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Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells

Nature Microbiology volume 2, Article number: 16250 (2017) Cite this article

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Abstract

Influenza A virus (IAV) causes annual epidemics and occasional pandemics, and is one of the best-characterized human RNA viral pathogens1. However, a physiologically relevant role for the RNA interference (RNAi) suppressor activity of the IAV non-structural protein 1 (NS1), reported over a decade ago2, remains unknown3. Plant and insect viruses have evolved diverse virulence proteins to suppress RNAi as their hosts produce virus-derived small interfering RNAs (siRNAs) that direct specific antiviral defence4–7 by an RNAi mechanism dependent on the slicing activity of Argonaute proteins (AGOs)8,9. Recent studies have documented induction and suppression of antiviral RNAi in mouse embryonic stem cells and suckling mice10,11. However, it is still under debate whether infection by IAV or any other RNA virus that infects humans induces and/or suppresses antiviral RNAi in mature mammalian somatic cells12–21. Here, we demonstrate that mature human somatic cells produce abundant virus-derived siRNAs co-immunoprecipitated with AGOs in response to IAV infection. We show that the biogenesis of viral siRNAs from IAV double-stranded RNA (dsRNA) precursors in infected cells is mediated by wild-type human Dicer and potently suppressed by both NS1 of IAV as well as virion protein 35 (VP35) of Ebola and Marburg filoviruses. We further demonstrate that the slicing catalytic activity of AGO2 inhibits IAV and other RNA viruses in mature mammalian cells, in an interferon-independent fashion. Altogether, our work shows that IAV infection induces and suppresses antiviral RNAi in differentiated mammalian somatic cells.

We found that 93.6% of the 41,324 virus reads, cloned by a protocol requiring the presence of monophosphates at the 5′ termini, were in the 21 to 23 nt size range of Dicer products, with 22 nt as the most dominant size for both positive and negative strands (Fig. 1a, left; Supplementary Table 1). The 22 nt RNAs of IAV exhibited a strong preference for uracil as the 5′-terminal nucleotide (1U) (71.5%, or 62.9% for 21–23 nt vsiRNAs, Supplementary Table 1) and were highly enriched for 20 nt perfect base-paired duplexes with 2 nt 3′ overhangs (Fig. 1a, left). The influenza vsiRNAs were abundant, representing 0.34% of the total sequenced reads and equal to 0.81% of the total mature miRNA content in the library (Supplementary Table 1). Moreover, 91.8% of the virus reads were derived from the terminal 100 nt regions of the eight virion RNA segments (Fig. 1c, Supplementary Table 1 and Supplementary Fig. 1), and these terminal virus reads formed successive (or phased) complementary pairs of vsiRNAs (Fig. 1d and Supplementary Fig. 2). These results suggest that the terminal viral dsRNA replicative intermediates served as the predominant precursors of the influenza vsiRNAs, similar to those terminal vsiRNAs produced by mouse embryonic stem cells to target encephalomyocarditis virus (EMCV)11. Approximately 65% of the vsiRNA reads were mapped to the shortest genome segment NS, which is 418 nt in length after the deletion of the NS1 gene. In particular, we measured 20,048 reads for the second pair of 22 nt vsiRNAs from the 3′ terminus, a number similar to those detected for microRNA 221-3p (20,206), the 22nd most abundant miRNA in the AGO co-IP library. Nevertheless, the total reads mapped to the remaining seven genome segments also exhibited the canonical properties of vsiRNAs (Supplementary Table 2 and Supplementary Figs 3 and 4).

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Figure 1: Production of viral siRNAs in mature human somatic cells.
Figure 2: Wild-type (WT) hDicer is necessary and sufficient for the biogenesis of human vsiRNAs in differentiated somatic cells.
Figure 3: Induction and suppression of influenza vsiRNA biogenesis in distinct human and monkey somatic cells.
Figure 4: AGO2 slicing activity restricts IAV, EMCV and VSV in mammalian somatic cells.

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  • 14 July 2017

    In the PDF version of this article previously published, the year of publication provided in the footer of each page and in the 'How to cite' section was erroneously given as 2017, it should have been 2016. This error has now been corrected. The HTML version of the article was not affected.

