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


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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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


  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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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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