a STAT1 deficient background eliminated escape in all 96 wells, and adenoviral reconstitution restored it. Moving the cassette to the phosphoprotein gene, which attenuates without exposing the genome, also permitted escape, with editing on the antigenome rather than the genome. Whether ADAR1 is recruited by the silencing complex or acts on duplex viral RNA is unresolved. ADAR1 orthologs track with interferon-based defense across species, and human ADAR1 suppressed endogenous silencing in Nicotiana benthamiana." }, "discoveries": [ "claim-09" ], "relationships": [ { "from": "2023-uhl-adar1-biology-can-hinder-effective", "to": "2010-varble-engineered-rna-viral-synthesis-of-", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2023-uhl-adar1-biology-can-hinder-effective" } ], "canonical_url": "https://tenoeverlab.us/publications/2023-uhl-adar1-biology-can-hinder-effective/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "sendai-virus" ], "technologies": [ "bulk-rna-seq", "reverse-genetics", "flow-cytometry", "mirna-target-site-insertion", "adenoviral-vector", "crispr-knockout", "phylogenetics", "amplicon-sequencing", "agroinfiltration", "high-content-imaging" ] } }, { "id": "2023-zhang-mouse-genome-rewriting-and-tailori", "slug": "2023-zhang-mouse-genome-rewriting-and-tailori", "url": "/publications/2023-zhang-mouse-genome-rewriting-and-tailori/", "title": "Mouse genome rewriting and tailoring of three important disease loci", "authors": [ "Weimin Zhang", "Ilona Golynker", "Ran Brosh", "Alvaro Fajardo", "Yinan Zhu", "Aleksandra M. Wudzinska", "Raquel Ordoñez", "André M. Ribeiro-dos-Santos", "Lucia Carrau", "Payal Damani-Yokota", "Stephen T. Yeung", "Camille Khairallah", "Antonio Vela Gartner", "Noor Chalhoub", "Emily Huang", "Hannah J. Ashe", "Kamal M. Khanna", "Matthew T. Maurano", "Sang Yong Kim", "Benjamin R. tenOever", "Jef D. Boeke" ], "author_count": 21, "first_author": "Weimin Zhang", "senior_authors": [ "Jef D. Boeke" ], "corresponding_authors": [ "Jef D. Boeke" ], "tenoever_position": 20, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2023, "journal": "Nature", "volume": "623", "issue": "7986", "pages": "423-431", "doi": "10.1038/s41586-023-06675-4", "doi_url": "https://doi.org/10.1038/s41586-023-06675-4", "pmid": "37914927", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/37914927/", "pmcid": "PMC10632133", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10632133/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10632133/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "pandemic-host-response", "programmable-virology" ], "themes": [ "synthetic-virology-as-method", "models-for-pandemic-virology" ], "pathogens": [ "SARS-CoV-2" ], "viral_families": [ "Coronaviridae" ], "host_species": [ "mouse", "human", "golden hamster", "yeast" ], "technologies": [ "mSwAP-In genome writing", "CRISPR-Cas9 assisted homologous recombination", "yeast assembly of large DNA", "bacterial artificial chromosomes", "tetraploid blastocyst complementation", "capture sequencing", "ATAC-seq", "CUT&RUN", "RNA sequencing", "unique molecular identifier amplicon sequencing", "plaque assay", "immunohistochemistry", "ELISA" ], "biological_systems": [ "mouse embryonic stem cells", "genetically engineered mouse models", "K18-hACE2 mouse", "golden hamster", "mouse lung", "mouse trachea", "mouse testis", "mouse small intestine", "yeast assembly vector" ], "key_concepts": [ "mammalian genome writing", "genomic humanization", "non-coding regulatory elements", "iterative genome rewriting", "biallelic engineering", "synonymous recoding", "alternative splicing", "animal models of COVID-19", "ACE2 receptor", "TMPRSS2", "p53 mutational hotspots" ], "keywords": [ "mSwAP-In", "GREAT-GEMM", "genome writing", "humanized ACE2 mouse", "TMPRSS2 humanization", "SARS-CoV-2 mouse model", "K18-hACE2", "synthetic Trp53", "mouse embryonic stem cells", "tetraploid complementation" ], "one_sentence_contribution": "An iterative, scarless and biallelic method for overwriting large mammalian genomic segments in mouse embryonic stem cells, used to build a recoded Trp53 locus and mice carrying the human ACE2 and TMPRSS2 loci in place of their mouse counterparts.", "summary_25": "A genome writing method replaces whole mouse loci with synthetic or human DNA, producing mice carrying the human ACE2 gene that catch SARS-CoV-2 and recover.", "summary_75": "Mouse models often miss human disease because regulatory DNA outside the coding sequence is left out. A new method swaps large genomic segments in mouse stem cells repeatedly and without scars, using paired selection markers that alternate with each round. It was used to build a mutation resistant p53 gene and to replace mouse Ace2 with the full human ACE2 locus. Those mice are infectable with SARS-CoV-2 and survive, unlike an existing transgenic model.", "summary_150": "mSwAP-In alternates two marker cassettes, each carrying positive and negative selection, so that every payload delivered into mouse embryonic stem cells selects for itself and against its predecessor, permitting iterative, scarless and biallelic overwriting of large genomic segments assembled in yeast. A synonymously recoded Trp53 lacking CG dinucleotides at five mutational hotspots retained transactivation, arrest and apoptosis while accumulating fewer hotspot mutations over passage. Mouse Ace2 was replaced with 116 or 180 kilobase human ACE2 loci, and the resulting animals, derived by tetraploid complementation, reproduced human tissue expression including testicular expression absent in mice, the interferon inducible dACE2 isoform, and human chromatin accessibility. These mice were infectable with SARS-CoV-2, mounted a moderate interferon response and a spike specific antibody response, and survived without weight loss, while all K18-hACE2 controls died. Mouse Tmprss2 was then replaced biallelically with human TMPRSS2 in the same line.", "citation": "Zhang W, Golynker I, Brosh R, Fajardo A, Zhu Y, Wudzinska AM, Ordoñez R, Ribeiro-dos-Santos AM, Carrau L, Damani-Yokota P, Yeung ST, Khairallah C, Vela Gartner A, Chalhoub N, Huang E, Ashe HJ, Khanna KM, Maurano MT, Kim SY, tenOever BR, Boeke JD. Mouse genome rewriting and tailoring of three important disease loci. Nature. 2023. Volume 623, issue 7986, pages 423-431. DOI 10.1038/s41586-023-06675-4. PMID 37914927. PMCID PMC10632133.", "sections": { "Citation": "Zhang W, Golynker I, Brosh R, Fajardo A, Zhu Y, Wudzinska AM, Ordoñez R, Ribeiro-dos-Santos AM, Carrau L, Damani-Yokota P, Yeung ST, Khairallah C, Vela Gartner A, Chalhoub N, Huang E, Ashe HJ, Khanna KM, Maurano MT, Kim SY, tenOever BR, Boeke JD. Mouse genome rewriting and tailoring of three important disease loci. Nature. 