Vertebrate somatic cells do not rely on small RNA silencing against viruses, and the reason is not that such a defence would fail in a mammalian cell

Status lab-led for Backes 2014, Benitez 2015 on engineered RNA interference, Aguado 2015 and the tenOever 2013 review, co-led with the Cherry laboratory for Backes 2012, and collaborative for Cullen 2013, a Minireview led by Bryan Cullen with Sara Cherry and tenOever Areas Small RNA Biology and the Limits of Antiviral Silencing

Plants, nematodes and arthropods process viral double-stranded RNA into small interfering RNAs that guide cleavage of viral transcripts, and their viruses encode suppressors of the pathway. Vertebrates detect the same double-stranded RNA and answer it transcriptionally while retaining most of the silencing machinery. Whether they also retain a functioning antiviral arm was genuinely open, and the question was reopened in 2013 by reports from the Voinnet and Ding groups. Cullen 2013 accepts the mouse embryonic stem cell evidence, declines to settle the somatic case, and names the reason the positive claims are not decisive, which is that the viral proteins they rest on bind double-stranded RNA and also antagonise interferon, so the two explanations have not been separated.

The negative half of the answer was obtained as a fitness experiment rather than by removing host components. Backes 2014 armed vesicular stomatitis virus with vaccinia VP55, the poly(A) polymerase subunit that Backes 2012 had identified as tailing RISC-loaded small RNAs for host-supplied decay, and compared it with a virus carrying influenza NS1 as a positive control for disabling a real antiviral system. The armed virus destroyed host microRNAs efficiently in fibroblasts, primary bone marrow-derived macrophages and mouse lung and gained no replication advantage in any of them. In wild-type mice it was attenuated by about a log, attributed by messenger RNA sequencing to derepression of interferon-stimulated transcripts normally held down by microRNAs, and in mice lacking both type I and type III interferon receptors all three viruses reached comparable titres, which removes the objection that a silencing contribution might be hidden beneath interferon. The authors state that absence of an activity is difficult to prove and treat the result as a strong argument rather than a demonstration.

Aguado 2015 then established what microRNAs do during the response. Delivering VP55 from a replication-incompetent adenovirus removed roughly ninety percent of abundant microRNAs from primary human fibroblasts within a day without inducing interferon-stimulated genes. Only 12 of 1,548 genes induced by double-stranded RNA changed, and 12 of 179 induced by six hours of interferon beta, while nine days of depletion changed more than 1,700 transcripts dominated by chemokines and cytokines, with IFIH1, IRF3, IRF7, RELA, RELB, IFNB, IFNAR1, STAT2 and IRF9 unchanged even then. MicroRNA function during infection is therefore confined to cytokine output rather than to the intrinsic antiviral program, and conflicting reports are reconciled by a timescale argument, since assays run over hours will find nothing even where real targets exist.

The complementary positive result is what makes the negative one interpretable. Benitez 2015 inserted perfectly complementary target sites for host microRNAs into influenza A virus, converting resident microRNAs into cleaving guides, and separately had the virus encode a small interfering RNA against its own nucleoprotein segment. A five-site virus grew in embryonated eggs but produced no plaques and no detectable nucleoprotein in mammalian cells and caused no morbidity in mice at 25,000 plaque-forming units, including in animals lacking the type I interferon receptor, a dose the authors note is more than 2,500 times the lethal dose 50 in that background. Protection there cannot be attributed to interferon, so the simplest explanation for the historical substitution is removed. The authors frame this as a possibility claim, that chordates could have used RNA interference in place of interferon, and state explicitly that the paper does not explain why they did not. Neither half of the pair supports a conclusion on its own, and the reconstructed system departs from an endogenous one in that guides are present before the virus arrives and targets are concentrated at one internal site. The evolutionary explanation remains a hypothesis by the explicit statement of tenOever 2016, and Backes 2014 lists the absence of stem cell work among its own limitations.

Substantiated by - Degradation of Host MicroRNAs by Poxvirus Poly(A) Polymerase Reveals Terminal RNA Methylation as a Protective Antiviral Mechanism, identifies VP55 as sufficient to tail and destroy RISC-loaded microRNAs and supplies the reagent the rest of the argument depends on - The Mammalian Response to Virus Infection Is Independent of Small RNA Silencing, shows that destroying host microRNAs gives a virus no fitness benefit, including in animals lacking both interferon receptors - microRNA Function Is Limited to Cytokine Control in the Acute Response to Virus Infection, shows that removing microRNAs leaves the intrinsic antiviral program intact and derepresses chemokines and cytokines instead - Engineered Mammalian RNAi Can Elicit Antiviral Protection that Negates the Requirement for the Interferon Response, shows that a reconstructed slicing defence protects mice with no contribution from type I interferon signalling - RNA viruses and the host microRNA machinery, argues from copy number, silencing capacity and kinetics that chordate microRNAs cannot be antiviral - Is RNA Interference a Physiologically Relevant Innate Antiviral Immune Response in Mammals?, led by Bryan Cullen, sets the evidentiary criteria and states why the positive claims then available did not meet them

Research areas

Supporting publications