1887

Abstract

The non-structural protein 1 (NS1) of the influenza A virus and the NS2 protein, which is also known as nuclear export protein, play important roles in the infectious life cycle of the virus. The objective of this study was to find the degree of conservation in the NS proteins and to identify conserved sites of functional or structural importance that may be utilized as potential drug target sites. The analysis was based on 2620 amino acid sequences for the NS1 protein and 1195 sequences for the NS2 protein. The degree of conservation and potential binding sites were mapped onto the protein structures obtained from a combination of experimentally available structure fragments with predicted threading models. In addition to high conservation in protein regions of known function, novel highly conserved sites have been identified, namely Glu159, Thr171, Val192, Arg200, Glu208 and Gln218 on the NS1 protein and Ser24, Leu28, Arg66, Arg84, Ser93, Ile97 and Leu103 on the NS2 protein. Using the Q-SiteFinder binding site prediction algorithm, several highly conserved binding sites were found, including two spatially close sites on the NS1 protein, which could be targeted with a bivalent ligand that would interfere with double-stranded RNA binding. Altogether, this work reveals novel universally conserved residues that are candidates for protein–protein interactions and provide the basis for designing universal anti-influenza drugs.

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2009-09-01
2024-03-28
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References

  1. Akarsu, H., Burmeister, W. P., Petosa, C., Petit, I., Muller, C. W., Ruigrok, R. W. H. & Baudin, F.(2003). Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2). EMBO J 22, 4646–4655.[CrossRef] [Google Scholar]
  2. Aragón, T., de la Luna, S., Novoa, I., Carrasco, L., Ortín, J. & Nieto, A.(2000). Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus. Mol Cell Biol 20, 6259–6268.[CrossRef] [Google Scholar]
  3. Baez, M., Zazra, J. J., Elliott, R. M., Young, J. F. & Palese, P.(1981). Nucleotide sequence of the influenza A/duck/Alberta/60/76 virus NS RNA: conservation of the NS1/NS2 overlapping gene structure in a divergent influenza virus RNA segment. Virology 113, 397–402.[CrossRef] [Google Scholar]
  4. Bao, Y., Bolotov, P., Dernovoy, D., Kiryutin, B., Zaslavsky, L., Tatusova, T., Ostell, J. & Lipman, D.(2008). The influenza virus resource at the National Center for Biotechnology Information. J Virol 82, 596–601.[CrossRef] [Google Scholar]
  5. Basu, D., Walkiewicz, M. P., Frieman, M., Baric, R. S., Auble, D. T. & Engel, D. A.(2009). Novel influenza virus NS1 antagonists block replication and restore innate immune function. J Virol 83, 1881–1891.[CrossRef] [Google Scholar]
  6. Bernstein, H. J.(2000). Recent changes to RasMol, recombining the variants. Trends Biochem Sci 25, 453–455.[CrossRef] [Google Scholar]
  7. Bornholdt, Z. A. & Prasad, B. V. V.(2008). X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus. Nature 456, 985–988.[CrossRef] [Google Scholar]
  8. 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] [Google Scholar]
  9. Claas, E. C. J., Osterhaus, A., van Beek, R., De Jong, J. C., Rimmelzwaan, G. F., Senne, D. A., Krauss, S., Shortridge, K. F. & Webster, R. G.(1998). Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351, 472–477.[CrossRef] [Google Scholar]
  10. Clamp, M., Cuff, J., Searle, S. M. & Barton, G. J.(2004). The Jalview Java alignment editor. Bioinformatics 20, 426–427.[CrossRef] [Google Scholar]
  11. Cox, N. J. & Subbarao, K.(2000). Global epidemiology of influenza: past and present. Annu Rev Med 51, 407–421.[CrossRef] [Google Scholar]
  12. 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] [Google Scholar]
  13. Edgar, R. C.(2004).muscle: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32, 1792–1797.[CrossRef] [Google Scholar]
  14. Ehrhardt, C. & Ludwig, S.(2009). A new player in a deadly game: influenza viruses and the PI3K/Akt signalling pathway. Cell Microbiol 11, 863–871.[CrossRef] [Google Scholar]
  15. Ehrhardt, C., Marjuki, H., Wolff, T., Nurnberg, 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] [Google Scholar]
  16. 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] [Google Scholar]
