Structures of influenza A virus RNA polymerase offer insight into viral genome replication

Article metrics

Abstract

Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans1,2. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPolA) composed of PB1, PB2 and PA subunits3,4,5. Although the high-resolution crystal structure of FluPolA of bat influenza A virus has previously been reported6, there are no complete structures available for human and avian FluPolA. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication—which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase7,8,9,10—remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPolA from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0–4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPolA forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPolA bound to the cRNA template reveals a binding site for the 3′ cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPolA dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPolA dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPolA, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPolA that could be targeted in the development of antiviral drugs.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Structures of human H3N2 and avian H5N1 FluPolA.
Fig. 2: Mutations at the interface of the FluPolA dimer inhibit cRNA to vRNA replication.
Fig. 3: Structures of H3N2 FluPolA bound to cRNA promoter.
Fig. 4: Nb8205, which binds FluPolA at the dimer interface, inhibits cRNA to vRNA replication and virus growth.

Data availability

All data are available from the corresponding authors and/or included in the manuscript or Supplementary Information. Atomic coordinates have been deposited in the PDB with accession codes 6QNW (H3N2 FluPolA), 6QPF (H5N1 FluPolA) and 6QPG (H3N2 FluPolA + Nb8205). Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank with accession codes EMD-4661 (monomeric H3N2 FluPolA + cRNA + Nb8205), EMD-4663 and EMD-4664 (dimeric H3N2 FluPolA + cRNA), EMD-4666 (dimeric H3N2 FluPolA + cRNA + Nb8205), EMD-4660 (monomeric FluPolB + cRNA) and EMD-4986 (monomeric H3N2 FluPolA + vRNA + capped RNA) with the corresponding atomic coordinates deposited in the PDB with accession numbers 6QX3, 6QX8, 6QXE, 6QWL and 6RR7, respectively.

References

  1. 1.

    Taubenberger, J. K. & Kash, J. C. Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe 7, 440–451 (2010).

  2. 2.

    Mostafa, A., Abdelwhab, E. M., Mettenleiter, T. C. & Pleschka, S. Zoonotic potential of influenza A viruses: a comprehensive overview. Viruses 10, 497 (2018).

  3. 3.

    Pflug, A., Lukarska, M., Resa-Infante, P., Reich, S. & Cusack, S. Structural insights into RNA synthesis by the influenza virus transcription-replication machine. Virus Res. 234, 103–117 (2017).

  4. 4.

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

  5. 5.

    Walker, A. P. & Fodor, E. Interplay between influenza virus and the host RNA polymerase II transcriptional machinery. Trends Microbiol. 27, 398–407 (2019).

  6. 6.

    Pflug, A., Guilligay, D., Reich, S. & Cusack, S. Structure of influenza A polymerase bound to the viral RNA promoter. Nature 516, 355–360 (2014).

  7. 7.

    Jorba, N., Coloma, R. & Ortín, J. Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication. PLoS Pathog. 5, e1000462 (2009).

  8. 8.

    York, A., Hengrung, N., Vreede, F. T., Huiskonen, J. T. & Fodor, E. Isolation and characterization of the positive-sense replicative intermediate of a negative-strand RNA virus. Proc. Natl Acad. Sci. USA 110, E4238–E4245 (2013).

  9. 9.

    Jorba, N., Area, E. & Ortín, J. Oligomerization of the influenza virus polymerase complex in vivo. J. Gen. Virol. 89, 520–524 (2008).

  10. 10.

    Moeller, A., Kirchdoerfer, R. N., Potter, C. S., Carragher, B. & Wilson, I. A. Organization of the influenza virus replication machinery. Science 338, 1631–1634 (2012).

  11. 11.

    Chang, S. et al. Cryo-EM structure of influenza virus RNA polymerase complex at 4.3 Å resolution. Mol. Cell 57, 925–935 (2015).

  12. 12.

