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Reply to ‘Questioning antiviral RNAi in mammals’

Nature Microbiology volume 2, Article number: 17053 (2017) Cite this article

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Jeffrey et al . reply — Benjamin tenOever purports to comment on our claim that “mammals elicit a small RNA-mediated response to RNA virus infection in somatic cells”. Our article1 is a follow-up of two published papers in 2013, which provided the first evidence for an antiviral function of RNA interference (RNAi) in mammals2,3. The 2013 studies demonstrated production of canonical virus-derived small interfering RNAs (siRNAs) in suckling mice and cultured mouse embryonic stem cells (mESCs) and hamster cells following infection with positive-strand RNA viruses. Production of the viral siRNAs in all three host systems was strongly inhibited by the B2 protein of Nodamura virus (NoV), known previously to suppress antiviral RNAi in insect cells and siRNA-induced RNAi in mammalian cells4–6. Notably, the suppressor activity of B2 is required for NoV infection in all three systems and deletion of Argonaute 2 (AGO2) in mESCs enhanced accumulation of the B2-deletion mutant of NoV significantly more than wild-type NoV, indicating B2 suppression of an AGO2-dependent antiviral RNAi mechanism in mammalian cells2,3. However, many questions remain to be addressed in mammalian antiviral RNAi. Our new study aimed firstly to understand why many previous deep sequencing studies were unable to detect viral siRNAs in mature human somatic cells infected with a range of RNA viruses. These unsuccessful attempts, including one by tenOever and colleagues, to deep sequence small RNAs from human A549 cells infected with wild-type A/Puerto Rico/8/1934(H1N1, PR8) strain of influenza A virus (IAV)7 have led to the idea that the conserved machinery of RNAi is unable to detect RNA virus infection in interferon (IFN)-competent mammalian somatic cells.

Our study1 has shown that human HEK-293T, A549 cells and monkey Vero cells produce highly abundant viral siRNAs after infection with either the same PR8 strain of IAV or a related WSN strain, A/WASN/1933(H1N1). Two technical improvements were critical for our success. Firstly, host cells needed to be infected with a mutant IAV lacking function of the viral non-structural protein 1 (NS1), known to suppress antiviral RNAi in insect cells and siRNA-induced RNAi in mammalian cells4,8. Secondly, the small RNAs that are not specifically associated with AGOs and the RNA-induced silencing complex (RISC) needed to be removed by only including AGO co-immunoprecipitation RNAs into the construction of small RNA libraries for sequencing. Using this approach, the relative abundance of influenza viral siRNAs in the mature human somatic cells is comparable to those found in mESCs3 and Drosophila cells9. Notably, single species positive- and negative-strand influenza viral siRNAs are readily detectable by northern blot hybridization using regular RNA probes1, a key criteria used in microRNA (miRNA) annotation10. We demonstrated that the influenza viral siRNAs become undetectable by either deep sequencing or northern blotting in Dicer knockout cells, and that the defect in the viral siRNA biogenesis was restored by ectopic expression of human Dicer. Drosophila Dicer-2, but not Dicer-1, which are responsible for the biogenesis of viral siRNAs and cellular miRNAs, respectively11, could also produce influenza viral siRNAs in human cells, although the predominant size shifts from 22 nucleotides (nt), made by human Dicer, to 21 nt by Drosophila Dicer-2 (ref. 1). Moreover, the sequenced influenza viral siRNAs are highly enriched for the canonical siRNA duplexes with 2-nt 3′-overhangs1, supporting the proposed model in which viral siRNAs are produced by human Dicer that uses double-stranded RNA (dsRNA) viral replicative intermediates as precursors. Consistent with the biogenesis of mammalian miRNAs by the same Dicer enzyme, Dicer-produced influenza viral siRNAs are abundantly loaded into AGOs and exhibit strong preference for uracil as the 5′-terminal nucleotide1.

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Authors and Affiliations

  1. Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, 02114, Massachusetts, USA

    Kate L. Jeffrey

  2. State Key Laboratory of Genetic Engineering, Collaborative Innovation Centre of Genetics and Development, School of Life Sciences, Fudan University, Shanghai, 200438, China

    Yang Li

  3. Department of Plant Pathology & Microbiology, and Institute for Integrative Genome Biology, University of California, Riverside, 92521, California, USA

    Shou-wei Ding

Authors
  1. Kate L. Jeffrey
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  2. Yang Li
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  3. Shou-wei Ding
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Correspondence to Kate L. Jeffrey, Yang Li or Shou-wei Ding.

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Jeffrey, K., Li, Y. & Ding, Sw. Reply to ‘Questioning antiviral RNAi in mammals’. Nat Microbiol 2, 17053 (2017). https://doi.org/10.1038/nmicrobiol.2017.53

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