  17. Enami, K., Sato, T. A., Nakada, S. & Enami, M.(1994). Influenza-virus NS1 protein stimulates translation of the M1 protein. J Virol 68, 1432–1437. [Google Scholar]
  18. Ferraris, O. & Lina, B.(2008). Mutations of neuraminidase implicated in neuraminidase inhibitors resistance. J Clin Virol 41, 13–19.[CrossRef] [Google Scholar]
  19. Fortes, P., Beloso, A. & Ortín, J.(1994). Influenza virus NS1 protein inhibits pre-messenger RNA splicing and blocks messenger RNA nucleocytoplasmic transport. EMBO J 13, 704–712. [Google Scholar]
  20. Glaser, F., Pupko, T., Paz, I., Bell, R. E., Bechor-Shental, D., Martz, E. & Ben-Tal, N.(2003). ConSurf: identification of functional regions in proteins by surface-mapping of phylogenetic information. Bioinformatics 19, 163–164.[CrossRef] [Google Scholar]
  21. Gog, J. R., Dos Santos Afonso, E., Dalton, R. M., Leclercq, I., Tiley, L., Elton, D., von Kirchbach, J. C., Naffakh, N., Escriou, N. & Digard, P.(2007). Codon conservation in the influenza A virus genome defines RNA packaging signals. Nucleic Acids Res 35, 1897–1907.[CrossRef] [Google Scholar]
  22. Greenspan, D., Krystal, M., Nakada, S., Arnheiter, H., Lyles, D. S. & Palese, P.(1985). Expression of influenza virus NS2 nonstructural proteins in bacteria and localization of NS2 in infected eucaryotic cells. J Virol 54, 833–843. [Google Scholar]
  23. Greenspan, D., Palese, P. & Krystal, M.(1988). Two nuclear localisation signals in the influenza virus NS1 nonstructural protein. J Virol 62, 3020–3026. [Google Scholar]
  24. 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] [Google Scholar]
  25. Hale, B. G., Batty, I. H., Downes, C. P. & Randall, R. E.(2008a). Binding of influenza A virus NS1 protein to the inter-SH2 domain of p85β suggests a novel mechanism for phosphoinositide 3-kinase activation. J Biol Chem 283, 1372–1380.[CrossRef] [Google Scholar]
  26. Hale, B. G., Randall, R. E., Ortín, J. & Jackson, D.(2008b). The multifunctional NS1 protein of influenza A viruses. J Gen Virol 89, 2359–2376.[CrossRef] [Google Scholar]
  27. Heikkinen, L. S., Kazlauskas, A., Melen, 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. [Google Scholar]
  28. Horimoto, T. & Kawaoka, Y.(2005). Influenza: lessons from past pandemics, warnings from current incidents. Nat Rev Microbiol 3, 591–600.[CrossRef] [Google Scholar]
  29. Johnson, N. P. & Mueller, J.(2002). Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 76, 105–115.[CrossRef] [Google Scholar]
  30. Kobasa, D. & Kawaoka, Y.(2005). Emerging influenza viruses: past and present. Curr Mol Med 5, 791–803.[CrossRef] [Google Scholar]
  31. Landau, M., Mayrose, I., Rosenberg, Y., Glaser, F., Martz, E., Pupko, T. & Ben-Tal, N.(2005). ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res 33, W299–W302.[CrossRef] [Google Scholar]
  32. Laurie, A. T. R. & Jackson, R. M.(2005). Q-SiteFinder: an energy-based method for the prediction of protein–ligand binding sites. Bioinformatics 21, 1908–1916.[CrossRef] [Google Scholar]
  33. Lin, Y. P., Shaw, M., Gregory, V., Cameron, K., Lim, W., Klimov, A., Subbarao, K., Guan, Y., Krauss, S. & other authors(2000). Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates. Proc Natl Acad Sci U S A 97, 9654–9658.[CrossRef] [Google Scholar]
  34. Lommer, B. S. & Luo, M.(2002). Structural plasticity in influenza virus protein NS2 (NEP). J Biol Chem 277, 7108–7117.[CrossRef] [Google Scholar]
  35. 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] [Google Scholar]
  36. Marión, R. M., Aragón, T., Beloso, A., Nieto, A. & Ortín, J.(1997). The N-terminal half of the influenza virus NS1 protein is sufficient for nuclear retention of mRNA and enhancement of viral mRNA translation. Nucleic Acids Res 25, 4271–4277.[CrossRef] [Google Scholar]
  37. Maroto, M., Fernandez, Y., Ortín, J., Pelaez, F. & Cabello, M. A.(2008). Development of an HTS assay for the search of anti-influenza agents targeting the interaction of viral RNA with the NS1 protein. J Biomol Screen 13, 581–590.[CrossRef] [Google Scholar]
  38. Min, J. Y., Li, S. D., 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.[CrossRef] [Google Scholar]