References

  1. Medina, R. A. & García-Sastre, A. Influenza A viruses: new research developments. Nat. Rev. Microbiol. 9, 590–603 (2011).

    Article  CAS  Google Scholar 

  2. Li, W. X. et al. Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc. Natl Acad. Sci. USA 101, 1350–1355 (2004).

    Article  CAS  Google Scholar 

  3. Marc, D. Influenza virus non-structural protein NS1: interferon antagonism and beyond. J. Gen. Virol. 95, 2594–2611 (2014).

    Article  CAS  Google Scholar 

  4. Lindenbach, B. D. & Rice, C. M. RNAi targeting an animal virus: news from the front. Mol. Cell 9, 925–927 (2002).

    Article  CAS  Google Scholar 

  5. Haasnoot, J., Westerhout, E. M. & Berkhout, B. RNA interference against viruses: strike and counterstrike. Nat. Biotechnol. 25, 1435–1443 (2007).

    Article  CAS  Google Scholar 

  6. Ding, S.-W. RNA-based antiviral immunity. Nat. Rev. Immunol. 10, 632–644 (2010).

    Article  CAS  Google Scholar 

  7. Csorba, T., Kontra, L. & Burgyán, J. Viral silencing suppressors: tools forged to fine-tune host–pathogen coexistence. Virology 479–480, 85–103 (2015).

    Article  Google Scholar 

  8. Carbonell, A. et al. Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. Plant Cell 24, 3613–3629 (2012).

    Article  CAS  Google Scholar 

  9. Marques, J. T. et al. Functional specialization of the small interfering RNA pathway in response to virus infection. PLoS Pathog. 9, e1003579 (2013).

    Article  CAS  Google Scholar 

  10. Li, Y., Lu, J., Han, Y., Fan, X. & Ding, S.-W. RNA interference functions as an antiviral immunity mechanism in mammals. Science 342, 231–234 (2013).

    Article  CAS  Google Scholar 

  11. Maillard, P. V. et al. Antiviral RNA interference in mammalian cells. Science 342, 235–238 (2013).

    Article  CAS  Google Scholar 

  12. Pfeffer, S. et al. Identification of microRNAs of the herpesvirus family. Nat. Methods 2, 269–276 (2005).

    Article  CAS  Google Scholar 

  13. Parameswaran, P. et al. Six RNA viruses and forty-one hosts: viral small RNAs and modulation of small RNA repertoires in vertebrate and invertebrate systems. PLoS Pathog. 6, e1000764 (2010).

    Article  Google Scholar 

  14. Umbach, J. L., Yen, H. L., Poon, L. L. & Cullen, B. R. Influenza A virus expresses high levels of an unusual class of small viral leader RNAs in infected cells. mBio 1, e00204-10 (2010).

    Article  Google Scholar 

  15. Perez, J. T. et al. Influenza A virus-generated small RNAs regulate the switch from transcription to replication. Proc. Natl Acad. Sci. USA 107, 11525–11530 (2010).

    Article  CAS  Google Scholar 

  16. Girardi, E., Chane-Woon-Ming, B., Messmer, M., Kaukinen, P. & Pfeffer, S. Identification of RNase L-dependent, 3′-end-modified, viral small RNAs in Sindbis virus-infected mammalian cells. mBio 4, e00698-13 (2013).

    Article  Google Scholar 

  17. Seo, G. J. et al. Reciprocal inhibition between intracellular antiviral signaling and the RNAi machinery in mammalian cells. Cell Host Microbe 14, 435–445 (2013).

    Article  CAS  Google Scholar 

  18. Bogerd, H. P. et al. Replication of many human viruses is refractory to inhibition by endogenous cellular microRNAs. J. Virol. 88, 8065–8076 (2014).

    Article  Google Scholar 

  19. Backes, S. et al. The mammalian response to virus infection is independent of small RNA silencing. Cell Rep. 8, 114–125 (2014).

    Article  CAS  Google Scholar 

  20. Tanguy, M. & Miska, E. A. Antiviral RNA interference in animals: piecing together the evidence. Nat. Struct. Mol. Biol. 20, 1239–1241 (2013).

