Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis

Nature Reviews Microbiology volume 14, pages 479–493 (2016)Cite this article

Subjects

Key Points

  • High-resolution structures have recently revealed the subunit and domain organization of the influenza A, influenza B and influenza C virus RNA polymerases. The polymerase basic 1 (PB1) subunit together with the C-terminal domain of polymerase acidic (PA) and the N-terminal one-third of PB2 form the core of the polymerase, which is decorated by several flexible domains, including the PB2 cap-binding and PA endonuclease domains.

  • Crystal structures obtained in the absence and presence of viral genomic RNA have captured the influenza virus RNA polymerase in transcriptionally inactive and transcription pre-initiation states, showing a different arrangement of the peripheral PB2 cap-binding and PA endonuclease domains and suggesting a mechanism for the activation of the cap-snatching function of the polymerase.

  • Structural and biochemical analyses have identified the binding sites of the 5′ and 3′ termini of viral RNAs, the template entry and exit channels, the substrate entry and product exit channels, the polymerase active site and a priming loop.

  • Based on the high-resolution structural information about the influenza virus RNA polymerases, the models for viral transcription and genome replication have been revised.

  • Viral and host factors participate in the regulation of viral transcription and genome replication. However, in many cases, the underlying molecular mechanisms remain unknown.

  • Understanding how the influenza virus RNA synthesis machine works at the molecular level and knowledge of its interaction with viral and host factors might lead to the identification of targets that could be exploited for the development of antivirals to prevent or treat influenza virus infections.

Abstract

The genomes of influenza viruses consist of multiple segments of single-stranded negative-sense RNA. Each of these segments is bound by the heterotrimeric viral RNA-dependent RNA polymerase and multiple copies of nucleoprotein, which form viral ribonucleoprotein (vRNP) complexes. It is in the context of these vRNPs that the viral RNA polymerase carries out transcription of viral genes and replication of the viral RNA genome. In this Review, we discuss our current knowledge of the structure of the influenza virus RNA polymerase, and insights that have been gained into the molecular mechanisms of viral transcription and replication, and their regulation by viral and host factors. Furthermore, we discuss how advances in our understanding of the structure and function of polymerases could help in identifying new antiviral targets.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The viral ribonucleoprotein complex and structures of the RNA polymerases of influenza A, influenza B and influenza C viruses.
Figure 2: Polymerase architecture and channels.
Figure 3: Models of viral transcription (mRNA synthesis) and replication (complementary RNA and viral genomic RNA synthesis).
Figure 4: Regulation of transcription and replication.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Krammer, F. & Palese, P. Advances in the development of influenza virus vaccines. Nat. Rev. Drug Discov. 14, 167–182 (2015).

    CAS  PubMed  Google Scholar 

  2. Watanabe, T. & Kawaoka, Y. Influenza virus–host interactomes as a basis for antiviral drug development. Curr. Opin. Virol. 14, 71–78 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Krug, R. & Fodor, E. in Textbook of Influenza (eds Webster, R., Monto, A., Braciale, T. & Lamb, R.) 57–66 (Wiley-Blackwell, 2013).

    Google Scholar 

  4. Fodor, E. The RNA polymerase of influenza a virus: mechanisms of viral transcription and replication. Acta Virol. 57, 113–122 (2013).

    CAS  PubMed  Google Scholar 

  5. Resa-Infante, P., Jorba, N., Coloma, R. & Ortin, J. The influenza virus RNA synthesis machine: advances in its structure and function. RNA Biol. 8, 207–215 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Arranz, R. et al. The structure of native influenza virion ribonucleoproteins. Science 338, 1634–1637 (2012). This paper describes the 3D structure of native RNPs that are derived from influenza virions.

    CAS  PubMed  Google Scholar 

  7. 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). This work determines the 3D structure of native RNPs that are derived from cells that express influenza virus RNP complex components.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Hutchinson, E. C. & Fodor, E. Transport of the influenza virus genome from nucleus to nucleus. Viruses 5, 2424–2446 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Plotch, S. J., Bouloy, M., Ulmanen, I. & Krug, R. M. 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).

    CAS  PubMed  Google Scholar 

  10. Dias, A. et al. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458, 914–918 (2009).

    CAS  PubMed  Google Scholar 

  11. Guilligay, D. et al. The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat. Struct. Mol. Biol. 15, 500–506 (2008).

    CAS  PubMed  Google Scholar 

  12. Yuan, P. et al. Crystal structure of an avian influenza polymerase PAN reveals an endonuclease active site. Nature 458, 909–913 (2009).

