Letter | Published:

Structural basis of an essential interaction between influenza polymerase and Pol II CTD

Nature volume 541, pages 117121 (05 January 2017) | Download Citation

Abstract

The heterotrimeric influenza polymerase (FluPol), comprising subunits PA, PB1 and PB2, binds to the conserved 5′ and 3′ termini (the ‘promoter’) of each of the eight single-stranded viral RNA (vRNA) genome segments and performs both transcription and replication of vRNA in the infected cell nucleus1,2,3. To transcribe viral mRNAs, FluPol associates with cellular RNA polymerase II (Pol II)4,5,6,7, which enables it to take 5′-capped primers from nascent Pol II transcripts8,9. Here we present a co-crystal structure of bat influenza A polymerase bound to a Pol II C-terminal domain (CTD) peptide mimic, which shows two distinct phosphoserine-5 (SeP5)-binding sites in the polymerase PA subunit, accommodating four CTD heptad repeats overall. Mutagenesis of the SeP5-contacting basic residues (PA K289, R454, K635 and R638) weakens CTD repeat binding in vitro without affecting the intrinsic cap-primed (transcription) or unprimed (replication) RNA synthesis activity of recombinant polymerase, whereas in cell-based minigenome assays the same mutations substantially reduce overall polymerase activity. Only recombinant viruses with a single mutation in one of the SeP5-binding sites can be rescued, but these viruses are severely attenuated and genetically unstable. Several previously described mutants that modulate virulence can be rationalized by our results, including a second site mutation (PA(C453R)) that enables the highly attenuated mutant virus (PA(R638A)) to revert to near wild-type infectivity10. We conclude that direct binding of FluPol to the SeP5 Pol II CTD is fine-tuned to allow efficient viral transcription and propose that the CTD-binding site on FluPol could be targeted for antiviral drug development.

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References

  1. 1.

    , & At the centre: influenza A virus ribonucleoproteins. Nat. Rev. Microbiol. 13, 28–41 (2015)

  2. 2.

    & The RNA synthesis machinery of negative-stranded RNA viruses. Virology 479-480, 532–544 (2015)

  3. 3.

    & Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis. Nat. Rev. Microbiol. 14, 479–493 (2016)

  4. 4.

    , & Inhibition of influenza virus replication by alpha-amanitin: mode of action. Proc. Natl Acad. Sci. USA 69, 1421–1424 (1972)

  5. 5.

    , & Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II. J. Virol. 79, 5812–5818 (2005)

  6. 6.

    et al. Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies. Virology 394, 154–163 (2009)

  7. 7.

    , , , & Influenza virus inhibits RNA polymerase II elongation. Virology 351, 210–217 (2006)

  8. 8.

    , , & A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell 23, 847–858 (1981)

  9. 9.

    et al. Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature 516, 361–366 (2014)

  10. 10.

    , , , & A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase promotes the generation of defective interfering RNAs. J. Virol. 77, 5017–5020 (2003)

  11. 11.

    et al. Influenza A virus preferentially snatches noncoding RNA caps. RNA 21, 2067–2075 (2015)

  12. 12.

    & Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation. Eur. J. Biochem . 270, 3859–3870 (2003)

  13. 13.

    , & Cap completion and C-terminal repeat domain kinase recruitment underlie the initiation-elongation transition of RNA polymerase II. Mol. Cell. Biol. 33, 3805–3816 (2013)

  14. 14.

    , & Function and control of RNA polymerase II C-terminal domain phosphorylation in vertebrate transcription and RNA processing. Mol. Cell. Biol. 34, 2488–2498 (2014)

  15. 15.

    et al. Molecular basis of transcription-coupled pre-mRNA capping. Mol. Cell 58, 1079–1089 (2015)

  16. 16.

    , & Influenza virus infection causes specific degradation of the largest subunit of cellular RNA polymerase II. J. Virol. 81, 5315–5324 (2007)

  17. 17.

    , , & Mechanisms and functional implications of the degradation of host RNA polymerase II in influenza virus infected cells. Virology 396, 125–134 (2010)

  18. 18.

    , , & Structure of influenza A polymerase bound to the viral RNA promoter. Nature 516, 355–360 (2014)

  19. 19.

    , , , & A structural perspective of CTD function. Genes Dev. 19, 1401–1415 (2005)

  20. 20.

    , & RNA-free and ribonucleoprotein-associated influenza virus polymerases directly bind the serine-5-phosphorylated carboxyl-terminal domain of host RNA polymerase II. J. Virol. 90, 6014–6021 (2016)

  21. 21.

    , , & NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome. J. Gen. Virol . 90, 1398–1407 (2009)

  22. 22.

    et al. Strand-specific real-time RT–PCR for distinguishing influenza vRNA, cRNA, and mRNA. J. Virol. Methods 173, 1–6 (2011)

  23. 23.

    et al. A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs. J. Virol. 76, 8989–9001 (2002)

  24. 24.

    , & Specific residues of PB2 and PA influenza virus polymerase subunits confer the ability for RNA polymerase II degradation and virus pathogenicity in mice. J. Virol. 88, 3455–3463 (2014)

  25. 25.

    et al. Adaptive mutations resulting in enhanced polymerase activity contribute to high virulence of influenza A virus in mice. J. Virol. 83, 6673–6680 (2009)

  26. 26.

    , , & Reassortment and mutation of the avian influenza virus polymerase PA subunit overcome species barriers. J. Virol. 86, 1750–1757 (2012)

  27. 27.

    et al. Functional constraint profiling of a viral protein reveals discordance of evolutionary conservation and functionality. PLoS Genet. 11, e1005310 (2015)

  28. 28.

    Reflections on the history of pre-mRNA processing and highlights of current knowledge: a unified picture. RNA 19, 443–460 (2013)

  29. 29.

    & The role of the influenza virus RNA polymerase in host shut-off. Virulence 1, 436–439 (2010)

  30. 30.

    Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D 66, 133–144 (2010)

  31. 31.

    , & Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

  32. 32.

    et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)

  33. 33.

    PyMOL Molecular Graphics System. (2002)

  34. 34.

    et al. Rescue of influenza A virus from recombinant DNA. J. Virol . 73, 9679–9682 (1999)

  35. 35.

    , , , & The generation of recombinant influenza A viruses expressing a PB2 fusion protein requires the conservation of a packaging signal overlapping the coding and noncoding regions at the 5′ end of the PB2 segment. Virology 341, 34–46 (2005)

  36. 36.

    , , & New low-viscosity overlay medium for viral plaque assays. Virol. J. 3, 63 (2006)

  37. 37.

    et al. Viral population analysis and minority-variant detection using short read next-generation sequencing. Phil. Trans. R. Soc. Lond. B 368, 20120205 (2013)

  38. 38.

    et al. The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc. Natl Acad. Sci. USA 102, 18590–18595 (2005)

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Acknowledgements

We thank ESRF for access to X-ray beamlines, the EMBL eukaryotic expression and crystallisation facilities and the biophysical platform within the Partnership for Structural Biology (PSB). D. Guilligay, M. Lethier and S. Gaudon helped with protein expression and crystallization. J. Ortin and T. Wolff supplied plasmids and members of the R. Pillai group (EMBL) provided advice for the minigenome assays. We thank V. Enouf and S. Leandri (Institut Pasteur, Pasteur International Bioresources network, Plateforme de Microbiologie Mutualisée) for the next-generation sequencing analysis and H. Varet (Institut Pasteur) for help with the statistical analysis. We acknowledge A. Politi, N. Daigle and J. Ellenberg for fluorescent microscopy experiments and discussions. This work was supported by ERC Advanced Grant V-RNA (322586) to S.C. and by the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence to N.N. The Institut Carnot Pasteur Maladies Infectieuses and the EU PREDEMICS project (278433) supported G.F.

Author information

Affiliations

  1. European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France

    • Maria Lukarska
    • , Alexander Pflug
    • , Patricia Resa-Infante
    • , Stefan Reich
    •  & Stephen Cusack
  2. Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Département de Virologie, F-75015 Paris, France

    • Guillaume Fournier
    •  & Nadia Naffakh
  3. CNRS, UMR3569, F-75015 Paris, France

    • Guillaume Fournier
    •  & Nadia Naffakh
  4. Université Paris Diderot, Sorbonne Paris Cité, Unité de Génétique Moléculaire des Virus à ARN, EA302, F-75015 Paris, France

    • Guillaume Fournier
    •  & Nadia Naffakh

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Contributions

M.L. performed protein expression, purification and crystallization with the help of A.P. and S.R. X-ray data collection and crystallographic analysis was performed by A.P., S.C. and M.L. Virus rescue experiments and RNA quantification were performed by G.F. under the supervision of N.N. M.L. performed peptide binding and polymerase activity assays, designed by S.R., who also helped with data analysis. P.R.-I. performed activity assays and primer extension assays. M.L. performed the minigenome experiments with the help of P.R-.I. S.C. and N.N. designed and supervised the project. S.C. and M.L wrote the manuscript with input from the other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Stephen Cusack.

Reviewer Information

Nature thanks K. Murakami, S. Shuman and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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    Supplementary Figures

    This file contains the uncropped gels with molecular markers for Figure 3b,d and Extended Data Figure 7a,b.

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DOI

https://doi.org/10.1038/nature20594

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