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.

  • Article
  • Published:

The structural basis for cap binding by influenza virus polymerase subunit PB2

Nature Structural & Molecular Biology volume 15, pages 500–506 (2008)Cite this article

Abstract

Influenza virus mRNAs are synthesized by the trimeric viral polymerase using short capped primers obtained by a 'cap-snatching' mechanism. The polymerase PB2 subunit binds the 5′ cap of host pre-mRNAs, which are cleaved after 10–13 nucleotides by the PB1 subunit. Using a library-screening method, we identified an independently folded domain of PB2 that has specific cap binding activity. The X-ray structure of the domain with bound cap analog m7GTP at 2.3-Å resolution reveals a previously unknown fold and a mode of ligand binding that is similar to, but distinct from, other cap binding proteins. Binding and functional studies with point mutants confirm that the identified site is essential for cap binding in vitro and cap-dependent transcription in vivo by the trimeric polymerase complex. These findings clarify the nature of the cap binding site in PB2 and will allow efficient structure-based design of new anti-influenza compounds inhibiting viral transcription.

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: Structure of PB2 cap binding domain with bound m7GTP.
Figure 2: Cap binding activity of wild-type (WT) or mutant recombinant polymerase complexes.
Figure 3: Replication and transcription activities of wild-type and mutant recombinant mini-RNPs.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Olsen, B. et al. Global patterns of influenza a virus in wild birds. Science 312, 384–388 (2006).

    Article  CAS  Google Scholar 

  2. Horimoto, T. & Kawaoka, Y. Influenza: lessons from past pandemics, warnings from current incidents. Nat. Rev. Microbiol. 3, 591–600 (2005).

    Article  CAS  Google Scholar 

  3. Elton, D., Digard, P., Tiley, L. & Ortín, J. Structure and function of the influenza virus RNP. in Current Topics in Influenza Virology (ed. Kawaoka, Y.) (Horizon Scientific, Norfolk, 2005).

    Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

  5. Li, M.L., Rao, P. & Krug, R.M. The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits. EMBO J. 20, 2078–2086 (2001).

    Article  CAS  Google Scholar 

  6. 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 

  7. 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 

  8. Honda, A., Mizumoto, K. & Ishihama, A. Two separate sequences of PB2 subunit constitute the RNA cap-binding site of influenza virus RNA polymerase. Genes Cells 4, 475–485 (1999).

    Article  CAS  Google Scholar 

  9. Fechter, P. et al. Two aromatic residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding. J. Biol. Chem. 278, 20381–20388 (2003).

    Article  CAS  Google Scholar 

  10. Fechter, P. & Brownlee, G.G. Recognition of mRNA cap structures by viral and cellular proteins. J. Gen. Virol. 86, 1239–1249 (2005).

    Article  CAS  Google Scholar 

  11. Area, E. et al. 3D structure of the influenza virus polymerase complex: localization of subunit domains. Proc. Natl. Acad. Sci. USA 101, 308–313 (2004).

    Article  CAS  Google Scholar 

  12. Torreira, E. et al. Three-dimensional model for the isolated recombinant influenza virus polymerase heterotrimer. Nucleic Acids Res. 35, 3774–3783 (2007).

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  Google Scholar 

  14. Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

    Article  CAS  Google Scholar 

  15. Marcotrigiano, J., Gingras, A.C., Sonenberg, N. & Burley, S.K. Cocrystal structure of the messenger RNA 5′ cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell 89, 951–961 (1997).

    Article  CAS  Google Scholar 

  16. Mazza, C., Segref, A., Mattaj, I.W. & Cusack, S. Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex. EMBO J. 21, 5548–5557 (2002).

    Article  CAS  Google Scholar 

  17. Hodel, A.E., Gershon, P.D. & Quiocho, F.A. Structural basis for sequence-nonspecific recognition of 5′-capped mRNA by a cap-modifying enzyme. Mol. Cell 1, 443–447 (1998).

    Article  CAS  Google Scholar 

  18. Niedzwiecka, A. et al. Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5′ cap structure and synthetic fragments of eIF4G and 4E–BP1 proteins. J. Mol. Biol. 319, 615–635 (2002).