    Hara, K., Schmidt, F. I., Crow, M. & Brownlee, G. G. Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding. J. Virol. 80, 7789–7798 (2006).

  13. 13.

    Mänz, B., Brunotte, L., Reuther, P. & Schwemmle, M. Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells. Nat. Commun. 3, 802 (2012).

  14. 14.

    Deng, T., Vreede, F. T. & Brownlee, G. G. Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viral RNA promoters during viral RNA replication. J. Virol. 80, 2337–2348 (2006).

  15. 15.

    Hengrung, N. et al. Crystal structure of the RNA-dependent RNA polymerase from influenza C virus. Nature 527, 114–117 (2015).

  16. 16.

    Thierry, E. et al. Influenza polymerase can adopt an alternative configuration involving a radical repacking of PB2 domains. Mol. Cell 61, 125–137 (2016).

  17. 17.

    Serna Martin, I. et al. A mechanism for the activation of the influenza virus transcriptase. Mol. Cell 70, 1101–1110 (2018).

  18. 18.

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

  19. 19.

    Gerlach, P., Malet, H., Cusack, S. & Reguera, J. Structural insights into bunyavirus replication and its regulation by the vRNA promoter. Cell 161, 1267–1279 (2015).

  20. 20.

    Oymans, J. & Te Velthuis, A. J. W. A mechanism for priming and realignment during influenza A virus replication. J. Virol. 92, e01773-17 (2018).

  21. 21.

    te Velthuis, A. J., Robb, N. C., Kapanidis, A. N. & Fodor, E. The role of the priming loop in influenza A virus RNA synthesis. Nat. Microbiol. 1, 16029 (2016).

  22. 22.

    Killip, M. J., Fodor, E. & Randall, R. E. Influenza virus activation of the interferon system. Virus Res. 209, 11–22 (2015).

  23. 23.

    te Velthuis, A. J. W. et al. Mini viral RNAs act as innate immune agonists during influenza virus infection. Nat. Microbiol. 3, 1234–1242 (2018).

  24. 24.

    Bieniossek, C., Imasaki, T., Takagi, Y. & Berger, I. MultiBac: expanding the research toolbox for multiprotein complexes. Trends Biochem. Sci. 37, 49–57 (2012).

  25. 25.

    Weissmann, F. et al. biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes. Proc. Natl Acad. Sci. USA 113, E2564–E2569 (2016).

  26. 26.

    Pardon, E. et al. A general protocol for the generation of nanobodies for structural biology. Nat. Protocols 9, 674–693 (2014).

  27. 27.

    Walter, T. S. et al. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr. D 61, 651–657 (2005).

  28. 28.

    Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010).

  29. 29.

    Tickle, I. J. et al. STARANISO. http://staraniso.globalphasing.org/cgi-bin/staraniso.cgi (2018).

  30. 30.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  31. 31.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

  32. 32.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

  33. 33.

    Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012).

  34. 34.

    Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011).

  35. 35.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

  36. 36.

    Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

  37. 37.

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

  38. 38.

    Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

  39. 39.

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

  40. 40.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

  41. 41.

    Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

  42. 42.

    Shkumatov, A. V. & Strelkov, S. V. DATASW, a tool for HPLC-SAXS data analysis. Acta Crystallogr. D 71, 1347–1350 (2015).

  43. 43.

    Deng, T., Sharps, J., Fodor, E. & Brownlee, G. G. In vitro assembly of PB2 with a PB1-PA dimer supports a new model of assembly of influenza A virus polymerase subunits into a functional trimeric complex. J. Virol. 79, 8669–8674 (2005).

  44. 44.

    Fodor, E. 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).

  45. 45.

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

  46. 46.

    Fodor, E. & Smith, M. The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the influenza A virus RNA polymerase complex. J. Virol. 78, 9144–9153 (2004).

  47. 47.