    CAS  PubMed  Google Scholar 

  13. Swale, C. et al. Structural characterization of recombinant IAV polymerase reveals a stable complex between viral PA-PB1 heterodimer and host RanBP5. Sci. Rep. 6, 24727 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Morin, B., Kranzusch, P. J., Rahmeh, A. A. & Whelan, S. P. J. The polymerase of negative-stranded RNA viruses. Curr. Opin. Virol. 3, 103–110 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Hengrung, N. et al. Crystal structure of the RNA-dependent RNA polymerase from influenza C virus. Nature 527, 114–117 (2015). This report presents the crystal structure of the RNA-free influenza C virus polymerase, revealing a configuration of the PB2 C-terminal domains that is radically different from that in the influenza A and influenza B virus polymerase structures that were obtained in the presence of vRNA. Small-angle X-ray scattering (SAXS) studies show that the polymerase can take various conformations.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Liang, B. et al. Structure of the L protein of vesicular stomatitis virus from electron cryomicroscopy. Cell 162, 314–327 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Pflug, A., Guilligay, D., Reich, S. & Cusack, S. Structure of influenza A polymerase bound to the viral RNA promoter. Nature 516, 355–360 (2014). This analysis obtains the crystal structure of a bat influenza A virus polymerase, demonstrating the architecture of the heterotrimeric complex and the structural basis for binding the vRNA promoter, which comprises the conserved 5′ and 3′ termini of the vRNA.

    CAS  PubMed  Google Scholar 

  19. Reich, S. et al. Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature 516, 361–366 (2014). This investigation determines the crystal structure of the influenza B virus polymerase and shows the rotation of the PB2 cap-binding domain that guides the 3′ end of the capped RNA primer into the polymerase active site.

    CAS  PubMed  Google Scholar 

  20. He, X. et al. Crystal structure of the polymerase PAC–PB1N complex from an avian influenza H5N1 virus. Nature 454, 1123–1126 (2008).

    CAS  PubMed  Google Scholar 

  21. Obayashi, E. et al. The structural basis for an essential subunit interaction in influenza virus RNA polymerase. Nature 454, 1127–1131 (2008).

    CAS  PubMed  Google Scholar 

  22. Sugiyama, K. et al. Structural insight into the essential PB1–PB2 subunit contact of the influenza virus RNA polymerase. EMBO J. 28, 1803–1811 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Tarendeau, F. et al. Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit. Nat. Struct. Mol. Biol. 14, 229–233 (2007).

    CAS  PubMed  Google Scholar 

  24. Tarendeau, F. et al. Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit. PLoS Pathog. 4, e1000136 (2008).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  26. te Velthuis, A. J. Common and unique features of viral RNA-dependent polymerases. Cell. Mol. Life Sci. 71, 4403–4420 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Subbarao, E. K., London, W. & Murphy, B. R. A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. J. Virol. 67, 1761–1764 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Thierry, E. et al. Influenza polymerase can adopt an alternative configuration involving a radical repacking of PB2 domains. Mol. Cell 61, 125–137 (2016). This paper presents structures of the influenza B virus polymerase, showing an arrangement of the PB2 C-terminal domains similar to that observed for the influenza C virus polymerase. Solution studies show the polymerase can take up various conformations.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Hsu, M. T., Parvin, J. D., Gupta, S., Krystal, M. & Palese, P. Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle. Proc. Natl Acad. Sci. USA 84, 8140–8144 (1987).

    CAS  PubMed  Google Scholar 

  30. Cianci, C., Tiley, L. & Krystal, M. Differential activation of the influenza virus polymerase via template RNA binding. J. Virol. 69, 3995–3999 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Fodor, E., Pritlove, D. C. & Brownlee, G. G. The influenza virus panhandle is involved in the initiation of transcription. J. Virol. 68, 4092–4096 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Fodor, E., Pritlove, D. C. & Brownlee, G. G. Characterization of the RNA-fork model of virion RNA in the initiation of transcription in influenza A virus. J. Virol. 69, 4012–4019 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Tiley, L. S., Hagen, M., Matthews, J. T. & Krystal, M. Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5′ ends of the viral RNAs. J. Virol. 68, 5108–5116 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Rao, P., Yuan, W. & Krug, R. M. Crucial role of CA cleavage sites in the cap-snatching mechanism for initiating viral mRNA synthesis. EMBO J. 22, 1188–1198 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Tomescu, A. I., Robb, N. C., Hengrung, N., Fodor, E. & Kapanidis, A. N. Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter. Proc. Natl Acad. Sci. USA 111, E3335–E3342 (2014).

