1887

Abstract

The influenza virus non-structural protein 1 (NS1) is a multifunctional virulence factor that plays a crucial role during infection by blocking the innate antiviral immune response of infected cells. In contrast to the well-studied NS1 protein of influenza A virus, knowledge about structure and functions of the influenza B virus homologue B/NS1, which shares less than 25 % sequence identity, is still limited. Here, we report on a reverse genetic analysis to study the role of a highly conserved class II Src homology 3 domain-binding motif matching the consensus PxxPx(K/R) that we identified at positions 122–127 of the B/NS1 protein. Surprisingly, glycine substitutions in the Src homology 3 domain-binding motif increased virus replication up to three orders of magnitude in human lung cells. Enhanced mutant virus propagation was accompanied by increased gene expression and apoptosis induction linking this motif to the control of programmed cell death. A MS-based interactome study revealed that the glycine substitutions facilitate binding of B/NS1 to heat shock protein 90-beta (HSP90β). Moreover, recruitment of the viral polymerase basic protein 2 to the B/NS1–HSP90β complex was observed. Pharmacological inhibition of HSP90 reduced mutant virus propagation suggesting that the mutation-induced involvement of HSP90β enhanced viral replication. This study not only functionally characterizes a conserved motif within the B/NS1 protein, but also illustrates a rare example in which mutation of a highly conserved sequence within a viral protein does not result in high fitness costs, but rather increases viral replication via recruitment of a host factor.

Keyword(s): HSP90 , influenza , NS1 and SH3-binding motif
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000604
2016-11-10
2021-07-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/97/11/2856.html?itemId=/content/journal/jgv/10.1099/jgv.0.000604&mimeType=html&fmt=ahah

References

  1. Agrawal V., Kishan K. V. 2002; Promiscuous binding nature of SH3 domains to their target proteins. Protein Pept Lett 9:185–193 [View Article][PubMed]
    [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410 [View Article][PubMed]
    [Google Scholar]
  3. Ashkenazy H., Erez E., Martz E., Pupko T., Ben-Tal N. 2010; ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res 38:W529–W533 [View Article][PubMed]
    [Google Scholar]
  4. Beare A. S., Sherwood J. E., Callow K. A., Craig J. W. 1977; Selection of influenza B virus recombinants and their testing in humans for attenuation and immunogenicity. Infect Immun 15:347–353[PubMed]
    [Google Scholar]
  5. Bergmann M., Garcia-Sastre A., Carnero E., Pehamberger H., Wolff K., Palese P., Muster T. 2000; Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication. J Virol 74:6203–6206 [View Article][PubMed]
    [Google Scholar]
  6. Berri F., Haffar G., V. B., Sadewasser A., Paki K., Lina B., Wolff T., Riteau B. 2014; Annexin V incorporated into influenza virus particles inhibits gamma interferon signaling and promotes viral replication. J Virol 88:11215–11228 [View Article][PubMed]
    [Google Scholar]
  7. Birge R. B., Kalodimos C., Inagaki F., Tanaka S. 2009; Crk and CrkL adaptor proteins: networks for physiological and pathological signaling. Cell Commun Signal 7:13 [View Article][PubMed]
    [Google Scholar]
  8. Biswas K. M., DeVido D. R., Dorsey J. G. 2003; Evaluation of methods for measuring amino acid hydrophobicities and interactions. J Chromatogr A 1000:637–655 [View Article][PubMed]
    [Google Scholar]
  9. Blagosklonny M. V., Toretsky J., Bohen S., Neckers L. 1996; Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90. Proc Natl Acad Sci U S A 93:8379–8383 [View Article][PubMed]
    [Google Scholar]
  10. Briedis D. J., Lamb R. A. 1982; Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1 and NS2 proteins. J Virol 42:186–193[PubMed]
    [Google Scholar]
  11. Chase G., Deng T., Fodor E., Leung B. W., Mayer D., Schwemmle M., Brownlee G. 2008; Hsp90 inhibitors reduce influenza virus replication in cell culture. Virology 377:431–439 [View Article][PubMed]
    [Google Scholar]
