1887

Abstract

The sites for fatty acylation, disulphide bond formation and phosphorylation of influenza C virus CM2 were investigated by site-specific mutagenesis. Cysteine 65 in the cytoplasmic tail was identified as the site for palmitoylation. Removal of one or more of three cysteine residues in the ectodomain showed that all of cysteines 1, 6 and 20 can participate in the formation of disulphide-linked dimers and/or tetramers, although cysteine 20 may play the most important role in tetramer formation. Furthermore, it was found that serine 78, located within the recognition motifs for mammary gland casein kinase and casein kinase I, is the predominant site for phosphorylation, although serine 103 is phosphorylated to a minor extent by proline-dependent protein kinase. The effects of acylation and phosphorylation on the formation of disulphide-linked oligomers were also studied. The results showed that, while palmitoylation has no role in oligomer formation, phosphorylation accelerates tetramer formation without influencing dimer formation. CM2 mutants defective in acylation, phosphorylation or disulphide bond formation were all transported to the cell surface, suggesting that none of these modifications is required for proper oligomerization. When proteins solubilized in detergent were analysed on sucrose gradients, however, the mutant lacking cysteines 1, 6 and 20 sedimented as monomers, raising the possibility that disulphide bond formation, although not essential for proper oligomerization, may stabilize the CM2 multimer. This was supported by the results of chemical cross-linking analysis, which showed that the triple-cysteine mutant can form multimers.

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2001-05-01
2024-12-14
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References

  1. Byrappa S., Gupta K. C. 1999; Human parainfluenza virus type 1 phosphoprotein is constitutively phosphorylated at Ser-120 and Ser-184. Journal of General Virology 80:1199–1209
    [Google Scholar]
  2. Byrappa S., Pan Y. B., Gupta K. C. 1996; Sendai virus P protein is constitutively phosphorylated at serine 249: high phosphorylation potential of the P protein. Virology 216:228–234
    [Google Scholar]
  3. Castrucci M. R., Hughes M., Calzoletti L., Donatelli I., Wells K., Takada A., Kawaoka Y. 1997; The cysteine residues of the M2 protein are not required for influenza A virus replication. Virology 238:128–134
    [Google Scholar]
  4. Ciampor F., Bayley P. M., Nermut M. V., Hirst E. M. A., Sugrue R. J., Hay A. J. 1992; Evidence that the amantadine-induced, M2-mediated conversion of influenza A virus hemagglutinin to the low pH conformation occurs in an acidic trans Golgi compartment. Virology 188:14–24
    [Google Scholar]
  5. Doms R. W., Lamb R. A., Rose J. K., Helenius A. 1993; Folding and assembly of viral membrane proteins. Virology 193:545–562
    [Google Scholar]
  6. Holsinger L. J., Lamb R. A. 1991; Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds. Virology 183:32–43
    [Google Scholar]
  7. Holsinger L. J., Shaughnessy M. A., Micko A., Pinto L. H., Lamb R. A. 1995; Analysis of the posttranslational modifications of the influenza virus M2 protein. Journal of Virology 69:1219–1225
    [Google Scholar]
  8. Hongo S., Sugawara K., Nishimura H., Muraki Y., Kitame F., Nakamura K. 1994; Identification of a second protein encoded by influenza C virus RNA segment 6. Journal of General Virology 75:3503–3510
    [Google Scholar]
  9. Hongo S., Sugawara K., Muraki Y., Kitame F., Nakamura K. 1997; Characterization of a second protein (CM2) encoded by RNA segment 6 of influenza C virus. Journal of Virology 71:2786–2792
    [Google Scholar]
  10. Hongo S., Gao P., Sugawara K., Muraki Y., Matsuzaki Y., Tada Y., Kitame F., Nakamura K. 1998; Identification of a 374 amino acid protein encoded by RNA segment 6 of influenza C virus. Journal of General Virology 79:2207–2213
    [Google Scholar]
  11. Hongo S., Sugawara K., Muraki Y., Matsuzaki Y., Takashita E., Kitame F., Nakamura K. 1999; Influenza C virus CM2 protein is produced from a 374-amino-acid protein (P42) by signal peptidase cleavage. Journal of Virology 73:46–50
    [Google Scholar]
  12. Kemp B. E., Pearson R. B. 1990; Protein kinase recognition sequence motifs. Trends in Biochemical Sciences 15:342–346
    [Google Scholar]
  13. Lamb R. A., Zebedee S. L., Richardson C. D. 1985; Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface. Cell 40:627–633
    [Google Scholar]
  14. Matsuzaki Y., Mizuta K., Kimura H., Sugawara K., Tsuchiya E., Suzuki H., Hongo S., Nakamura K. 2000; Characterization of antigenically unique influenza C virus strains isolated in Yamagata and Sendai Cites, Japan, during 1992–1993. Journal of General Virology 81:1447–1452
    [Google Scholar]
  15. Pekosz A., Lamb R. A. 1997; The CM2 protein of influenza C virus is an oligomeric integral membrane glycoprotein structurally analogous to influenza A virus M2 and influenza B virus NB proteins. Virology 237:439–451
    [Google Scholar]
  16. Pekosz A., Lamb R. A. 1998; Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence. Proceedings of the National Academy of Sciences, USA 95:13233–13238
    [Google Scholar]
  17. Pinto L. H., Holsinger L. J., Lamb R. A. 1992; Influenza virus M2 protein has ion channel activity. Cell 69:517–528
    [Google Scholar]
  18. Schmidt M. F. G. 1989; Fatty acylation of proteins. Biochimica et Biophysica Acta 988:411–426
    [Google Scholar]
  19. Sugawara K., Nishimura H., Kitame F., Nakamura K. 1986; Antigenic variation among human strains of influenza C virus detected with monoclonal antibodies to gp88 glycoprotein. Virus Research 6:27–32
    [Google Scholar]
  20. Sugrue R. J., Hay A. J. 1991; Structural characteristics of the M2 protein of influenza A viruses: evidence that it forms a tetrameric channel. Virology 180:617–624
    [Google Scholar]
  21. Tada Y., Hongo S., Muraki Y., Sugawara K., Kitame F., Nakamura K. 1997; Evolutionary analysis of influenza C virus M genes. Virus Genes 15:53–59
    [Google Scholar]
  22. Tada Y., Hongo S., Muraki Y., Matsuzaki Y., Sugawara K., Kitame F., Nakamura K. 1998; Phosphorylation of influenza C virus CM2 protein. Virus Research 58:65–72
    [Google Scholar]
  23. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. 1988; SRα promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Molecular and Cellular Biology 8:466–472
    [Google Scholar]
  24. Thomas J. M., Stevens M. P., Percy N., Barclay W. S. 1998; Phosphorylation of the M2 protein of influenza A virus is not essential for virus viability. Virology 252:54–64
    [Google Scholar]
  25. Yamashita M., Krystal M., Palese P. 1988; Evidence that the matrix protein of influenza C virus is coded for by a spliced mRNA. Journal of Virology 62:3348–3355
    [Google Scholar]
  26. Yokota M., Nakamura K., Sugawara K., Homma M. 1983; The synthesis of polypeptides in influenza C virus-infected cells. Virology 130:105–117
    [Google Scholar]
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