2023. Volume 623, issue 7986, pages 423-431.\n\nDOI 10.1038/s41586-023-06675-4. PMID 37914927. PMCID PMC10632133.", "One-sentence contribution": "An iterative, scarless and biallelic method for overwriting large mammalian genomic segments in mouse embryonic stem cells, used to build a recoded Trp53 locus and mice carrying the human ACE2 and TMPRSS2 loci in place of their mouse counterparts.", "Executive summary": "Mouse models often fail to reproduce human disease because the sequences that set where and when a gene is expressed, and how its transcripts are spliced, lie in non-coding regions that conventional transgenesis leaves behind. Overwriting a whole locus, regulatory sequence included, requires delivering DNA on a scale that existing methods handle poorly, and existing methods are generally not designed for repeated rounds. The authors describe mSwAP-In, adapted from a yeast genome rewriting strategy, in which two interchangeable marker cassettes carrying a fluorescent marker, a positive selection marker and a negative selection marker are alternated so that each incoming payload both selects for itself and selects against the preceding cassette. Payloads are assembled in yeast, delivered with CRISPR-Cas9, and integrated by homologous recombination in mouse embryonic stem cells. The method was applied to three loci. A synonymously recoded Trp53 with CG dinucleotides removed from five mutational hotspot codons retained p53 transactivation, growth arrest and apoptosis, and accumulated fewer hotspot mutations over thirty eight passages. Mouse Ace2 was replaced with either a 116 kilobase or a 180 kilobase human ACE2 locus, and the resulting mice reproduced human tissue expression, alternative splicing and chromatin accessibility patterns. These mice were susceptible to SARS-CoV-2 but survived infection without weight loss, unlike K18-hACE2 mice, which all died. Finally, mouse Tmprss2 was replaced biallelically with human TMPRSS2 in the ACE2 line, yielding double humanized animals.", "Scientific context": "Whole genome synthesis had been achieved for Escherichia coli, Mycoplasma and Saccharomyces cerevisiae, but mammalian genome synthesis remained out of reach because of genome size and complexity, so an intermediate goal is overwriting a full locus including its regulatory content. Combining assembly of DNA above one hundred kilobases with site specific recombinases had proved efficient for large scale mammalian modification, and the Big-IN method had largely solved the problem of scars left by earlier delivery approaches, but current methods were not generally designed for iterative delivery, which caps the total size that can be written. On the modeling side, ENCODE and genome wide association studies had established the importance of non-coding regulatory elements, making full genomic humanization preferable to transgenesis, in which a human coding sequence driven by a heterologous promoter gives non-physiological expression. Human bacterial artificial chromosome transgenes preserve full gene sequences but usually integrate randomly, producing position effects. In situ humanization of mouse immunoglobulin loci had been demonstrated, but with low per integration efficiency. For COVID-19 specifically, mice are naturally resistant because of coding differences in Ace2, mouse adapted viral strains change the virus being studied, and the K18-hACE2 transgenic model is uniformly lethal, which does not match human disease, lacks human regulatory elements around ACE2, may miss human specific splice isoforms, and retains an intact endogenous Ace2.", "Central question": "Can large native mammalian genomic segments be overwritten efficiently, scarlessly, iteratively and biallelically in mouse embryonic stem cells that retain full developmental potential, and does replacing an entire mouse locus with its human counterpart, regulatory sequence included, produce an animal whose expression, splicing and disease phenotype are more human-like than a transgenic model?", "Experimental strategy": "The method is built around a swap that is forced in both directions. Two marker cassettes each carry a fluorescent reporter, a positive selection marker and a negative selection marker, and a universal guide RNA target derived from GFP sits in front of each so that Cas9 cuts them specifically. One cassette is placed at a safe position adjacent to the target region. Each payload, assembled in yeast with roughly two kilobase homology arms and the alternate cassette, is co-delivered with guide RNAs that cut the universal target and the distal boundary of the region to be overwritten, and correct integrants are selected for the incoming cassette and against the outgoing one, which removes off target integrations. Alternating the two cassettes allows the process to repeat indefinitely, and the last cassette can be removed to leave a scarless product. Endogenous Hprt1 was deleted beforehand so that an HPRT1 minigene could be used as a selection marker later. The authors then chose three targets that test different demands. Trp53 tests whether a synthetically recoded mouse gene works and whether iterative writing is possible, using three downstream payloads of forty, seventy five and one hundred fifteen kilobases and orthogonal 28 base pair PCRTag watermarks to distinguish synthetic from native sequence. ACE2 tests whether entirely non homologous human DNA can replace a mouse locus, with two payload lengths chosen from DNase hypersensitivity and H3K27 acetylation to ask what the extra sequence contributes. TMPRSS2 tests serial and biallelic writing in an already engineered line. Mice were derived by blastocyst injection and by tetraploid complementation, which requires full pluripotency and therefore also tests whether repeated engineering damages the cells. Verification combined genotyping, copy number quantitative PCR, capture sequencing and a bamintersect analysis that detects reads spanning two references to find off target junctions.", "Key findings": "1. mSwAP-In rewrote the Trp53 locus efficiently. After delivering the recoded synTrp53 payload, 87.1 percent of colonies had switched cassettes, and of thirty eight genotype verified clones, twenty six carried recoded codons on one allele and three carried only recoded synTrp53 (Figures 2b and 2c). Capture sequencing confirmed hemizygosity in those three, and bamintersect detected no off target junctions in six sequenced clones, with yeast assembly vector backbone integration in one.