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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Protein Data Bank
Sequence Read Archive
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.
Extended data figures
This file contains the uncropped gels with molecular markers for Figure 3b,d and Extended Data Figure 7a,b.
About this article
BMC Biology (2017)