    Vreede, F. T., Jung, T. E. & Brownlee, G. G. Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates. J. Virol. 78, 9568–9572 (2004).

  48. 48.

    Nilsson-Payant, B. E., Sharps, J., Hengrung, N. & Fodor, E. The surface-exposed PA51–72-loop of the influenza A virus polymerase is required for viral genome replication. J. Virol. 92, e00687-18 (2018).

  49. 49.

    Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

  50. 50.

    Robb, N. C., Smith, M., Vreede, F. T. & Fodor, E. NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome. J. Gen. Virol. 90, 1398–1407 (2009).

  51. 51.

    Reich, S., Guilligay, D. & Cusack, S. An in vitro fluorescence based study of initiation of RNA synthesis by influenza B polymerase. Nucleic Acids Res. 45, 3353–3368 (2017).

  52. 52.

    Bussey, K. A. et al. PA residues in the 2009 H1N1 pandemic influenza virus enhance avian influenza virus polymerase activity in mammalian cells. J. Virol. 85, 7020–7028 (2011).

  53. 53.

    Hu, J. et al. The PA-gene-mediated lethal dissemination and excessive innate immune response contribute to the high virulence of H5N1 avian influenza virus in mice. J. Virol. 87, 2660–2672 (2013).

  54. 54.

    Ilyushina, N. A. et al. Adaptation of pandemic H1N1 influenza viruses in mice. J. Virol. 84, 8607–8616 (2010).

  55. 55.

    Kamiki, H. et al. A PB1-K577E mutation in H9N2 influenza virus increases polymerase activity and pathogenicity in mice. Viruses 10, 653 (2018).

  56. 56.

    Lee, C. Y. et al. Novel mutations in avian PA in combination with an adaptive mutation in PR8 NP exacerbate the virulence of PR8-derived recombinant influenza A viruses in mice. Vet. Microbiol. 221, 114–121 (2018).

  57. 57.

    Liedmann, S. et al. New virulence determinants contribute to the enhanced immune response and reduced virulence of an influenza A virus A/PR8/34 variant. J. Infect. Dis. 209, 532–541 (2014).

  58. 58.

    Mehle, A., Dugan, V. G., Taubenberger, J. K. & Doudna, J. A. Reassortment and mutation of the avian influenza virus polymerase PA subunit overcome species barriers. J. Virol. 86, 1750–1757 (2012).

  59. 59.

    Neumann, G., Macken, C. A. & Kawaoka, Y. Identification of amino acid changes that may have been critical for the genesis of A(H7N9) influenza viruses. J. Virol. 88, 4877–4896 (2014).

  60. 60.

    Peng, X. et al. Amino acid substitutions HA A150V, PA A343T, and PB2 E627K increase the virulence of H5N6 influenza virus in mice. Front. Microbiol. 9, 453 (2018).

  61. 61.

    Slaine, P. D. et al. Adaptive mutations in influenza A/California/07/2009 enhance polymerase activity and infectious virion production. Viruses 10, 272 (2018).

  62. 62.

    Wu, R. et al. Multiple amino acid substitutions are involved in the adaptation of H9N2 avian influenza virus to mice. Vet. Microbiol. 138, 85–91 (2009).

  63. 63.

    Xu, G. et al. Prevailing PA mutation K356R in avian influenza H9N2 virus increases mammalian replication and pathogenicity. J. Virol. 90, 8105–8114 (2016).

  64. 64.

    Yamaji, R. et al. Mammalian adaptive mutations of the PA protein of highly pathogenic avian H5N1 influenza virus. J. Virol. 89, 4117–4125 (2015).

  65. 65.

    Zhang, Z. et al. Multiple amino acid substitutions involved in enhanced pathogenicity of LPAI H9N2 in mice. Infect. Genet. Evol. 11, 1790–1797 (2011).

  66. 66.

    Zhong, G. et al. Mutations in the PA protein of avian H5N1 influenza viruses affect polymerase activity and mouse virulence. J. Virol. 92, e01557-17 (2018).