\n\n2. Recoding did not impair p53 function. Doxorubicin treated synTrp53 cells upregulated Mdm2, Pmaip1 and Cdkn1a comparably to wild type, showed similar global stress responses by transcript profiling, and underwent growth arrest and apoptosis (Figures 2d to 2f). Trp53 expression was thirty to forty percent lower in synTrp53 cells, which the authors relate to prior observations linking gene body methylation to higher expression.\n\n3. Recoding reduced hotspot mutation accumulation. After thirty eight passages, C to T and G to A mutations were frequent at wild type Trp53 hotspot codons but not at recoded synTrp53 hotspot codons, with no significant differences at other codons (Figure 2g and Extended Data Figure 3c).\n\n4. Iterative writing worked at increasing payload size. Forty, seventy five and one hundred fifteen kilobase downstream payloads all integrated with efficiency above fifty percent by genotyping, although total drug resistant colony number fell as payload length rose (Figures 2i, 2j and Extended Data Figure 4c). Final marker cassette removal was 47.6 percent efficient with a repair template and 36.4 percent without, and complete when using piggyBac (Figure 2k).\n\n5. Mouse Ace2 was replaced with human ACE2. Payloads of 116 and 180 kilobases were assembled from human bacterial artificial chromosomes and integrated at 61.5 percent and 60.8 percent efficiency by genotyping, with overall success rates after full sequence quality control of 15.4 percent and 22.8 percent (Figures 3d to 3f).\n\n6. Engineered cells retained developmental potential. Thirty one of forty five pups from blastocyst injection showed coat colour chimerism, several chimeric males gave complete germline transmission, and tetraploid complementation gave birth rates of 14 percent and 22.9 percent for the two payloads (Figure 4a and Supplementary Table 2).\n\n7. Expression followed human rather than mouse patterns in several respects. ACE2 mRNA was abundant in small intestine and kidney with moderate levels in testis and colon. ACE2 was readily detected in testis, where mouse Ace2 is not expressed, and lung ACE2 was lower than mouse Ace2 in wild type lung, both consistent with human against mouse transcriptome comparisons (Figure 4b and Extended Data Figure 6b). Immunohistochemistry showed ACE2 in Sertoli cells, spermatogonia and spermatocytes in humanized testis, against only a subset of spermatozoa in wild type (Figure 4c).\n\n8. The longer payload changed expression levels. ACE2 expression in the 180 kilobase model was roughly one hundred fold higher in brain, three to five fold higher in lung and liver, and two to three fold lower in small intestine and colon than in the 116 kilobase model, which the authors read as regulatory function residing in the additional 64 kilobases (Extended Data Figure 6c).\n\n9. Human specific splicing and chromatin accessibility were recapitulated. The interferon stimulated dACE2 isoform was detected in lung, kidney, small intestine and colon, and the long transcript variant 3 in small intestine, kidney, brain and testis (Figures 4d and 4e). ATAC-seq peaks in humanized small intestinal cells overlapped extensively with an ENCODE human small intestine DNase-seq track (Figure 4f).\n\n10. ACE2 mice were susceptible to SARS-CoV-2 but developed milder disease. At three days after intranasal challenge, viral RNA was undetectable in wild type lungs, high in K18-hACE2 lungs, and moderate in ACE2 mouse lungs at the higher inoculum, with plaque assay agreeing (Figures 5a and 5b). ACE2 mice expressed roughly seventy fold less ACE2 in lung than K18-hACE2 mice. Infected ACE2 lungs mounted a moderate type I and type III interferon response overlapping that of K18-hACE2 lungs and not that of wild type (Figures 5c and 5d). dACE2 transcript rose on infection, matching reports in humans (Figure 5e and Extended Data Figure 8a).\n\n11. At one hundred thousand plaque forming units, all five K18-hACE2 mice died by day eight after marked weight loss, while all four ACE2 mice survived fourteen days without weight loss and produced spike reactive serum IgG (Figures 5g to 5i). Histopathology showed pneumonia with monocyte infiltration in both, with substantially milder alveolar epithelial lesions in the humanized ACE2 mice (Figure 5f).\n\n12. Tropism outside the lung was limited. No viral RNA or infectious virus was detected in small intestine or kidney. Nucleocapsid protein was present mainly on Leydig cell membranes in testis, and the virus did not enter seminiferous tubules, unlike reports from severe human COVID-19, which the authors attribute tentatively to immune clearance in these immunocompetent animals (Extended Data Figures 8c to 8g).\n\n13. Compared against golden hamsters in a longitudinal infection, ACE2 mice had lower lung viral RNA at five days that declined by fourteen days, while a subset of ACE2 mice showed higher tracheal viral RNA than hamsters, which the authors relate to the limited Ace2 expression in hamster tracheal epithelium (Extended Data Figures 9a to 9c).\n\n14. Serial biallelic writing was demonstrated. An eighty kilobase human TMPRSS2 payload replaced both mouse Tmprss2 alleles in the ACE2 line at thirty to forty percent efficiency, about half the clones carried two TMPRSS2 copies, double humanized mice were obtained by tetraploid complementation, backcrossing gave complete heterozygous transmission, and both TMPRSS2 splice isoforms were detected across tissues (Figures 6c to 6g and Extended Data Figure 11e).", "Mechanistic model": "The study does not set out to establish a biological mechanism, and where it touches on mechanism it is careful. For the method, the causal logic is engineered rather than discovered, since alternating positive and negative selection between two cassettes is what enforces on target integration and permits iteration, and this is demonstrated directly by efficiency and sequencing data. For the recoded Trp53, the authors hypothesize that removing CG dinucleotides at mutational hotspots reduces the deamination of 5-methylcytosine and adduct binding that generate C to T changes, and the reduced mutation frequency after thirty eight passages is consistent with that hypothesis without isolating either process. For the phenotype of the humanized ACE2 mice, the data show a correlation between lung ACE2 expression level and disease severity, with roughly seventy fold lower expression than K18-hACE2 and markedly milder outcome, and the authors speculate that faster infection kinetics in the 180 kilobase model follow from its higher ACE2 expression. Whether the milder disease is caused by expression level, by the absence of the keratin 18 driven expression pattern, by the presence of human regulatory control, or by some combination is not resolved by these experiments. The