    CAS  PubMed  Google Scholar 

  36. Fodor, E., Seong, B. L. & Brownlee, G. G. Photochemical cross-linking of influenza-a polymerase to its virion RNA promoter defines a polymerase binding-site at residue-9 to residue-12 of the promoter. J. General Virol. 74, 1327–1333 (1993).

    CAS  Google Scholar 

  37. Flick, R., Neumann, G., Hoffmann, E., Neumeier, E. & Hobom, G. Promoter elements in the influenza vRNA terminal structure. RNA 2, 1046–1057 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Pritlove, D. C., Poon, L. L., Devenish, L. J., Leahy, M. B. & Brownlee, G. G. A hairpin loop at the 5′ end of influenza A virus virion RNA is required for synthesis of poly(A)+ mRNA in vitro. J. Virol. 73, 2109–2114 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Pritlove, D. C., Fodor, E., Seong, B. L. & Brownlee, G. G. In vitro transcription and polymerase binding studies of the termini of influenza A virus cRNA: evidence for a cRNA panhandle. J. Gen. Virol. 76, 2205–2213 (1995).

    CAS  PubMed  Google Scholar 

  40. Crow, M., Deng, T., Addley, M. & Brownlee, G. G. Mutational analysis of the influenza virus cRNA promoter and identification of nucleotides critical for replication. J. Virol. 78, 6263–6270 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Appleby, T. C. et al. Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase. Science 347, 771–775 (2015).

    CAS  PubMed  Google Scholar 

  42. Butcher, S. J., Grimes, J. M., Makeyev, E. V., Bamford, D. H. & Stuart, D. L. A mechanism for initiating RNA-dependent RNA polymerization. Nature 410, 235–240 (2001).

    CAS  PubMed  Google Scholar 

  43. Tao, Y. Z., Farsetta, D. L., Nibert, M. L. & Harrison, S. C. RNA synthesis in a cage — Structural studies of reovirus polymerase λ3. Cell 111, 733–745 (2002).

    CAS  PubMed  Google Scholar 

  44. te Velthuis, A. J. W., 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). This research finds that the priming loop of the influenza A virus polymerase is required for terminal de novo replication initiation on the vRNA template, but is not required for internal de novo replication initiation on the cRNA template and for capped RNA primer-dependent transcription initiation.

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Koppstein, D., Ashour, J. & Bartel, D. P. Sequencing the cap-snatching repertoire of H1N1 influenza provides insight into the mechanism of viral transcription initiation. Nucleic Acids Res. 43, 5052–5064 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Sikora, D., Rocheleau, L., Brown, E. G. & Pelchat, M. Deep sequencing reveals the eight facets of the influenza A/HongKong/1/1968 (H3N2) virus cap-snatching process. Sci. Rep. 4, 6181 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Beaton, A. R. & Krug, R. M. Selected host cell capped RNA fragments prime influenza viral RNA transcription in vivo. Nucleic Acids Res. 9, 4423–4436 (1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Shaw, M. W. & Lamb, R. A. A specific sub-set of host-cell mRNAs prime influenza virus mRNA synthesis. Virus Res. 1, 455–467 (1984).

    CAS  PubMed  Google Scholar 

  50. Poon, L. L., Pritlove, D. C., Fodor, E. & Brownlee, G. G. Direct evidence that the poly(A) tail of influenza A virus mRNA is synthesized by reiterative copying of a U track in the virion RNA template. J. Virol. 73, 3473–3476 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Poon, L. L., Fodor, E. & Brownlee, G. G. Polyuridylated mRNA synthesized by a recombinant influenza virus is defective in nuclear export. J. Virol. 74, 418–427 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Braam, J., Ulmanen, I. & Krug, R. M. Molecular model of a eucaryotic transcription complex: functions and movements of influenza P proteins during capped RNA-primed transcription. Cell 34, 609–618 (1983).

    CAS  PubMed  Google Scholar 

  53. Bier, K., York, A. & Fodor, E. Cellular cap-binding proteins associate with influenza virus mRNAs. J. Gen. Virol. 92, 1627–1634 (2011).

    CAS  PubMed  Google Scholar 

  54. York, A. & Fodor, E. Biogenesis, assembly, and export of viral messenger ribonucleoproteins in the influenza A virus infected cell. RNA Biol. 10, 1274–1282 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Vreede, F. T. & Brownlee, G. G. Influenza virion-derived viral ribonucleoproteins synthesize both mRNA and cRNA in vitro. J. Virol. 81, 2196–2204 (2007).