  12. Dauber B., Heins G., Wolff T. 2004; The influenza B virus nonstructural NS1 protein is essential for efficient viral growth and antagonizes beta interferon induction. J Virol 78:1865–1872 [View Article][PubMed]
    [Google Scholar]
  13. Dauber B., Schneider J., Wolff T. 2006; Double-stranded RNA binding of influenza B virus nonstructural NS1 protein inhibits protein kinase R but is not essential to antagonize production of alpha/beta interferon. J Virol 80:11667–11677 [View Article][PubMed]
    [Google Scholar]
  14. Dauber B., Martínez-Sobrido L., Schneider J., Hai R., Waibler Z., Kalinke U., García-Sastre A., Wolff T. 2009; Influenza B virus ribonucleoprotein is a potent activator of the antiviral kinase PKR. PLoS Pathog 5:e1000473 [View Article][PubMed]
    [Google Scholar]
  15. de Chassey B., Meyniel-Schicklin L., Aublin-Gex A., Navratil V., Chantier T., André P., Lotteau V. 2013; Structure homology and interaction redundancy for discovering virus–host protein interactions. EMBO Rep 14:938–944 [View Article][PubMed]
    [Google Scholar]
  16. Dhanasekaran D. N., Reddy E. P. 2008; JNK signaling in apoptosis. Oncogene 27:6245–6251 [View Article][PubMed]
    [Google Scholar]
  17. Donelan N. R., Dauber B., Wang X., Basler C. F., Wolff T., García-Sastre A. 2004; The N- and C-terminal domains of the NS1 protein of influenza B virus can independently inhibit IRF-3 and beta interferon promoter activation. J Virol 78:11574–11582 [View Article][PubMed]
    [Google Scholar]
  18. Ehrhardt C., Wolff T., Ludwig S. 2007a; Activation of phosphatidylinositol 3-kinase signaling by the nonstructural NS1 protein is not conserved among type A and B influenza viruses. J Virol 81:12097–12100 [View Article][PubMed]
    [Google Scholar]
  19. Ehrhardt C., Wolff T., Pleschka S., Planz O., Beermann W., Bode J. G., Schmolke M., Ludwig S. 2007b; Influenza A virus NS1 protein activates the PI3K/Akt pathway to mediate antiapoptotic signaling responses. J Virol 81:3058–3067 [View Article][PubMed]
    [Google Scholar]
  20. Forbes N. E., Ping J., Dankar S. K., Jia J. J., Selman M., Keleta L., Zhou Y., Brown E. G. 2012; Multifunctional adaptive NS1 mutations are selected upon human influenza virus evolution in the mouse. PLoS One 7:e31839 [View Article][PubMed]
    [Google Scholar]
  21. 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 [View Article][PubMed]
    [Google Scholar]
  22. Glezen W. P., Schmier J. K., Kuehn C. M., Ryan K. J., Oxford J. 2013; The burden of influenza B: a structured literature review. Am J Public Health 103:e43e51 [View Article][PubMed]
    [Google Scholar]
  23. 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 [View Article][PubMed]
    [Google Scholar]
  24. Hale B. G. 2014; Conformational plasticity of the influenza A virus NS1 protein. J Gen Virol 95:2099–2105 [View Article][PubMed]
    [Google Scholar]
  25. Hall T. A. 1999; BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98
    [Google Scholar]
  26. Heikkinen L. S., Kazlauskas A., Melén K., Wagner R., Ziegler T., Julkunen I., Saksela K. 2008; Avian and 1918 Spanish influenza A virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling. J Biol Chem 283:5719–5727 [View Article][PubMed]
    [Google Scholar]
  27. Hrincius E. R., Wixler V., Wolff T., Wagner R., Ludwig S., Ehrhardt C. 2010; CRK adaptor protein expression is required for efficient replication of avian influenza A viruses and controls JNK-mediated apoptotic responses. Cell Microbiol 12:831–843 [View Article][PubMed]
    [Google Scholar]
  28. Hrincius E. R., Liedmann S., Anhlan D., Wolff T., Ludwig S., Ehrhardt C. 2014; Avian influenza viruses inhibit the major cellular signalling integrator c-Abl. Cell Microbiol 16:1854–1874 [View Article][PubMed]
    [Google Scholar]
  29. Jirakanwisal K., Srisutthisamphan K., Thepparit C., Suptawiwat O., Auewarakul P., Paemanee A., Roytrakul S., Smith D. R. 2015; Identification of Hsp90 as a species independent H5N1 avian influenza A virus PB2 interacting protein. Comp Immunol Microbiol Infect Dis 43:28–35 [View Article][PubMed]
    [Google Scholar]
  30. Karagöz G. E., Rüdiger S. G. 2015; Hsp90 interaction with clients. Trends Biochem Sci 40:117–125 [View Article][PubMed]
    [Google Scholar]