interpretation that the additional 64 kilobases in the longer payload carries regulatory function rests on the expression difference between the two models and no direct element level test.", "Conceptual or technical advance": "mSwAP-In makes iterative, scarless, large scale genome writing routine enough in mouse embryonic stem cells that whole loci can be replaced and the cells can still make animals through tetraploid complementation, which the authors argue opens a path toward writing megabase scale synthetic DNA. Because a locus can be replaced together with its regulatory and intronic content, the resulting animals, which the authors call GREAT-GEMMs, allow questions about human specific regulation and splicing to be asked in vivo rather than inferred. The ACE2 model in particular provides a COVID-19 mouse that is infectable with an unmodified virus, survives, mounts a humoral response, and therefore supports study of medium and longer term consequences of infection, which the uniformly lethal K18-hACE2 model cannot. The double humanized ACE2 and TMPRSS2 animal shows that entry pathway components can be humanized together, which is relevant to testing therapies directed at TMPRSS2.", "Relationship to the broader research program": "The tenOever contribution to this study is the SARS-CoV-2 side. By the author contributions statement, Golynker, Fajardo and Carrau performed the SARS-CoV-2 infections and mouse tissue collection in the biosafety level 3 facility, tenOever participated in experimental design and in reviewing and editing the manuscript, and the study was conceptualized and led by Zhang and Boeke. The connection to the tenOever laboratory's own program is the question of what constitutes an adequate small animal model for SARS-CoV-2, and the paper cites the laboratory's golden hamster work as the comparator against which the humanized mouse is benchmarked. Category 3 synthesis, visible only when corpus papers are read together, is that the laboratory's recurring position is that model choice determines which parts of the host response can be seen at all, expressed here in the comparison of the humanized ACE2 mouse against both the K18-hACE2 transgenic and the golden hamster. That statement rests on more than this paper alone.", "Related publications": "- Hoagland et al. 2021, leveraging the antiviral type I interferon system as a first line of defense against SARS-CoV-2 pathogenicity, predecessor. Cited in this paper as the golden hamster model against which the humanized ACE2 mouse is compared, and produced by the tenOever laboratory.\n- Brosh et al. 2021, a versatile platform for locus scale genome rewriting and verification, methodological foundation. The Big-IN platform, the acceptor vector, the Capture-seq verification and the bamintersect analysis used here come from this work, and Brosh is a co-author.\n- Boeke et al. 2016, the Genome Project-write, conceptual extension. The cancer mutation resistant Trp53 was undertaken to address a challenge set by that project, and Boeke is the senior author of both.\n- Ribeiro-dos-Santos et al. 2022, genomic context sensitivity of insulator function, methodological foundation. Source of the unique molecular identifier based amplicon sequencing used to measure hotspot mutation frequencies, with shared authorship.\n- Mitchell et al. 2021, de novo assembly and delivery to mouse cells of a 101 kilobase functional human gene, predecessor. Source of the assemblon concept used for the payloads.", "Limitations and boundaries": "The genome writing demonstrations are confined to mouse embryonic stem cells, and the authors state that generalization to other mammalian species depends on those species having comparable homologous recombination efficiency. The payloads delivered in the Trp53 iterations were more than ninety nine percent identical to native mouse sequence, which the authors note may itself have contributed to the high efficiency, so efficiencies for non homologous human DNA are the lower figures reported for ACE2. Overall success rates after full sequence quality control were 15.4 and 22.8 percent, well below the raw genotyping efficiencies, and yeast assembly vector backbone integration was observed in one clone. Payload sequences carried single nucleotide polymorphisms present in the parental bacterial artificial chromosomes, so a humanized locus reflects one human haplotype. For the infection work, the authors state that the animals used were relatively young at ten to fifteen weeks and healthy, corresponding to people with mild or minimal COVID-19, and that older or comorbid models would be needed to address severe disease. Group sizes in the infection experiments are small, with four or five mice per arm. Male and female animals differed in lung viral RNA despite equal inoculum and no detected difference in ACE2 expression, which is unexplained. The two ACE2 models differ in both payload length and expression level, so the contribution of the extra sequence cannot be separated from the expression difference it produces. Comparison against the golden hamster rests on a single longitudinal experiment. Finally, humanizing ACE2 and TMPRSS2 does not humanize the rest of the mouse, so immune and physiological responses to infection remain those of a mouse.", "Audience summaries": "### 25 words\n\nA genome writing method replaces whole mouse loci with synthetic or human DNA, producing mice carrying the human ACE2 gene that catch SARS-CoV-2 and recover.\n\n### 75 words\n\nMouse models often miss human disease because regulatory DNA outside the coding sequence is left out. A new method swaps large genomic segments in mouse stem cells repeatedly and without scars, using paired selection markers that alternate with each round. It was used to build a mutation resistant p53 gene and to replace mouse Ace2 with the full human ACE2 locus. Those mice are infectable with SARS-CoV-2 and survive, unlike an existing transgenic model.