lab-ledInfluenza 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.
Andrew Varble; Mark A. Chua; Jasmine T. Perez; Balaji Manicassamy; Adolfo García-Sastre; Benjamin R. tenOever
2010 · Proceedings of the National Academy of Sciences · primary research
- Senior authors
- Benjamin R. tenOever
- Correspondence
- Benjamin R. tenOever
Research areas & themes
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.
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
- 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).
- 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.
- 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).
- 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).
- 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.
- 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.
- 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).
- 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.
- 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.
For 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.
Technically, 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.
Category 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.
- 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.
- 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.
- Makeyev and colleagues, 2007, on miR-124 and neuronal differentiation, cited as reference 23 and the basis for the CAD cell readout. Methodological foundation.
- 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.
- Varble and colleagues, 2013, An In Vivo RNAi Screening Approach to Identify Host Determinants of Virus Replication. Application of virus-encoded hairpin delivery.
- 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.
Every 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
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.
75 words
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.
150 words
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.
Discoveries supported by this paper
Discoverylab-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
Documented publication relationships
Pathogens
Technologies