  67. 67.

    Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).

  68. 68.

    Naydenova, K. & Russo, C. J. Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy. Nat. Commun. 8, 629 (2017).

Download references

Acknowledgements

We thank G. G. Brownlee and F. Vreede for plasmids; I. Berger for the MultiBac system; S. Cusack for sharing and discussing unpublished data on the 3′ promoter binding site; K. Harlos and T. Walter for assistance with crystallization; K. Dent and D. Clare for cryo-EM assistance; G. G. Brownlee, D. Stuart and A. te Velthuis, as well as members of the Fodor and Grimes laboratories, for helpful comments and discussions; and Instruct-ERIC, part of the European Strategy Forum on Research Infrastructures (ESFRI), Instruct-ULTRA (EU H2020 Grant 731005), and the Research Foundation - Flanders (FWO) for support with nanobody discovery. This work was supported by Medical Research Council (MRC) programme grants MR/K000241/1 and MR/R009945/1 (to E.F.), Wellcome Investigator Award 200835/Z/16/Z (to J.M.G.), MRC Studentships (to A.P.W. and I.S.M.) and Wellcome Studentship 092931/Z/10/Z (to N.H.). We thank Diamond Light source for beamtime (proposals MX10627, MX14744, and MX19946), and for access and support of the cryo-EM facilities at the UK National Electron Bio-Imaging Centre (eBIC) (proposals EM14856 and EM20233), funded by the Wellcome, MRC and BBSRC. Further electron microscopy provision was provided through the OPIC electron microscopy facility, which was funded by a Wellcome JIF award (060208/Z/00/Z) and is supported by a Wellcome equipment grant (093305/Z/10/Z). Computation used the Oxford Biomedical Research Computing (BMRC) facility, a joint development between the Wellcome Centre for Human Genetics and the Big Data Institute, supported by Health Data Research UK and the NIHR Oxford Biomedical Research Centre. Financial support was provided by a Wellcome Trust Core Award (203141/Z/16/Z). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. Part of this work was supported by Wellcome administrative support grant 203141/Z/16/Z.

Author information

H.F., A.P.W., L.C., J.R.K., J.M.G. and E.F. conceived and designed the study. H.F., L.C., J.R.K. and N.H. carried out cloning of recombinant baculoviruses and protein purification. H.F. and J.R.K. performed crystallizations, data collection and analysis, model building and refinement. L.C. and J.R.K. collected and processed electron microscopy data, and built and refined models with assistance from D.K. and I.S.M. A.P.W. performed functional assays and analysed data. J. Sharps performed dimerization assays in mammalian cells and E.F. analysed the data. E.P. and J. Steyaert designed and generated Nb8205 and Nb8210, and N.H. performed nanobody expression and purification. J.M.G. and E.F. supervised the structural and functional studies, respectively. H.F., A.P.W., L.C., J.R.K, J.M.G. and E.F. wrote the manuscript, with input from all co-authors.

Correspondence to Jonathan M. Grimes or Ervin Fodor.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Peer review information Nature thanks Seth Darst, Achilleas Frangakis and Peng Gong for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Subunit organization of FluPolA heterotrimers.

a, b, Views of the structure of human H3N2 (a) and avian H5N1 (b) FluPolA heterotrimers, coloured according to subunit. ce, Structures of the human H3N2 FluPolA subunits PA (c), PB1 (d) and PB2 (e), coloured and labelled by domain. f, Domain maps of each of the H3N2 FluPolA subunits. gi, The 2D Fo − Fc electron density maps of FluPolA dimer interface as shown in Fig. 1c (g, stereo view) and Fig. 1d (h, stereo view), as well as of the complete FluPolA dimer (i), are shown in grey mesh (contoured at 1.5σ, all from the structure model of H3N2 FluPolA).