\n\n### 150 words\n\nmSwAP-In alternates two marker cassettes, each carrying positive and negative selection, so that every payload delivered into mouse embryonic stem cells selects for itself and against its predecessor, permitting iterative, scarless and biallelic overwriting of large genomic segments assembled in yeast. A synonymously recoded Trp53 lacking CG dinucleotides at five mutational hotspots retained transactivation, arrest and apoptosis while accumulating fewer hotspot mutations over passage. Mouse Ace2 was replaced with 116 or 180 kilobase human ACE2 loci, and the resulting animals, derived by tetraploid complementation, reproduced human tissue expression including testicular expression absent in mice, the interferon inducible dACE2 isoform, and human chromatin accessibility. These mice were infectable with SARS-CoV-2, mounted a moderate interferon response and a spike specific antibody response, and survived without weight loss, while all K18-hACE2 controls died. Mouse Tmprss2 was then replaced biallelically with human TMPRSS2 in the same line." }, "discoveries": [], "relationships": [ { "from": "2023-zhang-mouse-genome-rewriting-and-tailori", "to": "2021-hoagland-leveraging-the-antiviral-type-i-in", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2023-zhang-mouse-genome-rewriting-and-tailori" } ], "canonical_url": "https://tenoeverlab.us/publications/2023-zhang-mouse-genome-rewriting-and-tailori/", "controlled_vocabulary": { "pathogens": [ "sars-cov-2" ], "technologies": [ "bulk-rna-seq", "plaque-assay", "elisa", "immunohistochemistry", "amplicon-sequencing", "atac-seq", "chip", "bac-recombineering", "targeted-capture", "mswap-in-genome-writing", "tetraploid-complementation" ] } }, { "id": "2025-manivasagam-transcriptional-repressor-capicua-", "slug": "2025-manivasagam-transcriptional-repressor-capicua-", "url": "/publications/2025-manivasagam-transcriptional-repressor-capicua-/", "title": "Transcriptional repressor Capicua is a gatekeeper of cell-intrinsic interferon responses", "authors": [ "Senthamizharasi Manivasagam", "Julianna Han", "Athmane Teghanemt", "Henry Keen", "Boopathi Sownthirarajan", "Boyang Cheng", "Abhiraj Singh", "Abigail Lewis", "Olivia A. Vogel", "Gayathri Loganathan", "Lei Huang", "Maryline Panis", "David K. Meyerholz", "Benjamin tenOever", "Jasmine T. Perez", "Santhakumar Manicassamy", "Priya D. Issuree", "Balaji Manicassamy" ], "author_count": 18, "first_author": "Senthamizharasi Manivasagam", "senior_authors": [ "Priya D. Issuree", "Balaji Manicassamy" ], "corresponding_authors": [ "Priya D. Issuree", "Balaji Manicassamy" ], "tenoever_position": 14, "tenoever_role": "middle", "contribution_character": "collaborative", "year": 2025, "journal": "Cell Host & Microbe", "volume": "33", "issue": "4", "pages": "512-528.e7", "doi": "10.1016/j.chom.2025.02.017", "doi_url": "https://doi.org/10.1016/j.chom.2025.02.017", "pmid": "40132591", "pmid_url": "https://pubmed.ncbi.nlm.nih.gov/40132591/", "pmcid": "PMC11985295", "pmc_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11985295/", "pmc_pdf_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11985295/pdf/", "publication_type": "primary research", "declared_conflicts": null, "research_areas": [ "innate-immune-signaling" ], "themes": [ "homeostatic-repression-of-isgs" ], "pathogens": [ "influenza A virus", "respiratory syncytial virus", "human parainfluenza virus type 3", "Sendai virus", "encephalomyocarditis virus", "Zika virus", "vesicular stomatitis virus" ], "viral_families": [ "Orthomyxoviridae", "Pneumoviridae", "Paramyxoviridae", "Picornaviridae", "Flaviviridae", "Rhabdoviridae" ], "host_species": [ "human", "mouse" ], "technologies": [ "CRISPR-Cas9 knockout", "RNA sequencing", "ATAC sequencing", "promoter reporter assays", "quantitative RT-PCR", "small interfering RNA knockdown", "immunofluorescence", "western blot", "tamoxifen-inducible conditional knockout mouse", "histopathology", "motif enrichment analysis" ], "biological_systems": [ "A549 cells", "HEK293T cells", "primary human airway basal cells", "mouse embryonic fibroblasts", "bone marrow derived dendritic cells", "bone marrow derived macrophages", "mouse lung" ], "key_concepts": [ "Capicua", "ATXN1L", "transcriptional repression", "CIC binding site motif", "interferon-stimulated genes", "endogenous double-stranded RNA", "MDA5 and MAVS signaling", "EGFR-MAPK signaling", "proteasomal degradation", "chromatin accessibility", "sterile inflammation" ], "keywords": [ "Capicua", "CIC", "ATXN1L", "interferon-stimulated genes", "influenza A virus", "MAPK", "EGFR", "transcriptional repressor", "ATAC-seq", "innate immunity" ], "one_sentence_contribution": "The Capicua and ATXN1L repressor complex binds an eight-nucleotide motif at interferon and interferon-stimulated gene loci to hold them repressed during homeostasis in human and mouse cells, and is degraded through EGFR-MAPK signaling early in respiratory viral entry, which relieves that repression.", "summary_25": "A repressor protein sits on interferon gene promoters and keeps them quiet, and respiratory viruses inadvertently destroy it within minutes of touching the cell surface.", "summary_75": "Interferon genes were thought to stay silent until activating factors switch them on. Here the repressor Capicua, with its partner ATXN1L, is shown to bind a short motif near these genes and hold them closed, in human and mouse cells. Respiratory viruses activate EGFR-MAPK signaling during entry, which destroys the complex within 40 minutes and releases the brake. Mice lacking Capicua controlled influenza better, with lower lung virus and less inflammation.", "summary_150": "Aberrant interferon expression causes inflammatory disease, and the safeguards described previously act on sensing and signaling proteins rather than at the DNA. Building on a CRISPR screen that flagged Capicua as an influenza host factor, the authors show that Capicua with ATXN1L represses interferon and interferon-stimulated genes during homeostasis. Knockout cells upregulated these genes globally, in a manner dependent on MAVS, indicating tonic sensing of host-derived ligands, and showed increased chromatin accessibility at the same loci. Most affected loci carried the eight-nucleotide CIC binding site motif, and reporters bearing native motifs were repressed by Capicua and activated by a synthetic Capicua carrying a transactivation domain, with motif mutation abolishing both. Influenza A virus, respiratory syncytial virus and parainfluenza virus triggered proteasomal degradation of the complex within 40 minutes through EGFR-MAPK signaling, reproduced by hemagglutinin or EGF alone. Capicua knockout mice showed lower viral burden and less pulmonary inflammation.", "citation": "Manivasagam S, Han J, Teghanemt A, Keen H, Sownthirarajan B, Cheng B, Singh A, Lewis A, Vogel OA, Loganathan G, Huang L, Panis M, Meyerholz DK, tenOever B, Perez JT, Manicassamy S, Issuree PD, Manicassamy B. Transcriptional repressor Capicua is a gatekeeper of cell-intrinsic interferon responses. Cell Host & Microbe. 2025. Volume 33, issue 4, pages 512-528.e7. DOI 10.1016/j.chom.2025.02.017. PMID 40132591. PMCID PMC11985295.", "sections": { "Citation": "Manivasagam S, Han J, Teghanemt A, Keen H, Sownthirarajan B, Cheng B, Singh A, Lewis A, Vogel OA, Loganathan G, Huang L, Panis M, Meyerholz DK, tenOever B, Perez JT, Manicassamy S, Issuree PD, Manicassamy B. Transcriptional repressor Capicua is a gatekeeper of cell-intrinsic interferon responses. Cell Host & Microbe. 