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

Nature volume 541, pages 117–121 (2017)Cite this article

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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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Figure 1: Structure of bat FluA polymerase bound to Ser5-phosphorylated CTD peptide.
Figure 2: Conservation of the CTD-binding site in influenza A and B strains.
Figure 3: In vitro and in vivo mutational analysis of the SeP5 binding pocket.
Figure 4: CTD-binding residues previously characterized as important for influenza replication and pathogenicity.

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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.

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Authors and Affiliations

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

    Maria Lukarska, Alexander Pflug, Patricia Resa-Infante, Stefan Reich & Stephen Cusack

  2. Département de Virologie, Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, F-75015 Paris, France

    Guillaume Fournier & Nadia Naffakh

  3. CNRS, UMR3569, Paris, F-75015, France

    Guillaume Fournier & Nadia Naffakh

  4. Université Paris Diderot, Sorbonne Paris Cité, Unité de Génétique Moléculaire des Virus à ARN, EA302, Paris, F-75015, 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.

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Correspondence to Stephen Cusack.

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Reviewer Information

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

Extended data figures and tables

Extended Data Figure 1 Fo − Fc omit map of the CTD peptides bound to bat FluA site 1 (left) and site 2 (right), contoured at 3 σ .

Extended Data Figure 2 Conformations of CTD peptides bound to bat FluA polymerase compared to other Pol-II-interacting factors.

Structures of proteins bound to SeP5 or SeP2-SeP5 CTD peptides are shown in grey cartoon, the peptides (stick representation) are coloured to highlight their position and zoomed in next to their corresponding structure. a, b, Binding site 1 (a) and site 2 (b) of bat FluA polymerase, with the corresponding parts of the CTD SeP5 peptides. c, SeP2–SeP5 CTD bound to the peptidyl-prolyl isomerase Pin1 (PDB: 1F8A). d, SeP5 CTD bound to Candida albicans capping-enzyme Cgt1 (PDB: 1P16). e, SeP2–SeP5 CTD bound to mammalian capping-enzyme Mce1 (PDB: 3RTX). f, SeP2–SeP5 CTD bound to Schizosaccharomyces pombe capping-enzyme Pce1 (PDB: 4PZ6). g, SeP5 CTD bound to human serine phosphatase Ssu72 (PDB: 3O2Q).

Extended Data Figure 3 Sequence alignment of the PA subunit of various influenza strains (bat A, human A, avian A, B, C, D).

Only the PA-C region starting from residue 220 is shown. Absolutely conserved residues are white on a red background and highly conserved residues are red letters boxed in blue. The amino acid numbering and secondary structure correspond to bat FluA polymerase. CTD binding site 1 residues are indicated with a cyan triangle (conserved in FluA and FluB strains) and site 2 with a yellow triangle (key residues are only conserved in FluA strains). Residues referred to in the discussion (site 1, C448 (H1N1, C453), site 2, I545 (H1N1, L550), T547 (H1N1, S552)) are shown with a black triangle.

Extended Data Figure 4 Fluorescence anisotropy data for bat FluA polymerase.

a, Displacement of a fluorescent SeP5 CTD peptide (four-repeat) bound to Pol–vRNA complex with non-labelled SeP5 peptide. The apparent Kd (Kd′) is shown. b, Binding of SeP5 peptide to FluA polymerase in the absence of the vRNA promotor. c, Interaction with non-phosphorylated four-repeat CTD peptide (Y1S2P3T4S5P6S7). The binding curve can be extrapolated to an estimated Kd of >10 μM. Error bars represent s.d. of three independent experiments, Kd values are shown ± the error of fit.

Extended Data Figure 5 In vitro data for CTD-binding-deficient mutants.

Binding data of SeP5 peptide to recombinant mutant proteins. a, Bat FluA site 2 double mutant K289A/R449A (left), site 1 double mutant K630A/R633A (right)). b, Influenza B site 1 double mutant K631A/R634A. Error bars represent s.d. of three independent experiments, Kd values are shown ± the error of the fit. c, Time courses of cap-primed transcription (left panel) and unprimed replication reactions in vitro with vRNA (middle panel) and cRNA (right panel) as template, comparing the site 1 and 2 double mutants with the wild-type bat FluA. Error bars show the s.d. from three different reactions. The tables show the measured rate constants ± the error of the fit.

Extended Data Figure 6 Minigenome assays for CTD-binding mutants of influenza A/WSN/33 in absence of NS2.

a, Overall RNP activity measured by a luciferase reporter assay (upper panel). Expression of PA, PB2 and nucleoprotein subunits is monitored by western blot (lower panel). b, Primer extension assay showing the different RNA levels produced by wild-type and mutant polymerases in the minigenome assay. Products of primer extension reactions were loaded on a 6% urea polyacrylamide gel, with their expected size indicated on the right and molecular weight markers on the left. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Figure 7 RNA quantification of the A/WSN/33 minigenome assay with NS2.

Normalized copy numbers of the different RNAs produced by wild-type and mutant polymerases, measured by quantitative RT–PCR. Error bars represent the s.d. of three independent experiments. Values lower than 105 are below the detection limit of the assay (indicated by a dashed line).

Source data

Extended Data Figure 8 Mutant A/WSN/33 virus rescue experiments.

The titers and plaque phenotypes of the recombinant A/WSN/33 viruses were determined on reverse genetics supernatants (left) or upon a single plaque purification followed by viral amplification (right), using a standard plaque assay on MDCK cells. Crystal violet staining of cell monolayers infected with the indicated viral dilutions is shown. WT, control performed with wild-type PA. For each recombinant virus, two independent viral stocks (denoted as P1 and P1′) derived from the same purified plaque were subjected to vRNA extraction, RT–PCR of the eight genomic segments and next-generation sequencing. The non-synonymous mutations detected in >30% of the reads upon alignment with the reference sequences for the wild-type virus are indicated. *Number of purified plaques leading to successful viral amplification per total number tested. †P1′ did not yield any detectable infectious virus for the R454A mutant. ‡P1 (for R454A and wild type) and P1′ (for K289A, K635A and R638A) were assayed in parallel.

Extended Data Figure 9 Overlap between a functionally important region of FluA PA defined by fitness profiling and the CTD-binding site.

a, b, A putative functional subdomain (a) identified in figure 5 of ref. 27 partially overlaps with the two CTD-binding sites (b). Amongst the residues shown to be functionally important in ref. 27, K559, R454 and K635 (K554, R449 and K630 bat FluA numbering) are directly involved in the interaction with the CTD peptide (see Extended Data Fig. 3). The authors also showed that R454 and K635 mutations essentially eliminated polymerase activity in a minigenome assay, consistent with our results.

Extended Data Table 1 Data collection and refinement statistics of bat FluA and human FluB polymerases bound to 28-amino-acid SeP5 CTD peptide
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Supplementary information

Supplementary Figures

This file contains the uncropped gels with molecular markers for Figure 3b,d and Extended Data Figure 7a,b. (PDF 927 kb)

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Lukarska, M., Fournier, G., Pflug, A. et al. Structural basis of an essential interaction between influenza polymerase and Pol II CTD. Nature 541, 117–121 (2017). https://doi.org/10.1038/nature20594

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