    Article  CAS  Google Scholar 

  19. Martin-Benito, J. et al. Three-dimensional reconstruction of a recombinant influenza virus ribonucleoprotein particle. EMBO Rep. 2, 313–317 (2001).

    Article  CAS  Google Scholar 

  20. Ortega, J. et al. Ultrastructural and functional analyses of recombinant influenza virus ribonucleoproteins suggest dimerization of nucleoprotein during virus amplification. J. Virol. 74, 156–163 (2000).

    Article  CAS  Google Scholar 

  21. Worch, R. et al. Specificity of recognition of mRNA 5′ cap by human nuclear cap-binding complex. RNA 11, 1355–1363 (2005).

    Article  CAS  Google Scholar 

  22. Hooker, L. et al. Quantitative analysis of influenza virus RNP interaction with RNA cap structures and comparison to human cap binding protein eIF4E. Biochemistry 42, 6234–6240 (2003).

    Article  CAS  Google Scholar 

  23. Bouloy, M., Plotch, S.J. & Krug, R.M. Both the 7-methyl and the 2′-O-methyl groups in the cap of mRNA strongly influence its ability to act as primer for influenza virus RNA transcription. Proc. Natl. Acad. Sci. USA 77, 3952–3956 (1980).

    Article  CAS  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

  25. 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).

    Article  CAS  Google Scholar 

  26. Penn, C.R. & Mahy, B.W. Capped mRNAs may stimulate the influenza virion polymerase by allosteric modulation. Virus Res. 1, 1–13 (1984).

    Article  CAS  Google Scholar 

  27. Kawakami, K., Mizumoto, K., Ishihama, A., Shinozaki-Yamaguchi, K. & Miura, K. Activation of influenza virus-associated RNA polymerase by cap-1 structure (m7GpppNm). J. Biochem. 97, 655–661 (1985).

    Article  CAS  Google Scholar 

  28. Cianci, C., Colonno, R.J. & Krystal, M. Differential effect of modified capped RNA substrates on influenza virus transcription. Virus Res. 50, 65–75 (1997).

    Article  CAS  Google Scholar 

  29. Li, M.L., Ramirez, B.C. & Krug, R.M. RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites. EMBO J. 17, 5844–5852 (1998).

    Article  CAS  Google Scholar 

  30. Ulmanen, I., Broni, B. & Krug, R.M. Influenza virus temperature-sensitive cap (m7GpppNm)-dependent endonuclease. J. Virol. 45, 27–35 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Yamanaka, K. et al. Characterization of a temperature-sensitive mutant in the RNA polymerase PB2 subunit gene of influenza A/WSN/33 virus. Arch. Virol. 114, 65–73 (1990).

    Article  CAS  Google Scholar 

  32. Luo, G., Danetz, S. & Krystal, M. Inhibition of influenza viral polymerases by minimal viral RNA decoys. J. Gen. Virol. 78, 2329–2333 (1997).

    Article  CAS  Google Scholar 

  33. De Clercq, E. Antiviral agents active against influenza A viruses. Nat. Rev. Drug Discov. 5, 1015–1025 (2006).

    Article  CAS  Google Scholar 

  34. DuBridge, R.B. et al. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol. Cell. Biol. 7, 379–387 (1987).

    Article  CAS  Google Scholar 

  35. Ortin, J., Najera, R., Lopez, C., Davila, M. & Domingo, E. Genetic variability of Hong Kong (H3N2) influenza viruses: spontaneous mutations and their location in the viral genome. Gene 11, 319–331 (1980).

    Article  CAS  Google Scholar 

  36. 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).

    Article  CAS  Google Scholar 

  37. Neumann, G. et al. Generation of influenza A viruses entirely from cloned cDNAs. Proc. Natl. Acad. Sci. USA 96, 9345–9350 (1999).

    Article  CAS  Google Scholar 

  38. de la Luna, S., Martinez, C. & Ortin, J. Molecular cloning and sequencing of influenza virus A/Victoria/3/75 polymerase genes: sequence evolution and prediction of possible functional domains. Virus Res. 13, 143–155 (1989).

    Article  CAS  Google Scholar 

  39. Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L. & Clardy, J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124 (1993).