    CAS  PubMed  Google Scholar 

  56. 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). This article reports the isolation and subsequent structural and functional characterization of the influenza A virus cRNP replicative intermediate. The authors propose that the cRNP-associated polymerase requires a trans -activating polymerase to be able to carry out vRNA synthesis.

    CAS  PubMed  Google Scholar 

  57. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Chan, W. H. et al. Functional analysis of the influenza virus H5N1 nucleoprotein tail loop reveals amino acids that are crucial for oligomerization and ribonucleoprotein activities. J. Virol. 84, 7337–7345 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Ng, A. K. et al. Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design. FASEB J. 22, 3638–3647 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Ye, Q., Krug, R. M. & Tao, Y. J. The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444, 1078–1082 (2006).

    CAS  PubMed  Google Scholar 

  61. Turrell, L., Lyall, J. W., Tiley, L. S., Fodor, E. & Vreede, F. T. The role and assembly mechanism of nucleoprotein in influenza A virus ribonucleoprotein complexes. Nat. Commun. 4, 1591 (2013).

    PubMed  PubMed Central  Google Scholar 

  62. Boulo, S. et al. Human importin-α and RNA do not compete for binding to influenza A virus nucleoprotein. Virology 409, 84–90 (2011).

    CAS  PubMed  Google Scholar 

  63. Chenavas, S. et al. Monomeric nucleoprotein of influenza A virus. PLoS Pathog. 9, e1003275 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Mondal, A., Potts, G. K., Dawson, A. R., Coon, J. J. & Mehle, A. Phosphorylation at the homotypic interface regulates nucleoprotein oligomerization and assembly of the influenza virus replication machinery. PLoS Pathog. 11, e1004826 (2015).

    PubMed  PubMed Central  Google Scholar 

  65. Turrell, L., Hutchinson, E. C., Vreede, F. T. & Fodor, E. Regulation of influenza A virus nucleoprotein oligomerization by phosphorylation. J. Virol. 89, 1452–1455 (2015).

    PubMed  Google Scholar 

  66. Jorba, N., Coloma, R. & Ortin, J. Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication. PLoS Pathog. 5, e1000462 (2009). This report describes a model for influenza virus transcription and genome replication. It proposes that transcription is carried out by the resident polymerase bound to the vRNA termini in the vRNP, whereas the cRNA-to-vRNA step of genome replication is carried out by a trans -acting polymerase that is not part of the cRNP.

    PubMed  PubMed Central  Google Scholar 

  67. Beaton, A. R. & Krug, R. M. Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5′ capped end. Proc. Natl Acad. Sci. USA 83, 6282–6286 (1986).

    CAS  PubMed  Google Scholar 

  68. Shapiro, G. I. & Krug, R. M. Influenza virus RNA replication in vitro: synthesis of viral template RNAs and virion RNAs in the absence of an added primer. J. Virol. 62, 2285–2290 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Resa-Infante, P., Recuero-Checa, M. A., Zamarreno, N., Llorca, O. & Ortin, J. Structural and functional characterization of an influenza virus RNA polymerase-genomic RNA complex. J. Virol. 84, 10477–10487 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Kawaguchi, A., Momose, F. & Nagata, K. Replication-coupled and host factor-mediated encapsidation of the influenza virus genome by viral nucleoprotein. J. Virol. 85, 6197–6204 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Newcomb, L. L. et al. Interaction of the influenza a virus nucleocapsid protein with the viral RNA polymerase potentiates unprimed viral RNA replication. J. Virol. 83, 29–36 (2009).

    CAS  PubMed  Google Scholar 

  73. Hutchinson, E. C. et al. Conserved and host-specific features of influenza virion architecture. Nat. Commun. 5, 4816 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Min, J. Y., Li, S., Sen, G. C. & Krug, R. M. A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. Virology 363, 236–243 (2007).

    CAS  PubMed  Google Scholar 

  75. Robb, N. C. et al. The influenza A virus NS1 protein interacts with the nucleoprotein of viral ribonucleoprotein complexes. J. Virol. 85, 5228–5231 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Marion, R. M., Zurcher, T., de la Luna, S. & Ortin, J. Influenza virus NS1 protein interacts with viral transcription–replication complexes in vivo. J. Gen. Virol. 78, 2447–2451 (1997).