  31. Karlas A., Machuy N., Shin Y., Pleissner K. P., Artarini A., Heuer D., Becker D., Khalil H., Ogilvie L. A. et al. 2010; Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 463:818–822 [View Article][PubMed]
    [Google Scholar]
  32. Kawaguchi A., Matsumoto K., Nagata K. 2012; YB-1 functions as a porter to lead influenza virus ribonucleoprotein complexes to microtubules. J Virol 86:11086–11095 [View Article][PubMed]
    [Google Scholar]
  33. Kim E.-H., Park S.-J., Kwon H.-I., Kim S. M., Kim Y. I., Song M.-S., Choi E.-J., Pascua P. N., Choi Y.-K. 2015; Mouse adaptation of influenza B virus increases replication in the upper respiratory tract and results in droplet transmissibility in ferrets. Sci Rep 5:15940 [View Article][PubMed]
    [Google Scholar]
  34. König R., Stertz S. 2015; Recent strategies and progress in identifying host factors involved in virus replication. Curr Opin Microbiol 26:79–88 [View Article][PubMed]
    [Google Scholar]
  35. Li S. S. C. 2005; Specificity and versatility of SH3 and other proline-recognition domains: structural basis and implications for cellular signal transduction. Biochem J 390:641–653 [View Article][PubMed]
    [Google Scholar]
  36. Lin L., Li Y., Pyo H. M., Lu X., Raman S. N., Liu Q., Brown E. G., Zhou Y. 2011; Identification of RNA helicase A as a cellular factor that interacts with influenza A virus NS1 protein and its role in the virus life cycle. J Virol 86:1942–1954 [View Article][PubMed]
    [Google Scholar]
  37. Liu D. 2014; The adaptor protein Crk in immune response. Immunol Cell Biol 92:80–89 [View Article][PubMed]
    [Google Scholar]
  38. Ludwig S., Pleschka S., Planz O., Wolff T. 2006; Ringing the alarm bells: signalling and apoptosis in influenza virus infected cells. Cell Microbiol 8:375–386 [View Article][PubMed]
    [Google Scholar]
  39. Marión R. M., Zürcher T., de la Luna S., Ortín J. 1997; Influenza virus NS1 protein interacts with viral transcription–replication complexes in vivo . J Gen Virol 78:2447–2451 [View Article][PubMed]
    [Google Scholar]
  40. Mayer B. J., Saksela K. 2004; SH3 domains. In Structure and Function of Modular Protein Domains pp 37–58 . Edited by Cesareni G., Gimona M., Sudol M., Yaffe M. Weinheim: Wiley-VCH;
    [Google Scholar]
  41. Meissner F., Mann M. 2014; Quantitative shotgun proteomics: considerations for a high-quality workflow in immunology. Nat Immunol 15:112–117 [View Article][PubMed]
    [Google Scholar]
  42. Min J. Y., Li S., Sen G. C., Krug R. M. 2007; 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 [View Article][PubMed]
    [Google Scholar]
  43. Momose F., Naito T., Yano K., Sugimoto S., Morikawa Y., Nagata K. 2002; Identification of Hsp90 as a stimulatory host factor involved in influenza virus RNA synthesis. J Biol Chem 277:45306–45314 [View Article][PubMed]
    [Google Scholar]
  44. Naito T., Momose F., Kawaguchi A., Nagata K. 2007; Involvement of Hsp90 in assembly and nuclear import of influenza virus RNA polymerase subunits. J Virol 81:1339–1349 [View Article][PubMed]
    [Google Scholar]
  45. 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 [View Article][PubMed]
    [Google Scholar]
  46. Robb N. C., Chase G., Bier K., Vreede F. T., Shaw P.-C., Naffakh N., Schwemmle M., Fodor E. 2011; The influenza A virus NS1 protein interacts with the nucleoprotein of viral ribonucleoprotein complexes. J Virol 85:5228–5231 [View Article][PubMed]
    [Google Scholar]
  47. Rolland M., Manocheewa S., Swain J. V., Lanxon-Cookson E. C., Kim M., Westfall D. H., Larsen B. B., Gilbert P. B., Mullins J. I. 2013; HIV-1 conserved-element vaccines: relationship between sequence conservation and replicative capacity. J Virol 87:5461–5467 [View Article][PubMed]
    [Google Scholar]
  48. Saksela K., Permi P. 2012; SH3 domain ligand binding: what's the consensus and where's the specificity?. FEBS Lett 586:2609–2614 [View Article][PubMed]
    [Google Scholar]
  49. Schneider J., Dauber B., Melén K., Julkunen I., Wolff T. 2009; Analysis of influenza B virus NS1 protein trafficking reveals a novel interaction with nuclear speckle domains. J Virol 83:701–711 [View Article][PubMed]
    [Google Scholar]
  50. Sen J., Jacobs A., Caffrey M. 2008; Role of the HIV gp120 conserved domain 5 in processing and viral entry. Biochem istry 47:7788–7795 [CrossRef]