Extended Data Fig. 2 Effect of mutations at the dimer interface on FluPolA dimerization and activity.

a, SEC-MALS analysis of wild-type and PA(352–356A) mutant H3N2 FluPolA (n = 1 experiment). Smooth lines reflect the relative UV signal of SEC, and dotted lines indicate estimated molecular mass for each frame. Note that monomeric FluPolA heterotrimer has an approximate molecular mass of 255 kDa. b, Effect of PA(352–356A) mutation on FluPolA dimerization in HEK293T cells. Data are mean ± s.e.m. n = 3 independent transfections. One-way ANOVA. P < 0.05 is considered significant. c, Effect of mutations designed to destabilize PB2, and PA loops, at the FluPolA dimer interface on FluPolA activity in a viral RNP reconstitution assay. Data are mean ± s.e.m. n = 3 independent transfections. Two-way ANOVA. P < 0.05 is considered significant. d, e, Effect of PA(352–356A) mutation on in vitro ApG-primer replication by FluPolA on a vRNA (d) and cRNA (e) template. f, Effect of an active-site polymerase mutant (PB1a) on in vitro ApG-primer replication by FluPolA on a cRNA template. Data are mean ± s.e.m. n = 4 independent reactions. g, Omitting UTP from in vitro ApG-primer replication by FluPolA on a cRNA template affects the synthesis of the 15-nucleotide, full-length vRNA but not of the 12-nucleotide, short vRNA, which indicates that the 12-nucleotide product is derived from internal initiation by the ApG dinucleotide at positions 4 and 5 of the cRNA template. The position in the template at which UTP is required is indicated in red. Representative data from n = 2 independent reactions. For gel source data, see Supplementary Fig. 2.

Extended Data Fig. 3 Single-particle cryo-EM analysis of human H3N2 FluPolA bound to cRNA promoter.

a, Representative micrograph of cRNA-bound FluPolA heterotrimer particles embedded in vitreous ice. b, Representative 2D class averages. c, Fourier shell correlation (FSC) curves for 3D reconstruction using gold-standard refinement in RELION, indicating overall map resolution of 4.07 Å and the model-to-map FSC. Curves are shown for phase-randomization, unmasked, masked and phase-randomization-corrected masked maps. d, A 3D reconstruction, locally filtered and coloured according to RELION local resolution. e, Angular distribution of particle projections with the cryo-EM map shown in grey. f, Cryo-EM density of the PA loop 352–356 at the dimer interface. g, Cryo-EM map of cRNA-bound FluPolA dimer refined without symmetry imposed (C1), which reveals an extra density (green) located next to the 3′ end of the 5′ cRNA close to the template entry channel. h, Close-up views highlighting extra density in the cryo-EM map (dark green) with the 3′ vRNA strand from the superimposed FluPolB structure51 (PDB 5MSG, light green) inserting into the polymerase active site. Localization of the 3′ vRNA shows that bases are positioned in the extra density, facing the density that corresponds to the 3′ end of the 5′ cRNA. This suggests the presence of a promoter RNA duplex region, as observed in the vRNA-bound FluPolB structure51. The extra density is consistent with the presence of a 3′ cRNA in one of the heterotrimers of the cRNA-bound FluPolA dimer, oriented towards the polymerase active site.

Extended Data Fig. 4 The effect of Nb8205 on FluPolA dimerization.

a, SDS–PAGE of purified nanobodies (n = 1 experiment). b, Analytical SEC of FluPolA in complex with nanobodies (n = 4 experiments for Nb8205 and n = 2 experiments or Nb8210, with similar results). c, Effect of nanobodies on FluPolA dimerization in HEK293T cells. Data are mean ± s.e.m. n = 4 independent transfections. One-way ANOVA. P < 0.05 is considered significant. For gel source data, see Supplementary Fig. 2. d, Crystal structure of H3N2 FluPolA in complex with Nb8205. e, Close-up view of FluPolA–Nb8205 interactions. Residues involved in hydrogen-bonding interactions are labelled, and hydrogen bonds are indicated with dashed lines. The complementarity determining regions (CDRs) are coloured individually, and labelled.