    PubMed  Google Scholar 

  77. Falcon, A. M. et al. Defective RNA replication and late gene expression in temperature-sensitive influenza viruses expressing deleted forms of the NS1 protein. J. Virol. 78, 3880–3888 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Garaigorta, U. & Ortin, J. Mutation analysis of a recombinant NS replicon shows that influenza virus NS1 protein blocks the splicing and nucleo-cytoplasmic transport of its own viral mRNA. Nucleic Acids Res. 35, 4573–4582 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Robb, N. C. & Fodor, E. The accumulation of influenza A virus segment 7 spliced mRNAs is regulated by the NS1 protein. J. Gen. Virol. 93, 113–118 (2012).

    CAS  PubMed  Google Scholar 

  80. Paterson, D. & Fodor, E. Emerging roles for the influenza A virus nuclear export protein (NEP). PLoS Pathog. 8, e1003019 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 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).

    CAS  PubMed  Google Scholar 

  82. Manz, 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).

    PubMed  Google Scholar 

  83. Perez, J. T. et al. Influenza A virus-generated small RNAs regulate the switch from transcription to replication. Proc. Natl Acad. Sci. USA 107, 11525–11530 (2010).

    CAS  PubMed  Google Scholar 

  84. Umbach, J. L., Yen, H. L., Poon, L. L. & Cullen, B. R. Influenza A virus expresses high levels of an unusual class of small viral leader RNAs in infected cells. mBio 1, e00204–10 (2010).

    PubMed  PubMed Central  Google Scholar 

  85. Perez, J. T. et al. A small-RNA enhancer of viral polymerase activity. J. Virol. 86, 13475–13485 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Brass, A. L. et al. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139, 1243–1254 (2009).

    PubMed  PubMed Central  Google Scholar 

  87. Hao, L. et al. Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature 454, 890–893 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Karlas, A. et al. Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 463, 818–822 (2010).

    CAS  PubMed  Google Scholar 

  89. Shapira, S. D. et al. A physical and regulatory map of host–influenza interactions reveals pathways in H1N1 infection. Cell 139, 1255–1267 (2009).

    PubMed  PubMed Central  Google Scholar 

  90. Naito, T. et al. An influenza virus replicon system in yeast identified Tat-SF1 as a stimulatory host factor for viral RNA synthesis. Proc. Natl Acad. Sci. USA 104, 18235–18240 (2007).

    CAS  PubMed  Google Scholar 

  91. Sui, B. et al. The use of Random Homozygous Gene Perturbation to identify novel host-oriented targets for influenza. Virology 387, 473–481 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Tripathi, S. et al. Meta- and orthogonal integration of influenza “OMICs” data defines a role for UBR4 in virus budding. Cell Host Microbe 18, 723–735 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Watanabe, T. et al. Influenza virus–host interactome screen as a platform for antiviral drug development. Cell Host Microbe 16, 795–805 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Konig, R. et al. Human host factors required for influenza virus replication. Nature 463, 813–817 (2010).

    PubMed  PubMed Central  Google Scholar 

  95. Jorba, N. et al. Analysis of the interaction of influenza virus polymerase complex with human cell factors. Proteomics 8, 2077–2088 (2008).

    CAS  PubMed  Google Scholar 

  96. York, A., Hutchinson, E. C. & Fodor, E. Interactome analysis of the influenza A virus transcription/replication machinery identifies protein phosphatase 6 as a cellular factor required for efficient virus replication. J. Virol. 88, 13284–13299 (2014).

    PubMed  PubMed Central  Google Scholar 

  97. Mayer, D. et al. Identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches. J. Proteome Res. 6, 672–682 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  98. Bradel-Tretheway, B. G. et al. Comprehensive proteomic analysis of influenza virus polymerase complex reveals a novel association with mitochondrial proteins and RNA polymerase accessory factors. J. Virol. 85, 8569–8581 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  99. Bortz, E. et al. Host- and strain-specific regulation of influenza virus polymerase activity by interacting cellular proteins. mBio 2, e00151–11 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  100. Cao, M. et al. DnaJA1/Hsp40 is co-opted by influenza A virus to enhance its viral RNA polymerase activity. J. Virol. 88, 14078–14089 (2014).

    PubMed  PubMed Central  Google Scholar 

  101. Deng, T. et al. Role of ran binding protein 5 in nuclear import and assembly of the influenza virus RNA polymerase complex. J. Virol. 80, 11911–11919 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. Fislova, T., Thomas, B., Graef, K. M. & Fodor, E. Association of the influenza virus RNA polymerase subunit PB2 with the host chaperonin CCT. J. Virol. 84, 8691–8699 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Hutchinson, E. C., Orr, O. E., Man Liu, S., Engelhardt, O. G. & Fodor, E. Characterization of the interaction between the influenza A virus polymerase subunit PB1 and the host nuclear import factor Ran-binding protein 5. J. Gen. Virol. 92, 1859–1869 (2011).