2025. Volume 33, issue 4, pages 512-528.e7. DOI 10.1016/j.chom.2025.02.017. PMID 40132591. PMCID PMC11985295.", "One-sentence contribution": "The Capicua and ATXN1L repressor complex binds an eight-nucleotide motif at interferon and interferon-stimulated gene loci to hold them repressed during homeostasis in human and mouse cells, and is degraded through EGFR-MAPK signaling early in respiratory viral entry, which relieves that repression.", "Executive summary": "Interferon and interferon-stimulated gene promoters were generally held to sit in a default off state until bound by activated interferon regulatory factors or by ISGF3, with negative regulation during homeostasis acting upstream of transcription on the sensing and signaling machinery. FOXO3 acting at the Irf7 promoter was the noted exception. The question here is whether a DNA-binding transcriptional repressor holds these loci off directly.\n\nStarting from a prior genome-wide CRISPR screen in which Capicua emerged as a host factor for influenza A virus, the authors compared control, Capicua knockout and ATXN1L knockout human lung epithelial cells, then extended to primary human airway basal cells, conditional knockout mice and three primary mouse cell types. Loss of either complex member raised interferon and interferon-stimulated gene transcripts under mock conditions and during infection, and restricted influenza A virus replication.\n\nRNA sequencing showed a global rise in innate immune genes rather than a few loci, ATAC sequencing showed matching increases in chromatin accessibility, and integrated analysis found CIC binding site motifs at the great majority of affected loci. Reporter assays with native and mutated motifs, and a synthetic transactivating Capicua, established motif dependence. During influenza A virus infection the complex was lost within 40 minutes in a proteasome-dependent manner, an effect reproduced by recombinant hemagglutinin or by EGF and blocked by MEK or ERK inhibitors. Capicua knockout mice lost less weight, carried 5 to 50-fold lower lung viral burden and showed smaller areas of inflammation.", "Scientific context": "Cell-intrinsic antiviral responses begin with RIG-I-like receptor detection of viral RNA, signaling through MAVS to activate IRF3 or IRF7, induction of type I interferon, and amplification through the interferon receptor, JAK-STAT signaling and ISGF3 acting at interferon-stimulated response elements. Aberrant activation in the absence of infection causes inflammatory disease, and the known safeguards act largely before transcription, through direct suppression of sensing components or post-translational modification, with ADAR1 editing endogenous double-stranded RNA to prevent MDA5 activation. FOXO3 repression of the Irf7 promoter was the one reported transcriptional-level exception. Capicua is an evolutionarily conserved HMG-box transcriptional repressor characterized in Drosophila, where it binds an eight-nucleotide site and is degraded through EGFR-MAPK signaling during development, and in humans it is studied mainly in cancer and neurodegeneration. The immediate precedent for this study is the authors' own genome-wide CRISPR screen for influenza A virus host factors, published by Han and colleagues in 2018 with tenOever as a co-author, in which Capicua knockout cells showed elevated interferon and interferon-stimulated genes and restricted replication of several RNA viruses.", "Central question": "Does a DNA-binding transcriptional repressor act directly at interferon and interferon-stimulated gene promoters to prevent their expression during homeostasis, and if the Capicua and ATXN1L complex does so, how is that repression lifted when a virus arrives?", "Experimental strategy": "The design proceeds from phenotype to mechanism to organism. Knockouts of Capicua and of its obligate partner ATXN1L in a human lung epithelial line establish that the phenotype belongs to the complex rather than to one protein, and complementation with each Capicua isoform confirms specificity. Separating whether the repressor acts on RIG-I-like receptor signaling or on interferon receptor signaling is done by stimulating with transfected viral RNA versus with recombinant interferon, and then genetically by making double knockouts with MAVS and with STAT1, which pins the source of the basal signal.\n\nWhether the effect is direct is addressed on three axes that are then intersected. Transcript changes come from RNA sequencing, chromatin changes from ATAC sequencing, and candidate direct targets from the presence of the Drosophila-defined CIC binding site motif. Promoter reporters with native and with mutated motifs test sufficiency, and a synthetic Capicua in which the repressor domain is swapped for VP16 transactivator repeats tests the converse, that the same motifs can be driven rather than silenced.\n\nThe relief mechanism is approached by timing the loss of the complex after synchronized infection, by pharmacology with proteasome, MEK and ERK inhibitors, by substituting recombinant hemagglutinin or EGF for virus, and by asking whether blocking degradation blunts interferon induction in control but not in knockout cells. Conservation and physiological relevance come from a tamoxifen-inducible conditional knockout mouse, three primary mouse cell types, lung RNA sequencing and a sublethal influenza A virus challenge.", "Key findings": "1. Capicua knockout and ATXN1L knockout human lung epithelial cells expressed higher IFNB1 and interferon-stimulated gene transcripts under mock conditions and after stimulation with viral RNA or with type I interferon, and restricted influenza A virus replication. Complementation with either Capicua isoform restored replication and lowered the transcripts (Figure 1C and 1D, Supplementary Figures 1 and 2). Knockdown in primary human airway basal cells reproduced the effect.\n\n2. RNA sequencing identified 800 differentially expressed genes under mock conditions and 3,608 during infection, with over 60 percent elevated in knockout cells and broad upregulation of restriction factors, antiviral transcription factors, nucleic acid sensors, cytokines and chemokines, and enrichment of IRF and ISRE motifs among the regulatory sequences (Figure 1E to 1G).