Letter | Published:

RNase III nucleases from diverse kingdoms serve as antiviral effectors

Nature volume 547, pages 114117 (06 July 2017) | Download Citation

Abstract

In contrast to the DNA-based viruses in prokaryotes, the emergence of eukaryotes provided the necessary compartmentalization and membranous environment for RNA viruses to flourish, creating the need for an RNA-targeting antiviral system1,2. Present day eukaryotes employ at least two main defence strategies that emerged as a result of this viral shift, namely antiviral RNA interference and the interferon system2. Here we demonstrate that Drosha and related RNase III ribonucleases from all three domains of life also elicit a unique RNA-targeting antiviral activity. Systemic evolution of ligands by exponential enrichment of this class of proteins illustrates the recognition of unbranched RNA stem loops. Biochemical analyses reveal that, in this context, Drosha functions as an antiviral clamp, conferring steric hindrance on the RNA-dependent RNA polymerases of diverse positive-stranded RNA viruses. We present evidence for cytoplasmic translocation of RNase III nucleases in response to virus in diverse eukaryotes including plants, arthropods, fish, and mammals. These data implicate RNase III recognition of viral RNA as an antiviral defence that is independent of, and possibly predates, other known eukaryotic antiviral systems.

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References

  1. 1.

    , , & The Big Bang of picorna-like virus evolution antedates the radiation of eukaryotic supergroups. Nat. Rev. Microbiol. 6, 925–939 (2008)

  2. 2.

    The evolution of antiviral defense systems. Cell Host Microbe 19, 142–149 (2016)

  3. 3.

    RNA viruses and the host microRNA machinery. Nat. Rev. Microbiol. 11, 169–180 (2013)

  4. 4.

    & On the origin and functions of RNA-mediated silencing: from protists to man. Curr. Genet. 50, 81–99 (2006)

  5. 5.

    , , & Natural selection drives extremely rapid evolution in antiviral RNAi genes. Curr. Biol. 16, 580–585 (2006)

  6. 6.

    , , & Evidence for a cytoplasmic microprocessor of pri-miRNAs. RNA 18, 1338–1346 (2012)

  7. 7.

    , , & Noncanonical cytoplasmic processing of viral microRNAs. RNA 16, 2068–2074 (2010)

  8. 8.

    , & Functional microRNA generated from a cytoplasmic RNA virus. Nucleic Acids Res. 38, 8328–8337 (2010)

  9. 9.

    , , , & Derivation and characterization of Dicer- and microRNA-deficient human cells. RNA 20, 923–937 (2014)

  10. 10.

    et al. The Drosha–DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027 (2004)

  11. 11.

    , , & Post-transcriptional control of DGCR8 expression by the Microprocessor. RNA 15, 1005–1011 (2009)

  12. 12.

    , , & Phosphorylation of the RNase III enzyme Drosha at serine300 or serine302 is required for its nuclear localization. Nucleic Acids Res. 38, 6610–6619 (2010)

  13. 13.

    et al. RNase III: genetics and function; structure and mechanism. Annu. Rev. Genet. 47, 405–431 (2013)

  14. 14.

    , & Modification of the 5′ terminus of Sindbis virus genomic RNA allows nsP4 RNA polymerases with nonaromatic amino acids at the N terminus to function in RNA replication. J. Virol. 77, 2301–2309 (2003)

  15. 15.

    , , & Kissing-loop interaction between 5′ and 3′ ends of tick-borne Langat virus genome ‘bridges the gap’ between mosquito- and tick-borne flaviviruses in mechanisms of viral RNA cyclization: applications for virus attenuation and vaccine development. Nucleic Acids Res. 44, 3330–3350 (2016)

  16. 16.

    et al. Expression of the zinc-finger antiviral protein inhibits alphavirus replication. J. Virol. 77, 11555–11562 (2003)

  17. 17.

    , & Requirement for the amino-terminal domain of sindbis virus nsP4 during virus infection. J. Virol. 85, 3449–3460 (2011)

  18. 18.

    et al. Stem-loop recognition by DDX17 facilitates miRNA processing and antiviral defense. Cell 158, 764–777 (2014)

  19. 19.

    et al. Drosha as an interferon-independent antiviral factor. Proc. Natl Acad. Sci. USA 111, 7108–7113 (2014)

  20. 20.

    et al. Dicer-2 processes diverse viral RNA species. PLoS ONE 8, e55458 (2013)

  21. 21.

    , & Evolution of MDA-5/RIG-I-dependent innate immunity: independent evolution by domain grafting. Proc. Natl Acad. Sci. USA 105, 17040–17045 (2008)

  22. 22.

    et al. Inactivation of the type I interferon pathway reveals long double-stranded RNA-mediated RNA interference in mammalian cells. EMBO J. 35, 2505–2518 (2016)

  23. 23.