Extended Data Fig. 5 Single-particle cryo-EM analysis of monomeric and dimeric cRNA-bound human H3N2 FluPolA heterotrimer in complex with Nb8205.

a, Representative micrograph of cRNA-bound FluPolA in complex with Nb8205, embedded in vitreous ice. b, Representative 2D class averages. c, FSC curves for the 3D reconstruction using gold-standard refinement in RELION, indicating an overall map resolution of 3.79 Å and 4.15 Å for the monomeric and dimeric FluPolA form, respectively, and the model-to-map FSC. Curves are shown for phase-randomization, unmasked, masked and phase-randomization-corrected masked maps. d, f, The 3D reconstructions, locally filtered and coloured according to RELION local resolution, for the dimeric (d) and monomeric (f) form. e, g, Angular distribution of particle projections for the dimeric (e) and monomeric (g) form, with the cryo-EM map shown in grey. h, Dimer of FluPolA heterotrimers bound to cRNA promoter and Nb8205 rigid-body-fitted into the cryo-EM map of dimeric cRNA-bound FluPolA heterotrimer in complex with Nb8205. i, Cryo-EM map of the dimeric cRNA-bound FluPolA heterotrimer in complex with Nb8205, revealing an extra density (green) located next to the 3′ end of the 5′ cRNA (as also observed for the cRNA-bound FluPolA dimer; Extended Data Fig. 3g, h).

Extended Data Fig. 6 Single-particle cryo-EM analysis of cRNA-bound FluPolB.

a, Representative micrograph of cRNA-bound FluPolB heterotrimer particles embedded in vitreous ice. b, Representative 2D class averages. c, A 3D reconstruction, locally filtered and coloured according to RELION local resolution. d, FSC curves for the 3D reconstruction using gold-standard refinement in RELION, indicating an overall map resolution of 4.18 Å and the model-to-map FSC. Curves are shown for the phase-randomization, unmasked, masked, phase-randomization-corrected masked maps. e, Angular distribution of particle projections according to cryoSPARC v.2.5 non-uniform refinement. f, Cryo-EM map of cRNA-bound FluPolB. g, Comparison of the dimerization interface and the 3ʹ cRNA binding site in H3N2 FluPolA (PDB 6QNW and 6QPG). h, 3′ cRNA binding site in FluPolA and FluPolB overlaps with the previously identified 3′ vRNA binding site in the La Crosse orthobunyavirus polymerase19 (PDB 5AMQ). Sites of 3′ vRNA binding at surface of the polymerase in FluPolB (PDB 4WRT) and in the polymerase active site for FluPolB (PDB 5MSG) are shown for comparison6,51. i, Comparison of the structure of dimeric FluPolA to monomeric FluPolB (PDB 5MSG)51 reveals a movement of the priming loop that protrudes from the thumb subdomain of PB1 into the polymerase active site. Resolved PB1 residues closest to the tip of the priming loop (residues E638 and M656) move away from the corresponding E637 and M655 residues in FluPolB and the polymerase active site (indicated by the end of the 3′ vRNA) by approximately 7 Å.

Extended Data Fig. 7 Single-particle cryo-EM analysis of human H3N2 FluPolA bound to vRNA promoter.