    [Google Scholar]
  51. Shaw. M. L., Palese P. 2013; Orthomyxoviridae: the viruses and their replication. In Fields Virology, 6th edn. pp 1151–1185 . Edited by Knipe D. M., Howley P. M. Philadelphia: Lippincott Williams & Wilkins;
    [Google Scholar]
  52. Shimizu K., Handa H., Nakada S., Nagata K. 1994; Regulation of influenza virus RNA polymerase activity by cellular and viral factors. Nucleic Acids Res 22:5047–5053 [View Article][PubMed]
    [Google Scholar]
  53. Sitbon E., Pietrokovski S. 2007; Occurrence of protein structure elements in conserved sequence regions. BMC Struct Biol 7:3 [View Article][PubMed]
    [Google Scholar]
  54. Talon J., Horvath C. M., Polley R., Basler C. F., Muster T., Palese P., García-Sastre A. 2000; Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein. J Virol 74:7989–7996 [View Article][PubMed]
    [Google Scholar]
  55. Trinquier G., Sanejouand Y. H. 1998; Which effective property of amino acids is best preserved by the genetic code?. Protein Eng 11:153–169 [View Article][PubMed]
    [Google Scholar]
  56. von Recum-Knepper J., Sadewasser A., Weinheimer V. K., Wolff T. 2015; Fluorescence-activated cell sorting-based analysis reveals an asymmetric induction of interferon-stimulated genes in response to seasonal influenza A virus. J Virol 89:6982–6993 [View Article][PubMed]
    [Google Scholar]
  57. Watanabe T., Kawakami E., Shoemaker J. E., Lopes T. J., Matsuoka Y., Tomita Y., Kozuka-Hata H., Gorai T., Kuwahara T. et al. 2014; Influenza virus-host interactome screen as a platform for antiviral drug development. Cell Host Microbe 16:795–805 [View Article][PubMed]
    [Google Scholar]
  58. Watkins R. L., Zou W., Denton P. W., Krisko J. F., Foster J. L., Garcia J. V. 2013; In vivo analysis of highly conserved Nef activities in HIV-1 replication and pathogenesis. Retrovirology 10:125 [View Article][PubMed]
    [Google Scholar]
  59. WHO 2014; Influenza (Seasonal), Fact sheet no. 211. Geneva World Health Organisation http://www.who.int/mediacentre/factsheets/fs211/en/
  60. Wright P. F., Neumann G., Kawaoka Y. 2013; Orthomyxoviruses. In Fields Virology, 6th edn. pp. 1186–1243 . Edited by Knipe D. M., Howley P. M. Philadelphia: Lippincott Williams & Wilkins;
    [Google Scholar]
  61. Wunderlich K., Juozapaitis M., Mänz B., Mayer D., Götz V., Zöhner A., Wolff T., Schwemmle M., Martin A. 2010; Limited compatibility of polymerase subunit interactions in influenza A and B viruses. J Biol Chem 285:16704–16712 [View Article][PubMed]
    [Google Scholar]
  62. Yin C., Khan J. A., Swapna G. V., Ertekin A., Krug R. M., Tong L., Montelione G. T. 2007; Conserved surface features form the double-stranded RNA binding site of non-structural protein 1 (NS1) from influenza A and B viruses. J Biol Chem 282:20584–20592 [View Article][PubMed]
    [Google Scholar]
  63. Ylösmäki L., Schmotz C., Ylösmäki E., Saksela K. 2015; Reorganization of the host cell Crk(L)-PI3 kinase signaling complex by the influenza A virus NS1 protein. Virology 484:146–152 [View Article][PubMed]
    [Google Scholar]
  64. Yuan W., Krug R. M. 2001; Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein. EMBO J 20:362–371 [View Article][PubMed]
    [Google Scholar]
  65. Yuan W., Aramini J. M., Montelione G. T., Krug R. M. 2002; Structural basis for ubiquitin-like ISG 15 protein binding to the NS1 protein of influenza B virus: a protein–protein interaction function that is not shared by the corresponding N-terminal domain of the NS1 protein of influenza A virus. Virology 304:291–301 [View Article][PubMed]
    [Google Scholar]
  66. Zhang C., Yang Y., Zhou X., Yang Z., Liu X., Cao Z., Song H., He Y., Huang P. 2011; The NS1 protein of influenza A virus interacts with heat shock protein Hsp90 in human alveolar basal epithelial cells: implication for virus-induced apoptosis. Virol J 8:181 [View Article][PubMed]
    [Google Scholar]
  67. Zhirnov O. P., Konakova T. E., Wolff T., Klenk H. D. 2002; NS1 protein of influenza A virus down-regulates apoptosis. J Virol 76:1617–1625 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000604
Loading
/content/journal/jgv/10.1099/jgv.0.000604
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

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