  39. Molinari, N. M., Ortega-Sanchez, I. R., Messonnier, M. L., Thompson, W. W., Wortley, P. M., Weintraub, E. & Bridges, C. B.(2007). The annual impact of seasonal influenza in the US: measuring disease burden and costs. Vaccine 25, 5086–5096.[CrossRef] [Google Scholar]
  40. 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] [Google Scholar]
  41. Nemeroff, M. E., Barabino, S. M. L., Li, Y. Z., 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] [Google Scholar]
  42. Neumann, G., Hughes, M. T. & Kawaoka, Y.(2000). Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1. EMBO J 19, 6751–6758.[CrossRef] [Google Scholar]
  43. Obenauer, J. C., Denson, J., Mehta, P. K., Su, X., Mukatira, S., Finkelstein, D. B., Xu, X., Wang, J., Ma, J. & other authors(2006). Large-scale sequence analysis of avian influenza isolates. Science 311, 1576–1580.[CrossRef] [Google Scholar]
  44. O'Neill, R. E., Talon, J. & Palese, P.(1998). The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J 17, 288–296.[CrossRef] [Google Scholar]
  45. Opitz, B., Rejaibi, A., Dauber, B., Eckhard, J., Vinzing, M., Schmeck, B., Hippenstiel, S., Suttorp, N. & Wolff, T.(2007). IFNβ induction by influenza A virus is mediated by RIG-I which is regulated by the viral NS1 protein. Cell Microbiol 9, 930–938.[CrossRef] [Google Scholar]
  46. Pichlmair, A., Schulz, O., Tan, C. P., Naslund, T. I., Liljestrom, P., Weber, F. & Sousa, C. R. E.(2006). RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates. Science 314, 997–1001.[CrossRef] [Google Scholar]
  47. Rahman, M., Bright, R. A., Kieke, B. A., Donahue, J. G., Greenlee, R. T., Vandermause, M., Balish, A., Foust, A., Cox, N. J. & other authors(2008). Adamantane-resistant influenza infection during the 2004–05 season. Emerg Infect Dis 14, 173–176.[CrossRef] [Google Scholar]
  48. Rice, P., Longden, I. & Bleasby, A.(2000).emboss: the European molecular biology open software suite. Trends Genet 16, 276–277.[CrossRef] [Google Scholar]
  49. Richardson, J. C. & Akkina, R. K.(1991). NS2 protein of influenza virus is found in purified virus and phosphorylated in infected cells. Arch Virol 116, 69–80.[CrossRef] [Google Scholar]
  50. Sayle, R. A. & Milnerwhite, E. J.(1995). RasMol – biomolecular graphics for all. Trends Biochem Sci 20, 374–376.[CrossRef] [Google Scholar]
  51. Schmitt, A. P. & Lamb, R. A.(2005). Influenza virus assembly and budding at the viral budozone. Adv Virus Res 64, 383–416. [Google Scholar]
  52. Schueler-Furman, O. & Baker, D.(2003). Conserved residue clustering and protein structure prediction. Proteins 52, 225–235.[CrossRef] [Google Scholar]
  53. Shin, Y. K., Liu, Q., Tikoo, S. K., Babiuk, L. A. & Zhou, Y.(2007). SH3 binding motif 1 in influenza A virus NS1 protein is essential for PI3K/Akt signalling pathway activation. J Virol 81, 12730–12739.[CrossRef] [Google Scholar]
  54. Subbarao, K., Klimov, A., Katz, J., Regnery, H., Lim, W., Hall, H., Perdue, M., Swayne, D., Bender, C. & other authors(1998). Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 279, 393–396.[CrossRef] [Google Scholar]
  55. Twu, K. Y., Noah, D. L., Rao, P., Kuo, R. L. & Krug, R. M.(2006). The CPSF30 binding site on the NS1A protein of influenza A virus is a potential antiviral target. J Virol 80, 3957–3965.[CrossRef] [Google Scholar]
  56. 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] [Google Scholar]
  57. Wang, X., Li, M., Zheng, H. Y., Muster, T., Palese, P., Beg, A. A. & Garcia-Sastre, A.(2000). Influenza A virus NS1 protein prevents activation of NF-κB and induction of alpha/beta interferon. J Virol 74, 11566–11573.[CrossRef] [Google Scholar]
  58. Ward, A. C., Castelli, L. A., Lucantoni, A. C., White, J. F., Azad, A. A. & Macreadie, I. G.(1995). Expression and analysis of the NS2 protein of influenza A virus. Arch Virol 140, 2067–2073.[CrossRef] [Google Scholar]
  59. World Health Organization(2004). Avian influenza A(H5N1). Wkly Epidemiol Rec 79, 65–70. [Google Scholar]
  60. Yin, C., Khan, J. A., Swapna, G. V. T., 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.[CrossRef] [Google Scholar]
  61. Zhang, Y.(2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9, 40[CrossRef] [Google Scholar]
  62. Zhang, Y. & Skolnick, J.(2004). Scoring function for automated assessment of protein structure template quality. Proteins 57, 702–710.[CrossRef] [Google Scholar]
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