1887

Abstract

The phosphorylation status of the small hydrophobic (SH) protein of respiratory syncytial virus (RSV) was examined in virus-infected Vero cells. The SH protein was isolated from [S]methionine- and [P]orthophosphate-labelled RSV-infected cells and analysed by SDS-PAGE. In each case, a protein product of the expected size for the SH protein was observed. Phosphoamino acid analysis and reactivity with the phosphotyrosine specific antibody PY20 showed that the SH protein was modified by tyrosine phosphorylation. The role of tyrosine kinase activity in SH protein phosphorylation was confirmed by the use of genistein, a broad-spectrum tyrosine kinase inhibitor, to inhibit SH protein phosphorylation. Further analysis showed that the different glycosylated forms of the SH protein were phosphorylated, as was the oligomeric form of the protein. Phosphorylation of the SH protein was specifically inhibited by the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580, suggesting that SH protein phosphorylation occurs via a MAPK p38-dependent pathway. Analysis of virus-infected cells using fluorescence microscopy showed that, although the SH protein was distributed throughout the cytoplasm, it appeared to accumulate, at low levels, in the endoplasmic reticulum/Golgi complex, confirming recent observations. However, in the presence of SB203580, an increased accumulation of the SH protein in the Golgi complex was observed, although other virus structures, such as virus filaments and inclusion bodies, remained largely unaffected. These results showed that during RSV infection, the SH protein is modified by an MAPK p38-dependent tyrosine kinase activity and that this modification influences its cellular distribution.

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2005-02-01
2019-11-12
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References

  1. Alconada, A., Bauer, U. & Hoflack, B. ( 1996; ). A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network. EMBO J 15, 6096–6110.
    [Google Scholar]
  2. Alconada, A., Bauer, U., Sodeik, B. & Hoflack, B. ( 1999; ). Intracellular traffic of herpes simplex virus glycoprotein gE: characterization of the sorting signals required for its trans-Golgi network localization. J Virol 73, 377–387.
    [Google Scholar]
  3. Anderson, K., King, A. M., Lerch, R. A. & Wertz, G. W. ( 1992; ). Polylactosaminoglycan modification of the respiratory syncytial virus small hydrophobic (SH) protein: a conserved feature among human and bovine respiratory syncytial viruses. Virology 191, 417–430.[CrossRef]
    [Google Scholar]
  4. Arumugham, R. G., Seid, R. C., Jr, Doyle, S., Hildreth, S. W. & Paradiso, P. R. ( 1989; ). Fatty acid acylation of the fusion glycoprotein of human respiratory syncytial virus. J Biol Chem 264, 10339–10342.
    [Google Scholar]
  5. Barr, F. A. & Short, B. ( 2003; ). Golgins in the structure and dynamics of the Golgi apparatus. Curr Opin Cell Biol 15, 405–413.[CrossRef]
    [Google Scholar]
  6. Bonifacino, J. S. & Traub, L. M. ( 2003; ). Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu Rev Biochem 72, 395–447.[CrossRef]
    [Google Scholar]
  7. Brown, G., Aitken, J., Rixon, H. W. McL. & Sugrue, R. J. ( 2002; ). Caveolin-1 is incorporated into mature RSV particles during virus assembly on the surface of virus-infected cells. J Gen Virol 83, 611–621.
    [Google Scholar]
  8. Bukreyev, A., Whitehead, S. S., Murphy, B. R. & Collins, P. L. ( 1997; ). Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse. J Virol 71, 8973–8982.
