1887

Abstract

The influenza A virus non-structural protein 1 (NS1) is a multifunctional protein and an important virulence factor. It is composed of two well-characterized domains linked by a short, but not well crystallographically defined, region of unknown function. To study the possible function of this region, we introduced alanine substitutions to replace the two highly conserved leucine residues at amino acid positions 69 and 77. The mutant L69,77A NS1 protein retained wild-type (WT)-comparable binding capabilities to dsRNA, cleavage and polyadenylation specificity factor 30 and the p85β subunit of PI3K. A mutant influenza A virus expressing the L69,77A NS1 protein was generated using reverse genetics. L69,77A NS1 virus infection induced significantly higher levels of beta interferon (IFN-β) expression in Madin–Darby canine kidney (MDCK) cells compared with WT NS1 virus. In addition, the replication rate of the L69,77A NS1 virus was substantially lower in MDCK cells but not in Vero cells compared with the WT virus, suggesting that the L69,77A NS1 protein does not fully antagonize IFN during viral replication. L69,77A NS1 virus infection was not able to activate the PI3K/Akt anti-apoptotic pathway, suggesting that the mutant NS1 protein may not be localized such that it has access to p85β during infection, which was supported by the altered subcellular localization pattern of the mutant NS1 compared with WT NS1 after transfection or virus infection. Our data demonstrate that this linker region between the two domains is critical for the functions of the NS1 protein during influenza A virus infection, possibly by determining the protein’s correct subcellular localization.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.031336-0
2011-08-01
2020-11-27
Loading full text...

Full text loading...

/deliver/fulltext/jgv/92/8/1832.html?itemId=/content/journal/jgv/10.1099/vir.0.031336-0&mimeType=html&fmt=ahah