\n\n3. Capicua knockout cells showed increased expression and nuclear localization of IRF2 and IRF9 without stimulation (Figure 2A). Deleting MAVS alongside Capicua returned basal IFNB1 to control levels, while deleting STAT1 lowered it only partially (Figure 2D). The authors interpret the MAVS dependence as tonic RIG-I-like receptor activation by endogenous double-stranded RNA ligands. The MAVS requirement is demonstrated. That the ligands are endogenous retroelement-derived double-stranded RNA is an inference drawn from the literature and is not directly shown here.\n\n4. Interferon treatment induced higher MX1 in the Capicua and MAVS double knockout than in control cells, indicating that Capicua also restrains genes induced through interferon receptor signaling (Figure 2E).\n\n5. ATAC sequencing found 6,992 differentially accessible peaks over 4,400 genes under mock conditions, with 3,904 showing increased accessibility in knockout cells at loci for sensors, transcription factors, interferons, interferon receptors, cytokines and restriction factors, and 11,004 peaks over 5,698 genes during infection. Peaks were predominantly intronic and distal intergenic (Figure 3A to 3C).\n\n6. Over 93 percent of the genes with differential accessibility carried the Drosophila-derived CIC binding site motif, and integrated analysis across transcript change, accessibility change and motif presence yielded 324 common genes under mock conditions and 1,108 during infection, with IRF and ISRE motifs co-enriched (Figure 3D and 3E). Motif presence is a computational assignment and is not by itself evidence of occupancy.\n\n7. Capicua with ATXN1 repressed luciferase from a synthetic interferon beta promoter carrying six copies of the motif under RIG-I or Sendai virus stimulation, and from native MX1, IFIT1 and TRIM22 promoters, while mutation of the motifs abolished repression (Figure 4A to 4D). A synthetic Capicua with VP16 transactivator repeats in place of the repressor domain activated IRF1, IRF7, MX1, IFIT1 and TRIM22 promoters without any stimulus, and failed to do so when the motifs were mutated (Figure 4E). Together these are the strongest evidence in the paper for motif-dependent action at these promoters.\n\n8. Capicua and ATXN1L protein fell from 40 minutes after synchronized influenza A virus infection, was blocked by MG132 or bortezomib, and occurred in primary human airway basal cells on the same timescale, before genome replication. Levels recovered gradually between 8 and 24 hours (Figure 5C to 5F). Type I interferon treatment caused transient loss at 40 to 60 minutes with recovery by 120 minutes (Figure 5G). The authors suggest the later recovery participates in restoring homeostasis after clearance. That is an interpretation.\n\n9. Degradation occurred with multiple influenza A virus subtypes and with respiratory syncytial virus, human parainfluenza virus type 3 and Sendai virus, but not with Zika virus, encephalomyocarditis virus or Ebola glycoprotein pseudotyped vesicular stomatitis virus (Figure 6A and 6B).\n\n10. MEK or ERK inhibition blocked infection-induced degradation, recombinant H1 or H5 hemagglutinin alone was sufficient to trigger it, and recombinant EGF caused degradation within five minutes (Figure 6C, 6D and 6F).\n\n11. MEK or ERK inhibition reduced IFNB1 and MX1 induction by Sendai virus, interferon or viral RNA in control cells but not in Capicua knockout cells, which places the inhibitor effect on the Capicua axis (Figure 6E, Supplementary Figure 4). Adding EGF during encephalomyocarditis virus or pseudotyped vesicular stomatitis virus infection increased interferon and interferon-stimulated gene induction (Figure 6G).\n\n12. In conditional knockout mice, mouse embryonic fibroblasts, bone marrow derived dendritic cells and macrophages all showed higher Ifnb1 and interferon-stimulated gene induction, and lung RNA sequencing found 5,296 differentially expressed genes under mock conditions with antiviral gene ontology enrichment, but only 61 differentially expressed genes at day 5 of infection, since control mice had by then induced these genes (Figure 7A to 7C, Supplementary Figures 5 and 6).\n\n13. Following sublethal influenza A virus challenge, Capicua knockout mice lost less weight and recovered earlier, carried 5 to 50-fold lower lung viral burden, expressed 5 to 10-fold more Ifnb1 and interferon-stimulated genes, and showed smaller areas of inflammation on histopathology (Figure 7D to 7G). In mice, MEK inhibition reduced poly(inosinic-cytidylic) acid-induced interferon-stimulated gene expression in control but not in knockout animals.", "Mechanistic model": "The model the data support is that the Capicua and ATXN1L complex occupies CIC binding site motifs at and near interferon and interferon-stimulated gene loci, holding chromatin at those loci less accessible and basal transcription low, so that tonic RIG-I-like receptor signaling driven by host-derived ligands does not escalate into an interferon response. When a respiratory virus engages its receptor, EGFR-MAPK signaling is activated within minutes, the complex is degraded by the proteasome, and the repression is lifted before the viral genome has replicated, which primes the cell for a faster and larger response.\n\nSeveral links in this chain are inferred rather than demonstrated. Direct occupancy of endogenous loci by Capicua was not measured, for example by chromatin immunoprecipitation. Motif presence was assigned computationally from a Drosophila consensus, and the functional evidence for motif dependence comes from transfected reporters rather than from the native chromatin context. The kinase or ubiquitin ligase that connects ERK activity to degradation of the complex is not identified, and the model that MAPK activation is the direct cause rests on inhibitor pharmacology plus the EGF and hemagglutinin sufficiency experiments. Why non-respiratory viruses fail to trigger degradation is attributed to differences in engagement with the MAPK pathway, which the authors state as a likely explanation rather than a finding. The identity of the endogenous double-stranded RNA ligands driving basal signaling is taken from prior literature. Finally, the causal chain from lower viral burden to milder disease in knockout mice is a correlation the authors draw, and the contribution of any developmental or immune-cell-intrinsic effects of Capicua loss is not separated.", "Conceptual or technical advance": "A negative regulator of the interferon system is placed at the DNA, rather than on the sensing and signaling proteins. That changes the default-off account of interferon and interferon-stimulated gene promoters into an actively repressed state with an identified repressor, a binding motif and a defined removal mechanism. It also connects an RTK-MAPK derepression circuit conserved from Drosophila development to vertebrate antiviral defense, and it supplies a rationale, which the authors