    [Google Scholar]
  9. Chen, M. D., Vazquez, M., Buonocore, L. & Kahn, J. S. ( 2000; ). Conservation of the respiratory syncytial virus SH gene. J Infect Dis 182, 1228–1233.[CrossRef]
    [Google Scholar]
  10. Collins, P. L. & Wertz, G. W. ( 1985; ). The 1A protein gene of human respiratory syncytial virus: nucleotide sequence of the mRNA and a related polycistronic transcript. Virology 141, 283–291.[CrossRef]
    [Google Scholar]
  11. Collins, P. L. & Mottet, G. ( 1993; ). Membrane orientation and oligomerization of the small hydrophobic protein of human respiratory syncytial virus. J Gen Virol 74, 1445–1450.[CrossRef]
    [Google Scholar]
  12. Collins, P. L., Olmsted, R. A. & Johnson, P. R. ( 1990; ). The small hydrophobic protein of human respiratory syncytial virus: comparison between antigenic subgroups A and B. J Gen Virol 71, 1571–1576.[CrossRef]
    [Google Scholar]
  13. Cottin, V., Van Linden, A. & Riches, D. W. ( 1999; ). Phosphorylation of tumor necrosis receptor CD120a (p55) by p42(mapk/erk2) induces changes in its subcellular localization. J Biol Chem 274, 32975–32987.[CrossRef]
    [Google Scholar]
  14. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J. & Saltiel, A. R. ( 1995; ). A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A 92, 7686–7689.[CrossRef]
    [Google Scholar]
  15. Feldman, S. A., Crim, R. L., Audet, S. A. & Beeler, J. A. ( 2001; ). Human respiratory syncytial virus surface glycoproteins F, G and SH form an oligomeric complex. Arch Virol 146, 2369–2383.[CrossRef]
    [Google Scholar]
  16. Gallagher, T. F., Seibel, G. L., Kassis, S. & 20 other authors ( 1997; ). Regulation of stress-induced cytokine production by pyridinylimidazoles: inhibition of CSBP kinase. Bioorg Med Chem 5, 49–64.[CrossRef]
    [Google Scholar]
  17. Gruber, C. & Levine, S. ( 1985; ). Respiratory syncytial virus polypeptides. IV. The oligosaccharides of the glycoproteins. J Gen Virol 66, 417–432.[CrossRef]
    [Google Scholar]
  18. Heineman, T. C. & Hall, S. L. ( 2001; ). VZV gB endocytosis and Golgi localization are mediated by YXXΦ motifs in its cytoplasmic domain. Virology 285, 42–49.[CrossRef]
    [Google Scholar]
  19. Heminway, B. R., Yu, Y., Tanaka, Y., Perrine, K. G., Gustafson, E., Bernstein, J. M. & Galinski, M. S. ( 1994; ). Analysis of respiratory syncytial virus F, G, and SH proteins in cell fusion. Virology 200, 801–805.[CrossRef]
    [Google Scholar]
  20. Kochva, U., Leonov, H. & Arkin, I. T. ( 2003; ). Modeling the structure of the respiratory syncytial virus small hydrophobic protein by silent-mutation analysis of global searching molecular dynamics. Protein Sci 12, 2668–2674.[CrossRef]
    [Google Scholar]
  21. Kong, X., San Juan, H., Behera, A., Peeples, M. E., Wu, J., Lockey, R. F. & Mohapatra, S. S. ( 2004; ). ERK-1/2 activity is required for efficient RSV infection. FEBS Lett 559, 33–38.[CrossRef]
    [Google Scholar]
  22. Lambert, D. M. & Pons, M. W. ( 1983; ). Respiratory syncytial virus glycoproteins. Virology 130, 204–214.[CrossRef]
    [Google Scholar]
  23. Lambert, D. M., Hambor, J., Diebold, M. & Galinski, B. ( 1988; ). Kinetics of synthesis and phosphorylation of respiratory syncytial virus polypeptides. J Gen Virol 69, 313–323.[CrossRef]
    [Google Scholar]
  24. Meusel, T. R. & Imani, F. ( 2003; ). Viral induction of inflammatory cytokines in human epithelial cells follows a p38 mitogen-activated protein kinase-dependent but NF-κB-independent pathway. J Immunol 171, 3768–3774.[CrossRef]
    [Google Scholar]
  25. Monick, M. M., Cameron, K., Powers, L. S., Butler, N. S., McCoy, D., Mallampalli, R. K. & Hunninghake, G. W. ( 2004; ). Sphingosine kinase mediates activation of extracellular signal-related kinase and Akt by respiratory syncytial virus. Am J Respir Cell Mol Biol 30, 844–852.[CrossRef]
    [Google Scholar]
  26. Olmsted, R. A. & Collins, P. L. ( 1989; ). The 1A protein of respiratory syncytial virus is an integral membrane protein present as multiple, structurally distinct species. J Virol 63, 2019–2029.