    Article  CAS  Google Scholar 

  21. Sagan, S. M. & Sarnow, P. Molecular biology. RNAi, antiviral after all. Science 342, 207–208 (2013).

    Article  CAS  Google Scholar 

  22. Delgadillo, M. O., Saenz, P., Salvador, B., Garcia, J. A. & Simon-Mateo, C. Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants. J. Gen. Virol. 85, 993–999 (2004).

    Article  CAS  Google Scholar 

  23. Bucher, E., Hemmes, H., de Haan, P., Goldbach, R. & Prins, M. The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants. J. Gen. Virol. 85, 983–991 (2004).

    Article  CAS  Google Scholar 

  24. de Vries, W., Haasnoot, J., Fouchier, R., de Haan, P. & Berkhout, B. Differential RNA silencing suppression activity of NS1 proteins from different influenza A virus strains. J. Gen. Virol. 90, 1916–1922 (2009).

    Article  CAS  Google Scholar 

  25. Kennedy, E. M. et al. Production of functional small interfering RNAs by an amino-terminal deletion mutant of human Dicer. Proc. Natl Acad. Sci. USA 112, E6945–E6954 (2015).

    Article  CAS  Google Scholar 

  26. Aliyari, R. et al. Mechanism of induction and suppression of antiviral immunity directed by virus-derived small RNAs in Drosophila. Cell Host Microbe 4, 387–397 (2008).

    Article  CAS  Google Scholar 

  27. Garcia-Sastre, A. et al. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252, 324–330 (1998).

    Article  CAS  Google Scholar 

  28. Bogerd, H. P., Whisnant, A. W., Kennedy, E. M., Flores, O. & Cullen, B. R. Derivation and characterization of Dicer- and microRNA-deficient human cells. RNA 20, 923–937 (2014).

    Article  CAS  Google Scholar 

  29. Liu, Q. & Paroo, Z. Biochemical principles of small RNA pathways. Annu. Rev. Biochem. 79, 295–319 (2010).

    Article  CAS  Google Scholar 

  30. Galiana-Arnoux, D., Dostert, C., Schneemann, A., Hoffmann, J. A. & Imler, J. L. Essential function in vivo for Dicer-2 in host defense against RNA viruses in drosophila. Nat. Immunol. 7, 590–597 (2006).

    Article  CAS  Google Scholar 

  31. Wang, X. H. et al. RNA interference directs innate immunity against viruses in adult Drosophila. Science 312, 452–454 (2006).

    Article  CAS  Google Scholar 

  32. Lee, Y. S. et al. Distinct roles for Drosophila dicer-1 and dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69–81 (2004).

    Article  CAS  Google Scholar 

  33. Girardi, E. et al. Cross-species comparative analysis of dicer proteins during Sindbis virus infection. Sci. Rep. 5, 10693 (2015).

    Article  Google Scholar 

  34. Flemr, M. et al. A retrotransposon-driven dicer isoform directs endogenous small interfering RNA production in mouse oocytes. Cell 155, 807–816 (2013).

    Article  CAS  Google Scholar 

  35. Haasnoot, J. et al. The Ebola virus VP35 protein is a suppressor of RNA silencing. PLoS Pathog. 3, e86 (2007).

    Article  Google Scholar 

  36. Gurtan, A. M., Lu, V., Bhutkar, A. & Sharp, P. A. In vivo structure–function analysis of human Dicer reveals directional processing of precursor miRNAs. RNA 18, 1116–1122 (2012).

    Article  CAS  Google Scholar 

  37. Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).

    Article  CAS  Google Scholar 

  38. Cheloufi, S., Dos Santos, C. O., Chong, M. M. & Hannon, G. J. A Dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 465, 584–589.

  39. O'Carroll, D. et al. A slicer-independent role for Argonaute 2 in hematopoiesis and the microRNA pathway. Genes Dev. 21, 1999–2004 (2007).

    Article  CAS  Google Scholar 

  40. Quinlivan, M. et al. Attenuation of equine influenza viruses through truncations of the NS1 protein. J. Virol. 79, 8431–8439 (2005).

    Article  CAS  Google Scholar 

  41. Otsuka, M. et al. Hypersusceptibility to vesicular stomatitis virus infection in Dicer1-deficient mice is due to impaired miR24 and miR93 expression. Immunity 27, 123–134 (2007).