    Article  CAS  Google Scholar 

  40. Perrakis, A., Morris, R. & Lamzin, V.S. Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463 (1999).

    Article  CAS  Google Scholar 

  41. Murshudov, G.N. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  42. Lovell, S.C. et al. Structure validation by Cα geometry: φ, ψ and Cβ deviation. Proteins 50, 437–450 (2003).

    Article  CAS  Google Scholar 

  43. Barcena, J. et al. Monoclonal antibodies against influenza virus PB2 and NP polypeptides interfere with the initiation step of viral mRNA synthesis in vitro. J. Virol. 68, 6900–6909 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Perales, B., de la Luna, S., Palacios, I. & Ortin, J. Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit. J. Virol. 70, 1678–1686 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank P. Mas for help with ESPRIT screening; H. Belrhali, A. McCarthy and M. Walsh (MRC-France) for help in data collection and structure determination; Y. Fernández (CSIC) for help in the polymerase assays; and B. Dublet, L. Signor and E. Forest (IBS) for MS measurements. We acknowledge the ESRF, EMBL and MRC-France for access to synchrotron facilities, the Partnership for Structural Biology for an integrated structural biology environment and the EU for funding of the FLUPOL contract (SP5B-CT-2007-044263). Work at CNB was also supported by the Spanish Ministry of Education and Science (Ministerio de Educación y Ciencia) (BFU2004-491) and the Research Programme VIRHOST-CM from Comunidad de Madrid (S-SAL/0185/2006). Thanks to Catherine for inspiration.

Author information

Author notes

  1. Delphine Guilligay and Franck Tarendeau: These authors contributed equally to this work.

Authors and Affiliations

  1. Grenoble Outstation, European Molecular Biology Laboratory, 6 rue Jules Horowitz, BP181, Grenoble, 38042, Cedex 9, France

    Delphine Guilligay, Franck Tarendeau, Thibaut Crepin, Darren J Hart & Stephen Cusack

  2. Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMR 5233, 6 rue Jules Horowitz, Grenoble, 38042, Cedex 9, France

    Delphine Guilligay, Franck Tarendeau, Thibaut Crepin, Rob W H Ruigrok, Darren J Hart & Stephen Cusack

  3. Centro Nacional de Biotecnología (CSIC), Darwin 3, Campus de Cantoblanco, 28049, Madrid, Spain

    Patricia Resa-Infante, Rocío Coloma & Juan Ortin

  4. CIBER de Enfermedades Respiratorias,

    Patricia Resa-Infante, Rocío Coloma & Juan Ortin

  5. Chemical Biology Core Facility, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117, Heidelberg, Germany

    Peter Sehr & Joe Lewis

Authors
  1. Delphine Guilligay
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Franck Tarendeau
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Patricia Resa-Infante
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rocío Coloma
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thibaut Crepin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peter Sehr
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Joe Lewis
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rob W H Ruigrok
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Juan Ortin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Darren J Hart
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Stephen Cusack
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.J.H. and F.T. devised and implemented the extension of ESPRIT to internal domains and identified soluble central fragments of PB2. D.G. and F.T. purified and biochemically characterized soluble central fragments of PB2. D.G. identified and crystallized the minimal cap binding domain, made and crystallized selenomethionated protein and made and assayed cap binding of domain mutants. S.C. performed the crystal structure analysis with T.C. P.R.-I. established conditions for binding of polymerase to cap-analog resins. P.R.-I. and R.C. contributed equally to the generation of and functional characterization of mutants in the context of recombinant polymerase and RNPs. J.O. designed and supervised the mutant phenotype analyses. S.C. compiled the text with contributions from J.O. and D.H. P.S. and J.L. performed and analysed the SPR measurements. J.O., R.W.H.R. and S.C. are principal investigators of the EU FP6 FLUPOL project.

Corresponding author

Correspondence to Stephen Cusack.

Ethics declarations

Competing interests

D.J.H. and F.T. have applied for a patent on ESPRIT technology. S.C., D.G., D.J.H. and F.T. have applied for a patent on the use of recombinantly expressed constructs of the PB2 cap binding domain and the three-dimensional structure for anti-influenza drug design.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 3271 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guilligay, D., Tarendeau, F., Resa-Infante, P. et al. The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat Struct Mol Biol 15, 500–506 (2008). https://doi.org/10.1038/nsmb.1421

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1421

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