    [Google Scholar]
  27. Pazdrak, K., Olszewska-Pazdrak, B., Liu, T., Takizawa, R., Brasier, A. R., Garofalo, R. P. & Casola, A. ( 2002; ). MAPK activation is involved in posttranscriptional regulation of RSV-induced RANTES gene expression. Am J Physiol Lung Cell Mol Physiol 283, L364–L372.[CrossRef]
    [Google Scholar]
  28. Perez, M., Garcia-Barreno, B., Melero, J. A., Carrasco, L. & Guinea, R. ( 1997; ). Membrane permeability changes induced in Escherichia coli by the SH protein of human respiratory syncytial virus. Virology 235, 342–351.[CrossRef]
    [Google Scholar]
  29. Rixon, H. W., Brown, G., Aitken, J., McDonald, T., Graham, S. & Sugrue, R. J. ( 2004; ). The small hydrophobic (SH) protein accumulates within lipid-raft structures of the Golgi complex during respiratory syncytial virus infection. J Gen Virol 85, 1153–1165.[CrossRef]
    [Google Scholar]
  30. Roux, P. P. & Blenis, J. ( 2004; ). ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68, 320–344.[CrossRef]
    [Google Scholar]
  31. Santhamma, K. R., Sadhukhan, R., Kinter, M., Chattopadhyay, S., McCue, B. & Sen, I. ( 2004; ). Role of tyrosine phosphorylation in the regulation of cleavage–secretion of angiotensin-converting enzyme. J Biol Chem 279, 40227–40236.[CrossRef]
    [Google Scholar]
  32. Shiratori, T., Miyatake, S., Ohno, H., Nakaseko, C., Isono, K., Bonifacino, J. S. & Saito, T. ( 1997; ). Tyrosine phosphorylation controls internalization of CTLA-4 by regulating its interaction with clathrin-associated adaptor complex AP-2. Immunity 6, 583–589.[CrossRef]
    [Google Scholar]
  33. Short, B. & Barr, F. A. ( 2003; ). Membrane traffic: a glitch in the Golgi matrix. Curr Biol 13, R311–R313.[CrossRef]
    [Google Scholar]
  34. Stokoe, D., Engel, K., Campbell, D. G., Cohen, P. & Gaestel, M. ( 1992; ). Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 313, 307–313.[CrossRef]
    [Google Scholar]
  35. 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.[CrossRef]
    [Google Scholar]
  36. Taylor, G., Stott, E. J., Furze, J., Ford, J. & Sopp, P. ( 1992; ). Protective epitopes on the fusion protein of respiratory syncytial virus recognized by murine and bovine monoclonal antibodies. J Gen Virol 73, 2217–2223.[CrossRef]
    [Google Scholar]
  37. Techaarpornkul, S., Barretto, N. & Peeples, M. E. ( 2001; ). Functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene. J Virol 75, 6825–6834.[CrossRef]
    [Google Scholar]
  38. Van Linden, A. A., Cottin, V., Leu, C. & Riches, D. W. ( 2000; ). Phosphorylation of the membrane proximal region of tumor necrosis factor receptor CD120a (p55) at ERK consensus sites. J Biol Chem 275, 6996–7003.[CrossRef]
    [Google Scholar]
  39. Ward, B. M. & Moss, B. ( 2000; ). Golgi network targeting and plasma membrane internalization signals in vaccinia virus B5R envelope protein. J Virol 74, 3771–3780.[CrossRef]
    [Google Scholar]
  40. Wright, C., Oliver, K. C., Fenwick, F. I., Smith, N. M. & Toms, G. L. ( 1997; ). A monoclonal antibody pool for routine immunohistochemical detection of human respiratory syncytial virus antigens in formalin-fixed, paraffin-embedded tissue. J Pathol 182, 238–244.[CrossRef]
    [Google Scholar]
  41. Zhang, Y. & Allison, J. P. ( 1997; ). Interaction of CTLA-4 with AP50, a clathrin-coated pit adaptor protein. Proc Natl Acad Sci U S A 94, 9273–9278.[CrossRef]
    [Google Scholar]
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