    Article  CAS  Google Scholar 

  42. Prins, K. C. et al. Mutations abrogating VP35 interaction with double-stranded RNA render Ebola virus avirulent in guinea pigs. J. Virol. 84, 3004–3015 (2010).

    Article  CAS  Google Scholar 

  43. Liu, X., Jiang, F., Kalidas, S., Smith, D. & Liu, Q. Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes. RNA 12, 1514–1520 (2006).

    Article  CAS  Google Scholar 

  44. Liu, X. et al. Dicer-1, but not Loquacious, is critical for assembly of miRNA-induced silencing complexes. RNA 13, 2324–2329 (2007).

    Article  CAS  Google Scholar 

  45. Li, Y., Anderson, D. H., Liu, Q. & Zhou, Y. Mechanism of influenza A virus NS1 protein interaction with the p85β, but not the p85α, subunit of phosphatidylinositol 3-kinase (PI3K) and up-regulation of PI3K activity. J. Biol. Chem. 283, 23397–23409 (2008).

    Article  CAS  Google Scholar 

  46. Pall, G. S., Codony-Servat, C., Byrne, J., Ritchie, L. & Hamilton, A. Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res. 35, e60 (2007).

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank L.A. Ball, C. Basler, B.R. Cullen, A. Garcia-Sastre, C. Rice, M. McDonald, K.L. Johnson, Q. Liu and P. Palese for providing materials, A. Tarakhovsky for scientific discussions and support, and F. Uhl and A.E. Handte-Reinecker for technical assistance. G. Hannon provided Ago2D587A MEF lines. This study was supported by NIH grants R01AI107087 (to K.L.J.), MGH Executive Committee on Research (ECOR) funds (to K.L.J.), R01AI52447 (to S.W.D.) and R56AI110579 (to S.W.D.), CNAS of UC Riverside (to S.W.D.), AI113333 and DK068181 (to H.C.R.), a Department of Defense PRCRP fellowship CA120212 (to S.C.) and NHMRC grants 1027020 and 1083596 (to P.H.).

Author information

Author notes

  1. Yang Li, Megha Basavappa, Jinfeng Lu and Shuwei Dong: These authors contributed equally to this work.

Authors and Affiliations

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

    Yang Li, Jinfeng Lu, Shuwei Dong, Yanhong Han, Wan-Xiang Li & Shou-Wei Ding

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

    Yang Li

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

    Megha Basavappa, D. Alexander Cronkite, John T. Prior, Hans-Christian Reinecker & Kate L. Jeffrey

  4. Graduate Program in Genetics, Genomics, and Bioinformatics, University of California, Riverside, 92521, California, USA

    Jinfeng Lu & Shou-Wei Ding

  5. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, 3168, Victoria, Australia

    Paul Hertzog

  6. Massachusetts General Hospital, Cancer Center and Center for Regenerative Medicine and Harvard Stem Cell Institute, 185 Cambridge Street, Boston, 02114, Massachusetts, USA

    Sihem Cheloufi

  7. Department of Cell Biology and Neuroscience, University of California, Riverside, 92521, California, USA

    Fedor V. Karginov

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Contributions

Y.L. and S.D. performed all virus infection experiments in 293T, A549 and Vero cells. M.B. performed and analysed all virus infection experiments in Ago2D597A cells. J.L. performed all bioinformatic analyses of small RNA libraries. Y.H., W.-X.L. and F.V.K. assisted with cloning of small RNAs. D.A.C. and J.T.P. assisted with viral infections. H.C.R and P.H. provided reagents and interpreted results. S.C. provided Ago2D597A MEFs. S.W.D. and K.L.J conceived of the study, designed experiments, interpreted results and wrote the final manuscript.

Corresponding authors

Correspondence to Shou-Wei Ding or Kate L. Jeffrey.

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Supplementary Tables 1 and 2, Supplementary Figures 1–11. (PDF 14391 kb)

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Li, Y., Basavappa, M., Lu, J. et al. Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells. Nat Microbiol 2, 16250 (2017). https://doi.org/10.1038/nmicrobiol.2016.250

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