    CAS  PubMed  Google Scholar 

  104. Chase, G. P. et al. Influenza virus ribonucleoprotein complexes gain preferential access to cellular export machinery through chromatin targeting. PLoS Pathog. 7, e1002187 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. Jackson, D. A., Caton, A. J., McCready, S. J. & Cook, P. R. Influenza virus RNA is synthesized at fixed sites in the nucleus. Nature 296, 366–368 (1982).

    CAS  PubMed  Google Scholar 

  106. Lopez-Turiso, J. A., Martinez, C., Tanaka, T. & Ortin, J. The synthesis of influenza virus negative-strand RNA takes place in insoluble complexes present in the nuclear matrix fraction. Virus Res. 16, 325–337 (1990).

    CAS  PubMed  Google Scholar 

  107. Takizawa, N., Watanabe, K., Nouno, K., Kobayashi, N. & Nagata, K. Association of functional influenza viral proteins and RNAs with nuclear chromatin and sub-chromatin structure. Microbes Infect. 8, 823–833 (2006).

    CAS  PubMed  Google Scholar 

  108. Alfonso, R. et al. CHD6 chromatin remodeler is a negative modulator of influenza virus replication that relocates to inactive chromatin upon infection. Cell. Microbiol. 13, 1894–1906 (2011).

    CAS  PubMed  Google Scholar 

  109. Marcos-Villar, L., Pazo, A. & Nieto, A. Influenza virus and chromatin: role of the CHD1 chromatin remodeler in the virus life cycle. J. Virol. 90, 3694–3707 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  110. Rodriguez, A., Perez-Gonzalez, A. & Nieto, A. Cellular human CLE/C14orf166 protein interacts with influenza virus polymerase and is required for viral replication. J. Virol. 85, 12062–12066 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. Ver, L. S., Marcos-Villar, L., Landeras-Bueno, S., Nieto, A. & Ortin, J. The cellular factor NXP2/MORC3 is a positive regulator of influenza virus multiplication. J. Virol. 89, 10023–10030 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. Martinez-Alonso, M., Hengrung, N. & Fodor, E. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Engelhardt, O. G., Smith, M. & Fodor, E. Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II. J. Virol. 79, 5812–5818 (2005). This is the first study to demonstrate the interaction between the influenza virus RNA polymerase and the host Pol II transcriptional machinery. The authors propose that cap-snatching is carried out by the viral polymerase bound to the serine-5-phosphorylated form of the CTD of Pol II.

    CAS  PubMed  PubMed Central  Google Scholar 

  114. Bentley, D. L. Coupling mRNA processing with transcription in time and space. Nat. Rev. Genet. 15, 163–175 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. Yamayoshi, S., Watanabe, M., Goto, H. & Kawaoka, Y. Identification of a novel viral protein expressed from the PB2 segment of influenza A virus. J. Virol. 90, 444–456 (2015).

    PubMed  PubMed Central  Google Scholar 

  116. Fournier, G. et al. Recruitment of RED–SMU1 complex by influenza A virus RNA polymerase to control viral mRNA splicing. PLoS Pathog. 10, e1004164 (2014).

    PubMed  PubMed Central  Google Scholar 

  117. Landeras-Bueno, S., Jorba, N., Perez-Cidoncha, M. & Ortin, J. The splicing factor proline-glutamine rich (SFPQ/PSF) is involved in influenza virus transcription. PLoS Pathog. 7, e1002397 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  118. Shih, S. R. & Krug, R. M. Novel exploitation of a nuclear function by influenza virus: the cellular SF2/ASF splicing factor controls the amount of the essential viral M2 ion channel protein in infected cells. EMBO J. 15, 5415–5427 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  119. Tsai, P. L. et al. Cellular RNA binding proteins NS1-BP and hnRNP K regulate influenza A virus RNA splicing. PLoS Pathog. 9, e1003460 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  120. Kawaguchi, A. & Nagata, K. De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM. EMBO J. 26, 4566–4575 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. Sugiyama, K., Kawaguchi, A., Okuwaki, M. & Nagata, K. pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA. eLife 4, e08939 (2015). This analysis identifies ANP32A and ANP32B as host factors that are required for the replication of the influenza A virus cRNA replicative intermediate into vRNA.