offer as speculation, for why MEK and ERK inhibitors limit inflammation in severe respiratory viral infection, namely that they preserve the repressor. The observation that Capicua loss improves outcome in a sublethal influenza A virus challenge makes the axis a candidate for pharmacological tuning in either direction.", "Relationship to the broader research program": "The tenOever contribution is listed under investigation, alongside Maryline Panis of the same laboratory, and the study descends from a collaboration between the Manicassamy and tenOever groups, the genome-wide CRISPR screen for influenza A virus host factors published as Han and colleagues 2018, which is where Capicua first appeared. The substance of the paper touches a question the tenOever laboratory has pursued independently, namely which transcription factors set the interferon-stimulated gene program and how much of that program can run without interferon signaling. Category 3 synthesis, visible only when this paper is set beside Schmid and colleagues 2010 from the tenOever laboratory, is that the regulatory logic at interferon-stimulated response elements involves both redundancy among activating factors and, as shown here, an independent repressive layer acting through a distinct motif, so that the output at a given promoter reflects both which activators are present and whether the repressor has been removed. That combination is not drawn in either paper alone.", "Related publications": "- Han and colleagues 2018, Cell Reports, Genome-wide CRISPR/Cas9 screen identifies host factors essential for influenza virus replication. Predecessor. The screen, co-authored by tenOever, that identified Capicua and reported the elevated interferon phenotype followed up here.\n- Vogel and colleagues 2020, PLoS Pathogens, on the p150 isoform of ADAR1 and sustained RLR signaling. Conceptual extension. From the same group, addressing the parallel safeguard against endogenous double-stranded RNA, cited here for that role.\n- Schmid and colleagues 2010, Journal of Biological Chemistry. Conceptual extension. Addresses the activating side of the same promoters through IRF7 and ISGF3 motif specificity.", "Limitations and boundaries": "Direct binding of Capicua to the proposed motifs in native chromatin is not shown, so the mechanism rests on motif prediction, accessibility changes and transfected reporters. The motif itself is the Drosophila consensus applied to mammalian genomes, and 93 percent of differentially accessible genes carrying it raises the question of how discriminating the motif is. The human cellular work is centered on one lung epithelial line, with primary airway basal cells used only for knockdown and degradation timing. Degradation kinetics are inferred from western blots and immunofluorescence at a small number of time points, and no ubiquitin ligase or ERK substrate site is identified. The MAPK conclusion depends on two inhibitors whose selectivity is not established here, although the absence of effect in knockout cells substantially strengthens the interpretation. The virus panel that fails to trigger degradation is small and mechanistically unexplained. In mice, Capicua deletion is whole-body and tamoxifen-induced rather than lung-restricted, so cell-intrinsic and systemic contributions are not separated, and the infection data come from one strain at one sublethal dose, with group sizes of two to three animals for some RNA sequencing comparisons. Improved outcome in knockout mice was measured over a sublethal challenge and does not address whether chronic loss of this repression is harmful, which the authors themselves raise in connection with autoimmunity and cancer as speculation. The sequencing-based claims of global change are from n equal to two per group in the cell line experiments.", "Audience summaries": "### 25 words\n\nA repressor protein sits on interferon gene promoters and keeps them quiet, and respiratory viruses inadvertently destroy it within minutes of touching the cell surface.\n\n### 75 words\n\nInterferon genes were thought to stay silent until activating factors switch them on. Here the repressor Capicua, with its partner ATXN1L, is shown to bind a short motif near these genes and hold them closed, in human and mouse cells. Respiratory viruses activate EGFR-MAPK signaling during entry, which destroys the complex within 40 minutes and releases the brake. Mice lacking Capicua controlled influenza better, with lower lung virus and less inflammation.\n\n### 150 words\n\nAberrant interferon expression causes inflammatory disease, and the safeguards described previously act on sensing and signaling proteins rather than at the DNA. Building on a CRISPR screen that flagged Capicua as an influenza host factor, the authors show that Capicua with ATXN1L represses interferon and interferon-stimulated genes during homeostasis. Knockout cells upregulated these genes globally, in a manner dependent on MAVS, indicating tonic sensing of host-derived ligands, and showed increased chromatin accessibility at the same loci. Most affected loci carried the eight-nucleotide CIC binding site motif, and reporters bearing native motifs were repressed by Capicua and activated by a synthetic Capicua carrying a transactivation domain, with motif mutation abolishing both. Influenza A virus, respiratory syncytial virus and parainfluenza virus triggered proteasomal degradation of the complex within 40 minutes through EGFR-MAPK signaling, reproduced by hemagglutinin or EGF alone. Capicua knockout mice showed lower viral burden and less pulmonary inflammation." }, "discoveries": [ "claim-05" ], "relationships": [ { "from": "2025-manivasagam-transcriptional-repressor-capicua-", "to": "2018-han-genome-wide-crispr-cas9-screen-ide", "relationship": "predecessor", "evidence": "stated in the Related publications section of 2025-manivasagam-transcriptional-repressor-capicua-" }, { "from": "2025-manivasagam-transcriptional-repressor-capicua-", "to": "2010-schmid-transcription-factor-redundancy-en", "relationship": "conceptual extension", "evidence": "stated in the Related publications section of 2025-manivasagam-transcriptional-repressor-capicua-" } ], "canonical_url": "https://tenoeverlab.us/publications/2025-manivasagam-transcriptional-repressor-capicua-/", "controlled_vocabulary": { "pathogens": [ "influenza-a-virus", "vsv", "sendai-virus", "emcv", "hpiv3", "rsv", "zika-virus" ], "technologies": [ "rt-qpcr", "bulk-rna-seq", "immunofluorescence-microscopy", "sirna-knockdown", "luciferase-promoter-reporter", "immunoblotting", "histopathology", "crispr-knockout", "atac-seq", "conditional-knockout", "motif-analysis" ] } } ]