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

  24. 24.

    et al. An in vivo RNAi screening approach to identify host determinants of virus replication. Cell Host Microbe 14, 346–356 (2013)

  25. 25.

    , , , & Bax-independent inhibition of apoptosis by Bcl-XL. Nature 379, 554–556 (1996)

  26. 26.

    & A novel procedure for the localization of viral RNAs in protoplasts and whole plants. Plant J. 35, 665–673 (2003)

  27. 27.

    et al. MicroRNA-mediated species-specific attenuation of influenza A virus. Nat. Biotechnol. 27, 572–576 (2009)

  28. 28.

    & Entry is a rate-limiting step for viral infection in a Drosophila melanogaster model of pathogenesis. Nat. Immunol. 5, 81–87 (2004)

  29. 29.

    , , & The RNAseIII enzyme Drosha is critical in T cells for preventing lethal inflammatory disease. J. Exp. Med. 205, 2005–2017 (2008)

  30. 30.

    , , , & FAK nuclear export signal sequences. FEBS Lett. 582, 2402–2406 (2008)

  31. 31.

    & Improved northern blot method for enhanced detection of small RNA. Nat. Protocols 3, 1077–1084 (2008)

  32. 32.

    et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011)

  33. 33.

    & Hepatitis C virus subverts liver-specific miR-122 to protect the viral genome from exoribonuclease Xrn2. Cell Host Microbe 16, 257–264 (2014)

  34. 34.

    SELEX to identify protein-binding sites on RNA. Cold Spring Harb. Protoc. 2013, 156–163 (2013)

  35. 35.

    Electrophoretic mobility shift assays for RNA-protein complexes. Cold Spring Harb. Protoc. 2014, 435–440 (2014)

  36. 36.

    et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003)

  37. 37.

    & Requirements at the 3′ end of the sindbis virus genome for efficient synthesis of minus-strand RNA. J. Virol. 79, 4630–4639 (2005)

  38. 38.

    & in Current Protocols in Microbiology Ch. 16, Unit 16D.1 (Wiley, 2006)

  39. 39.

    , , & Turbo FISH: a method for rapid single molecule RNA FISH. PLoS ONE 8, e75120 (2013)

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Acknowledgements

We thank J. K. Lim and A. G. Pletnev for Langat virus reagents, B. Lee for Sendai virus reagents, R. W. Hardy for SINV reagents, B. R. Cullen for NoDice cells, K. K. Conzelmann for BSR-T7 cells, and B. Ramratnam for pEGFP–Drosha. Recombinant IFN-β was provided by the National Institute of Health’s Biodefense and Emerging Infections Research Resources Repository (HuIFN-β, NR-3080). This material is based upon work supported in part by the Burroughs Wellcome Fund, which provides support for both S.C. and B.R.T. S.C. is also supported by the National Institute of Allergy and Infectious Diseases (NIAID) (R01A1074951). J.P.L. is supported by the DIM Malinf, Conseil Regional d’Ile-de-France. L.C.A. is partly supported by the American Heart Association (15PRE24930012). A.E.S. is supported by National Science Foundation (MCB-1411836) and NIAID (R21AI117882). J.M. is supported by National Institute of General Medicine (F32 GM119235). B.R.T. is also supported by NIAID (R01AI110575).

Author information

Author notes

    • Lauren C. Aguado
    •  & Sonja Schmid

    These authors contributed equally to this work.

Affiliations

  1. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA

    • Lauren C. Aguado
    • , Sonja Schmid
    • , Maryline Panis
    • , Daniel Blanco-Melo
    •  & Benjamin R. tenOever
  2. Department of Cell Biology and Molecular Genetics, University of Maryland College Park, College Park, Maryland 20742, USA

    • Jared May
    •  & Anne E. Simon
  3. Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    • Leah R. Sabin
    •  & Sara Cherry
  4. Department of Pharmacology and Therapeutics, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA

    • Jaehee V. Shim
  5. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA

    • David Sachs
  6. Macrophages et Développement de l’Immunité, Institut Pasteur, CNRS UMR3738, 25–28 rue du Dr. Roux, 75724 Paris Cedex 15, France

    • Jean-Pierre Levraud

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Contributions

L.C.A. and S.S. designed and conducted experiments. M.P. performed SELEX. J.M., L.C.A., and A.E.S. were responsible for plant data. L.S. and S.C. generated the Drosophila data. J.V.S. generated the RNaseIII−/− cells. J.P.L. and L.C.A. performed the zebrafish work. D.S. and D.B.M. were responsible for all bioinformatics. B.R.T., L.C.A., and S.S. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Benjamin R. tenOever.

Reviewer Information Nature thanks B. Cullen, L. Maraffini and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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DOI

https://doi.org/10.1038/nature22990

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