Status lab-led for Perez 2009, Varble 2010, Langlois 2012 in PNAS, Langlois 2012 in Molecular Therapy, Pham 2012, Varble 2013, Schmid 2014, Benitez 2015 on in vivo screening, Møller 2018 and the tenOever 2019 review, and co-led for Langlois 2013, whose ferret transmission work sits with the Perez and García-Sastre groups. Daniloski 2021 in Cell is co-led with the Sanjana laboratory Areas Programmable Virology, Innate Immune Signaling and the Interferon Response
Asking what one cell type contributes to an infection is normally done by removing that cell type or a gene it needs, and both perturbations remove far more than the infection. A depleted compartment is not an animal in which the compartment is present but uninfected. Screens for host factors had converged on cultured transformed cells with surrogate readouts, and meta-analyses of nine genome-wide influenza screens found little overlap beyond the vacuolar ATPase subunits. The move that answers both problems is to put the perturbation in the pathogen, where it travels with the infection, reaches whatever cells the pathogen reaches, and is subject to the same selection.
The enabling element is a fully complementary target site for a host microRNA placed in a viral transcript, which turns a resident microRNA into a virus-specific silencing guide. Perez 2009 built such sites into the influenza nucleoprotein open reading frame at positions preserving the side chain class of the encoded amino acid, using miR-93 because published profiles place it in mouse and human and not chicken, and obtained a virus attenuated by more than two logs in mice while reaching roughly 10^8 plaque-forming units per millilitre in eggs. Its control set, a pairing-disrupted parental strain, Dicer-deficient cells and an antimiR rescue, defines what such a virus must show before any phenotype can be read, and its report that neither influenza infection nor NS1 disturbs microRNA biogenesis or silencing in mammalian cells is the licensing observation for everything after it. Langlois 2012 in PNAS turned the design into an instrument, silencing influenza only in hematopoietic cells and finding that mice cleared the virus and made normal nucleoprotein- and polymerase acidic-specific CD8 T cell responses while whole lung interferon beta and IRF-7 induction fell, a deficit its authors state they cannot explain from the size of the compartment silenced. Pham 2012 ran the same subtraction against dengue dissemination. Langlois 2013 moved the sites into a duplicated packaging region so the safety layer costs no fitness and selected miR-192 from small RNA sequencing of the exact tissues that mattered, giving a virus causing no disease in mice at ten times a lethal dose while an H3N2 version transmitted in ferrets indistinguishably from controls. Møller 2018 made an essential cytomegalovirus gene conditionally dispensable, so IE2 could be studied in macrophages despite being required in the fibroblasts used for rescue, and found that essentiality determined in fibroblasts does not transfer to the myeloid lineage.
The reciprocal line had the virus produce small RNAs rather than respond to them. Varble 2010 removed the assumed prohibition on an RNA virus encoding a microRNA by splitting the overlapping NS1 and NEP reading frames of segment 8, so the hairpin is excised in the spliced lariat rather than from the genome, and mature miR-124 appeared within four hours at levels comparable to abundant endogenous microRNAs with growth matching wild type. Langlois 2012 in Molecular Therapy carried that to a cytoplasmic negative-sense vector, reaching an estimated 25,000 to 35,000 copies per cell and five organs after intravenous delivery, and Schmid 2014 made the vector replication-incompetent and its output adjustable by installing the miR-93-targeted nucleoprotein segment from Perez 2009. The screening format follows from the same space. Varble 2013 passaged roughly 10,000 hairpin-encoding viruses through mice with a matched barcode library quantifying how much apparent reproducibility a bottlenecked passage generates on its own, and recovered Zfx and Mga as transcriptional maintenance factors. Benitez 2015 changed the selective pressure to an NS1 mutant crippled by the host response itself, so silencing a gene that contributes to that response restores fitness directly, and recovered Ifih1 with more than fiftyfold enrichment in four independent screens, lost in Ifih1 knockout mice and absent in canine cells. That result revises something two prior studies had treated as settled, since interferon beta induction during influenza infection requires RIG-I and not MDA5, which Benitez 2015 confirms, and yet MDA5 is required for full induction of Irf7, OAS isoforms, Ifit1, Stat1 and Isg15 and its loss relieves restriction. An interferon-induction readout can miss a sensor's contribution.
Boundaries are substantial and mostly stated by the papers themselves. MicroRNA silencing is potent but incomplete, so a lineage-restricted phenotype is a strong knockdown and not a null, and because miR-142 is expressed across hematopoietic lineages none of the compartment papers can assign a phenotype to one subset. Escape is bounded rather than excluded, since Perez 2009 and Langlois 2013 recovered no revertants within their sampling while Pham 2012 shows total excision under pressure. The screens are cell-autonomous, cannot report on constitutively abundant restriction factors, which the papers demonstrate with RIG-I itself, and their target assignment in Varble 2013 is computational. The delivery line reached intranasal delivery in Schmid 2014 with in vivo silencing left to future studies and no later paper here demonstrates it, the biocontainment proposal to generalise to filoviruses, coronaviruses and henipaviruses was not taken up, and tenOever 2019 records that only the screening application became a sustained program. The two CRISPR screens in this area, Han 2018 led by the Manicassamy laboratory and Daniloski 2021 co-led with the Sanjana laboratory, share the logic of fitness as readout but sit in cell culture, which is the constraint the animal platform was built to escape, so their relation to this work is convergence rather than descent.
Substantiated by - MicroRNA-mediated species-specific attenuation of influenza A virus, founds the design and the control set, and shows that infection leaves the silencing machinery available - Hematopoietic-specific targeting of influenza A virus reveals replication requirements for induction of antiviral immune responses, converts attenuation into an instrument and locates much of the in vivo interferon response in a numerically minor compartment - Replication in Cells of Hematopoietic Origin Is Necessary for Dengue Virus Dissemination, applies the subtraction to dengue dissemination and shows what escape looks like in an animal - MicroRNA-based strategy to mitigate the risk of gain-of-function influenza studies, moves the element into engineered noncoding space and demonstrates a host-conditional containment layer at no measurable fitness cost - miRNA-mediated targeting of human cytomegalovirus reveals biological host and viral targets of IE2, extends the element to a large DNA virus and turns it into lineage-restricted conditional genetics - Engineered RNA viral synthesis of microRNAs, removes the prohibition on an RNA virus encoding a microRNA and creates the segment 8 insertion space the later work occupies - In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs, delivers a functional microRNA to five organs and contains the proposal that becomes the screening platform - A Versatile RNA Vector for Delivery of Coding and Noncoding RNAs, makes the vector replication-incompetent and its output adjustable by the host silencing machinery - An In Vivo RNAi Screening Approach to Identify Host Determinants of Virus Replication, implements selection inside an animal as the assay and supplies the neutral drift control that makes it interpretable - In Vivo RNAi Screening Identifies MDA5 as a Significant Contributor to the Cellular Defense against Influenza A Virus, recovers MDA5 and shows that a sensor can be required for restriction without being required for interferon beta induction - Synthetic Virology: Building Viruses to Better Understand Them, states the premise and sorts the designs by what each can ask and whether it preserves viral fitness - Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells, co-led with the Sanjana laboratory, applies survival as selection at genome scale in culture and converges on endosomal machinery
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.