    PubMed  PubMed Central  Google Scholar 

  122. Long, J. S. et al. Species difference in ANP32A underlies influenza A virus polymerase host restriction. Nature 529, 101–104 (2016). This work finds that ANP32A is the host factor that underlies the influenza A virus polymerase host restriction mediated by residue 627 of the PB2 subunit.

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Zhou, Z. et al. Fragile X mental retardation protein stimulates ribonucleoprotein assembly of influenza A virus. Nat. Commun. 5, 3259 (2014).

    PubMed  Google Scholar 

  124. Resa-Infante, P. et al. The host-dependent interaction of α-importins with influenza PB2 polymerase subunit is required for virus RNA replication. PLoS ONE 3, e3904 (2008).

    PubMed  PubMed Central  Google Scholar 

  125. Nemeroff, M. E., Barabino, S. M., Li, Y., Keller, W. & Krug, R. M. Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3′ end formation of cellular pre-mRNAs. Mol. Cell 1, 991–1000 (1998).

    CAS  PubMed  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  127. Vreede, F. T., Chan, A. Y., Sharps, J. & Fodor, E. Mechanisms and functional implications of the degradation of host RNA polymerase II in influenza virus infected cells. Virology 396, 125–134 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  128. Llompart, C. M., Nieto, A. & Rodriguez-Frandsen, A. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  130. Vasin, A. V. et al. Molecular mechanisms enhancing the proteome of influenza A viruses: an overview of recently discovered proteins. Virus Res. 185, 53–63 (2014).

    CAS  PubMed  Google Scholar 

  131. Monod, A. et al. Learning from structure-based drug design and new antivirals targeting the ribonucleoprotein complex for the treatment of influenza. Expert Opin. Drug Discov. 10, 345–371 (2015).

    CAS  PubMed  Google Scholar 

  132. Byrn, R. A. et al. Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit. Antimicrob. Agents Chemother. 59, 1569–1582 (2015).

    PubMed  PubMed Central  Google Scholar 

  133. Clark, M. P. et al. Discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (VX-787) of influenza PB2. J. Med. Chem. 57, 6668–6678 (2014).

    CAS  PubMed  Google Scholar 

  134. DuBois, R. M. et al. Structural and biochemical basis for development of influenza virus inhibitors targeting the PA endonuclease. PLoS Pathog. 8, e1002830 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  135. Kowalinski, E. et al. Structural analysis of specific metal chelating inhibitor binding to the endonuclease domain of influenza pH1N1 polymerase. PLoS Pathog. 8, e1002831 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  136. Pautus, S. et al. New 7-methylguanine derivatives targeting the influenza polymerase PB2 cap-binding domain. J. Med. Chem. 56, 8915–8930 (2013).

    CAS  PubMed  Google Scholar 

  137. Sagong, H. Y. et al. Phenyl substituted 4-hydroxypyridazin-3(2H)-ones and 5-hydroxypyrimidin-4(3H)-ones: inhibitors of influenza A endonuclease. J. Med. Chem. 57, 8086–8098 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. Yuan, S. et al. A novel small-molecule inhibitor of influenza A virus acts by suppressing PA endonuclease activity of the viral polymerase. Sci. Rep. 6, 22880 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  139. Sangawa, H. et al. Mechanism of action of T-705 ribosyl triphosphate against influenza virus RNA polymerase. Antimicrob. Agents Chemother. 57, 5202–5208 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  140. Sleeman, K. et al. In vitro antiviral activity of favipiravir (T-705) against drug-resistant influenza and 2009 A(H1N1) viruses. Antimicrob. Agents Chemother. 54, 2517–2524 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. Tisdale, M. et al. Inhibition of influenza A and B viruses by 2′-deoxy-2′-fluororibosides. Antiviral Chem. Chemother. 4, 281–287 (1993).

    CAS  Google Scholar 

  142. Ghanem, A. et al. Peptide-mediated interference with influenza A virus polymerase. J. Virol. 81, 7801–7804 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  143. Massari, S. et al. A broad anti-influenza hybrid small molecule that potently disrupts the interaction of polymerase acidic protein–basic protein 1 (PA-PB1) subunits. J. Med. Chem. 58, 3830–3842 (2015).

    CAS  PubMed  Google Scholar 

  144. Wunderlich, K. et al. Identification of high-affinity PB1-derived peptides with enhanced affinity to the PA protein of influenza A virus polymerase. Antimicrob. Agents Chemother. 55, 696–702 (2011).