References

  1. Bornholdt Z. A., Prasad B. V. 2006; X-ray structure of influenza virus NS1 effector domain. Nat Struct Mol Biol 13:559–560 [CrossRef][PubMed]
    [Google Scholar]
  2. Bornholdt Z. A., Prasad B. V. 2008; X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus. Nature 456:985–988 [CrossRef][PubMed]
    [Google Scholar]
  3. Bridges C. B., Harper S. A., Fukuda K., Uyeki T. M., Cox N. J.,, Singleton J. A.Advisory Committee on Immunization Practices 2003; Prevention and control of influenza. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 52(RR-8):1–34
    [Google Scholar]
  4. Chen Z., Li Y., Krug R. M. 1999; Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3′-end processing machinery. EMBO J 18:2273–2283 [CrossRef][PubMed]
    [Google Scholar]
  5. Donelan N. R., Basler C. F., García-Sastre A. 2003; A recombinant influenza A virus expressing an RNA-binding-defective NS1 protein induces high levels of beta interferon and is attenuated in mice. J Virol 77:13257–13266 [CrossRef][PubMed]
    [Google Scholar]
  6. Ehrhardt C., Marjuki H., Wolff T., Nürnberg B., Planz O., Pleschka S., Ludwig S. 2006; Bivalent role of the phosphatidylinositol-3-kinase (PI3K) during influenza virus infection and host cell defence. Cell Microbiol 8:1336–1348 [CrossRef][PubMed]
    [Google Scholar]
  7. Ehrhardt C., Wolff T., Pleschka S., Planz O., Beermann W., Bode J. G., Schmolke M., Ludwig S. 2007; Influenza A virus NS1 protein activates the PI3K/Akt pathway to mediate antiapoptotic signaling responses. J Virol 81:3058–3067 [CrossRef][PubMed]
    [Google Scholar]
  8. Emeny J. M., Morgan M. J. 1979; Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production. J Gen Virol 43:247–252 [CrossRef][PubMed]
    [Google Scholar]
  9. Franke T. F., Kaplan D. R., Cantley L. C. 1997; PI3K: downstream AKTion blocks apoptosis. Cell 88:435–437 [CrossRef][PubMed]
    [Google Scholar]
  10. Gack M. U., Albrecht R. A., Urano T., Inn K. S., Huang I. C., Carnero E., Farzan M., Inoue S., Jung J. U., García-Sastre A. 2009; Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I. Cell Host Microbe 5:439–449 [CrossRef][PubMed]
    [Google Scholar]
  11. García-Sastre A., Egorov A., Matassov D., Brandt S., Levy D. E., Durbin J. E., Palese P., Muster T. 1998; Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252:324–330 [CrossRef][PubMed]
    [Google Scholar]
  12. George R. A., Heringa J. 2002; An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng 15:871–879 [CrossRef][PubMed]
    [Google Scholar]
  13. Gonfloni S., Williams J. C., Hattula K., Weijland A., Wierenga R. K., Superti-Furga G. 1997; The role of the linker between the SH2 domain and catalytic domain in the regulation and function of Src. EMBO J 16:7261–7271 [CrossRef][PubMed]
    [Google Scholar]
  14. Greenspan D., Palese P., Krystal M. 1988; Two nuclear location signals in the influenza virus NS1 nonstructural protein. J Virol 62:3020–3026[PubMed]
    [Google Scholar]
  15. Hale B. G., Jackson D., Chen Y. H., Lamb R. A., Randall R. E. 2006; Influenza A virus NS1 protein binds p85β and activates phosphatidylinositol-3-kinase signaling. Proc Natl Acad Sci U S A 103:14194–14199 [CrossRef][PubMed]
    [Google Scholar]
  16. Hale B. G., Randall R. E., Ortín J., Jackson D. 2008; The multifunctional NS1 protein of influenza A viruses. J Gen Virol 89:2359–2376 [CrossRef][PubMed]
    [Google Scholar]
  17. Hirt B. 1967; Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 26:365–369 [CrossRef][PubMed]
    [Google Scholar]
  18. Jackson D., Hossain M. J., Hickman D., Perez D. R., Lamb R. A. 2008; A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity. Proc Natl Acad Sci U S A 105:4381–4386 [CrossRef][PubMed]
    [Google Scholar]
  19. Jackson D., Killip M. J., Galloway C. S., Russell R. J., Randall R. E. 2010; Loss of function of the influenza A virus NS1 protein promotes apoptosis but this is not due to a failure to activate phosphatidylinositol 3-kinase (PI3K). Virology 396:94–105 [CrossRef][PubMed]
    [Google Scholar]
  20. Kochs G., García-Sastre A., Martínez-Sobrido L. 2007; Multiple anti-interferon actions of the influenza A virus NS1 protein. J Virol 81:7011–7021 [CrossRef][PubMed]
    [Google Scholar]
  21. Li Z., Jiang Y., Jiao P., Wang A., Zhao F., Tian G., Wang X., Yu K., Bu Z., Chen H. 2006a; The NS1 gene contributes to the virulence of H5N1 avian influenza viruses. J Virol 80:11115–11123 [CrossRef][PubMed]
    [Google Scholar]
  22. Li S., Min J. Y., Krug R. M., Sen G. C. 2006b; Binding of the influenza A virus NS1 protein to PKR mediates the inhibition of its activation by either PACT or double-stranded RNA. Virology 349:13–21 [CrossRef][PubMed]
    [Google Scholar]
  23. Liu J., Lynch P. A., Chien C. Y., Montelione G. T., Krug R. M., Berman H. M. 1997; Crystal structure of the unique RNA-binding domain of the influenza virus NS1 protein. Nat Struct Biol 4:896–899 [CrossRef][PubMed]
    [Google Scholar]
  24. Lu Y., Wambach M., Katze M. G., Krug R. M. 1995; Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor. Virology 214:222–228 [CrossRef][PubMed]
    [Google Scholar]
  25. Mattison K., Oropeza R., Kenney L. J. 2002; The linker region plays an important role in the interdomain communication of the response regulator OmpR. J Biol Chem 277:32714–32721 [CrossRef][PubMed]
    [Google Scholar]
  26. Min J. Y., Krug R. M. 2006; The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: Inhibiting the 2′-5′ oligo (A) synthetase/RNase L pathway. Proc Natl Acad Sci U S A 103:7100–7105 [CrossRef][PubMed]
    [Google Scholar]
  27. Nemeroff M. E., Qian X. Y., Krug R. M. 1995; The influenza virus NS1 protein forms multimers in vitro and in vivo. Virology 212:422–428 [CrossRef][PubMed]
    [Google Scholar]
  28. Nemeroff M. E., Barabino S. M., Li Y., Keller W., Krug R. M. 1998; 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 [CrossRef][PubMed]
    [Google Scholar]
  29. Noah D. L., Twu K. Y., Krug R. M. 2003; Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the 3′ end processing of cellular pre-mRNAs. Virology 307:386–395 [CrossRef][PubMed]
    [Google Scholar]
  30. Noah J. W., Severson W., Noah D. L., Rasmussen L., White E. L., Jonsson C. B. 2007; A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals. Antiviral Res 73:50–59 [CrossRef][PubMed]
    [Google Scholar]
  31. Shin Y. K., Liu Q., Tikoo S. K., Babiuk L. A., Zhou Y. 2007a; Influenza A virus NS1 protein activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by direct interaction with the p85 subunit of PI3K. J Gen Virol 88:13–18 [CrossRef][PubMed]
    [Google Scholar]
  32. Shin Y. K., Li Y., Liu Q., Anderson D. H., Babiuk L. A., Zhou Y. 2007b; SH3 binding motif 1 in influenza A virus NS1 protein is essential for PI3K/Akt signaling pathway activation. J Virol 81:12730–12739 [CrossRef][PubMed]
    [Google Scholar]
  33. Wang W., Riedel K., Lynch P., Chien C. Y., Montelione G. T., Krug R. M. 1999; RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids. RNA 5:195–205 [CrossRef][PubMed]
    [Google Scholar]
  34. Yu J., Zhang Y., McIlroy J., Rordorf-Nikolic T., Orr G. A., Backer J. M. 1998; Regulation of the p85/p110 phosphatidylinositol 3′-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit. Mol Cell Biol 18:1379–1387[PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.031336-0
Loading
/content/journal/jgv/10.1099/vir.0.031336-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error