a, Representative micrograph of vRNA-bound FluPolA heterotrimer particles embedded in vitreous ice. b, Representative 2D class averages. c, FSC curves for 3D reconstruction using gold-standard refinement in RELION, indicating an overall map resolution of 3.01 Å and the model-to-map FSC. Curves are shown for phase-randomization, unmasked, masked and phase-randomization-corrected masked maps. d, A 3D reconstruction, locally filtered and coloured according to RELION local resolution. e, Angular distribution of particle projections with the cryo-EM map shown in grey. f, Cryo-EM map of vRNA-bound FluPolA heterotrimer, revealing the presence of a fully resolved priming loop. g, Close-up views highlighting the stacking of the 3′ vRNA by the priming loop. h, Cartoon of the role of polymerase dimerization in template realignment during replication initiation on a cRNA template. Base-pairing between the 5′ and 3′ cRNAs positions bases 4 and 5 of the 3′ cRNA next to the catalytic aspartates (residues D445 and D446 of PB1) in the active site to enable internal replication initiation by the synthesis of a pppApG dinucleotide. The priming loop stacks the cRNA template through P651 of PB1 (left). Rotation of the thumb subdomain of PB1 and the N1 subdomain of PB2 triggered by polymerase dimerization results in a movement of the priming loop and backtracking of the stacked template (arrows). Backtracking is also facilitated by an interaction of residue R46 of PB2 with the 3′ cRNA, which introduces a ‘kink’ into the template. Backtracking positions bases 1 and 2 of the cRNA template opposite the pppApG dinucleotide that remains coordinated by the catalytic aspartates. The resulting replication complex is ready to extend the pppApG dinucleotide by incorporating the next incoming NTP (right).

Extended Data Fig. 8 Effect of Nb8205 on FluPolA activity, and mapping of host adaptive mutations at the FluPolA dimer interface.

a, b, Effect of Nb8205 on in vitro ApG-primer replication by FluPolA on a vRNA (a) and cRNA (b) template. Data are mean ± s.e.m. n = 3 independent reactions. c, Omitting UTP from in vitro ApG-primer replication by FluPolA on a cRNA template affects the synthesis of the 15-nucleotide, full-length vRNA but not of the 12-nucleotide, short vRNA. The position in the template at which UTP is required is indicated in red. Representative data from n = 2 independent reactions. For gel source data, see Supplementary Fig. 2. d, Crystal structure of H3N2 FluPolA with amino acid residues implicated in avian-to-mammalian host adaptation of influenza A viruses indicated52,53,54,55,56,57,58,59,60,61,62,63,64,65,66.

Extended Data Table 1 Crystallographic data collection and refinement statistics
Extended Data Table 2 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-2. Supplementary Fig. 1 Sequence similarity of FluPol from different influenza virus genera. a-c, Sequence alignment of polymerase subunits PA (a), PB1 (b) and PB2 (c) from A/NT/60/1968 (H3N2), A/duck/Fujian/01/2002 (H5N1), A/bat/Guatemala/060/2010 (H17N10), B/Panama/45/1990, and C/Johannesburg/1/1966. Residues involved in hydrogen bonding interactions at the FluPolA dimer interface are indicated in cyan, residues involved in 3ʹ cRNA promoter binding site are indicated in orange, and residues involved in binding Nb8205 are shown in pink. The figure was prepared with Espript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Supplementary Fig. 2 Source data (gels). a, Source data for main figures. b, Source data for extended data figures.

Reporting Summary

Video 1

Structure of human H3N2 FluPolA bound to cRNA and Nb8205. This video shows a close-up of the 3′ cRNA binding site.

Video 2

Comparison of monomeric and dimeric FluPolA structures in complex with Nb8205. The video shows that dimerisation induces a movement of a helical bundle formed by the PB1 thumb and PB2 N1 subdomains. Dimerisation leads to the opening of the 3′ cRNA binding site which is incompatible with 3′ cRNA binding.

Video 3

Comparison of the structure of dimeric FluPolA to monomeric FluPolB (PDB: 5MSG). Comparison of the structure of dimeric FluPolA to monomeric FluPolB (PDB: 5MSG).

Video 4

Comparison of the structure of dimeric FluPolA to monomeric vRNA-bound FluPolA. This video shows the movement of the priming loop in the polymerase active site triggered by dimerisation.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.