    CAS  PubMed  Google Scholar 

  145. Yuan, S. et al. Identification of a small-molecule inhibitor of influenza virus via disrupting the subunits interaction of the viral polymerase. Antiviral Res. 125, 34–42 (2016).

    CAS  PubMed  Google Scholar 

  146. Manz, B. et al. Disruption of the viral polymerase complex assembly as a novel approach to attenuate influenza A virus. J. Biol. Chem. 286, 8414–8424 (2011).

    PubMed  Google Scholar 

  147. Muratore, G. et al. Small molecule inhibitors of influenza A and B viruses that act by disrupting subunit interactions of the viral polymerase. Proc. Natl Acad. Sci. USA 109, 6247–6252 (2012).

    CAS  PubMed  Google Scholar 

  148. Tintori, C. et al. High-throughput docking for the identification of new influenza A virus polymerase inhibitors targeting the PA–PB1 protein–protein interaction. Bioorg. Med. Chem. Lett. 24, 280–282 (2014).

    CAS  PubMed  Google Scholar 

  149. Li, C. et al. A peptide derived from the C-terminus of PB1 inhibits influenza virus replication by interfering with viral polymerase assembly. FEBS J. 280, 1139–1149 (2013).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors are grateful to F. Vreede for his comments on the manuscript. This work was supported by the UK Medical Research Council grant MR/K000241/1 (to E.F.), the Wellcome Trust grant 098721/Z/12/Z (to A.T.), the Netherlands Organization for Scientific Research (NWO) grant 825.11.029 (to A.T.) and a Kemp postdoctoral fellowship from Lincoln College, University of Oxford, UK (to A.T.).

Author information

Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK

    Aartjan J. W. te Velthuis & Ervin Fodor

Authors
  1. Aartjan J. W. te Velthuis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ervin Fodor
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ervin Fodor.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Related links

PowerPoint slides

Supplementary information

Supplementary information S1 (movie)

Conformational flexibility of the influenza virus RNA polymerase. Morph between the structure of the RNA-free influenza C virus polymerase (PDB: 5D98) and that of vRNA promoter-bound influenza A virus polymerase (PDB: 4WSB). PB1 and PA are coloured orange and blue, respectively. PB2 is coloured according to its domain structure with the N terminal one third shown in green, the cap-binding domain in dark yellow, the mid-link in light yellow, the 627 domain in red and the C terminal NLS in brown. Promoter RNA is shown in red. Modified from Hengrung et al. (MOV 14521 kb)

Glossary

Antigenic variation

Changes in the antigenicity of a protein of interest (here, the haemagglutinin and neuraminidase proteins) owing to antigenic drift or shift.

Haemagglutinin

(HA). A type I integral membrane glycoprotein of influenza viruses that binds to cell surface receptors in the host and facilitates fusion between the viral envelope and endosomal membrane. It is the main antigen that is targeted by the humoral immune response of the host.

Neuraminidase

(NA). A type II integral membrane glycoprotein of influenza viruses that facilitates viral release from cells by removing sialic acid from sialyloligosaccharides on the cell and viral surface.

Cap-snatching

A process in which a cellular capped RNA is cleaved a few nucleotides downstream of the 5′-cap by an endonuclease activity that is encompassed within a viral RNA-dependent RNA polymerase.

Host range determinant

A characteristic of a pathogen that enables it to replicate in a particular host.

Panhandle structure

A double-stranded RNA structure that is formed by the conserved 5′ and 3′ RNA termini of negative-sense viral RNA genomes.

Apo form

A protein with no ligand bound.

Helicase

An enzyme that catalyses the unwinding and separation of double-stranded DNA or RNA using energy from ATP hydrolysis.

De novo initiation

The initiation step of nucleic acid synthesis that occurs by means of a primer-independent mechanism.

Pol II pausing

(Cellular DNA-dependent RNA polymerase II pausing). A control step in gene transcription, in which Pol II pauses at certain sites and requires specific stimuli and elongation factors to overcome this pausing block and enter productive elongation.

Pol II large subunit CTD

(Cellular DNA-dependent RNA polymerase II large subunit C-terminal domain). An unstructured, evolutionarily conserved domain at the C terminus of the largest Pol II subunit; this domain comprises tandem copies of the consensus heptapeptide YSPTSPS, and phosphorylation of these repeats is crucial for the regulation of Pol II function.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

te Velthuis, A., Fodor, E. Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis. Nat Rev Microbiol 14, 479–493 (2016). https://doi.org/10.1038/nrmicro.2016.87

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrmicro.2016.87

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing