Phosphorylation of NS Protein by the L Protein of Vesicular Stomatitis virus Free

Abstract

SUMMARY

The structural proteins L and NS of vesicular stomatitis virus were obtained from purified viral ribonucleoprotein complex followed by phosphocellulose column chromatography and assayed for protein kinase activity using [-P]ATP as the phosphate donor. The fractions containing purified L protein phosphorylated NS protein . 8-Azido-ATP, a photoreactive analogue of ATP, was also used as the phosphate donor for phosphorylation of NS protein by the L protein. In the presence of ultraviolet light, only L protein was specifically cross-linked with 8-azido-[-P]ATP. In the absence of u.v. light 8-azido ATP did no inhibit RNA transcription in a reconstituted reaction or substitute ATP for RNA synthesis . The above results, taken together, suggest that 8-azido-ATP was bound to the kinase site and phosphorylation of NS protein was mediated by th L protein. Exogenous phosphate acceptor proteins such as phosvitin and casein were also phosphorylated by the L protein fraction. However, addition of an excess of phosvitin failed to compete with the phosphorylation of NS by L, indicating that the protein kinase activity possessed higher affinity for NS. The phosphorylation of NS was strongly inhibited by photoreaction of L protein with 8-azido-ATP with concomitant inhibition of transcription . These results suggest that phosphorylation of NS protein by L may have a role in the regulation of the virus genome transcription .

Keyword(s): phosphorylation , transcription and VSV
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1985-05-01
2024-03-28
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References

  1. Akusjӓrvi G., Philipson L., Petterson U. 1978; A protein kinase associated with adenovirus type 2. Virology 87:276–286
    [Google Scholar]
  2. Banerjee R. K. Racker E. 1977; Solubilization, purification, and characterization of a nucleoside triphosphatase from avian myeloblastosis virus. Journal of Biological Chemistry 252:6700–6706
    [Google Scholar]
  3. Banerjee A. K., Moyer S. A., Rhodes D. P. 1974; Studies on the in vitro adenylation of RNA by vesicular stomatitis virus. Virology 61:547–558
    [Google Scholar]
  4. Bell J. C., Brown E. G., Takayesu D., Prevec L. 1984; Protein kinase activity associated with immunoprecipitates of the vesicular stomatitis virus phosphoprotein NS. Virology 132:229–238
    [Google Scholar]
  5. Clinton G. M., Huang A. S. 1981; Distribution of phosphoserine, phosphothreonine and phosphotyrosine in proteins of vesicular stomatitis virus. Virology 108:510–514
    [Google Scholar]
  6. Clinton G. M., Burge B. W., Huang A. S. 1978; Effect of phosphorylation and pH on the association of protein with vesicular stomatitis virus cores. Journal of Virology 27:340–346
    [Google Scholar]
  7. Clinton G. M., Burge B. W., Huang A. S. 1979; Phosphoproteins of vesicular stomatitis virus: identity and interconversion of phosphorylated forms. Virology 99:84–94
    [Google Scholar]
  8. Clinton G. M., Guerina N. G., Guo H., Huang A. S. 1982; Host-dependent phosphorylation and kinase activity associated with vesicular stomatitis virus. Journal of Biological Chemistry 257:3313–3319
    [Google Scholar]
  9. DE B. P., Thornton G. B., Luk D., Banerjee A. K. 1982; Purified matrix protein of vesicular stomatitis virus blocks viral transcription in vitro . Proceedings of the National Academy of Sciences, U. S. A 79:7137–7141
    [Google Scholar]
  10. Downer D. N., Rogers H. W., Randall C. C. 1973; Endogenous protein kinase and phosphate acceptor proteins in vaccinia virus. Virology 52:13–21
    [Google Scholar]
  11. Eckhart W., Hutchinson M. A., Hunter T. 1979; An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell 18:925–933
    [Google Scholar]
  12. Emerson S. U., Yu Y. -H. 1975; Both NS and L proteins are required for in vitro RNA synthesis by vesicular stomatitis virus. Journal of Virology 15:1348–1356
    [Google Scholar]
  13. Grubman M. J., Baxt B., La Torre J. L., Bachrach H. L. 1981; Identification of a protein kinase activity in purified foot-and-mouth disease virus. Journal of Virology 39:455–462
    [Google Scholar]
  14. Haley B. E. 1983; Development and utilization of 8-azido-purine nucleotide photoaffinity probes. Federation Proceedings 42:2831–2836
    [Google Scholar]
  15. Haley B. E., Hoffman J. F. 1974; Interactions of a photoaffinity ATP analog with cation-stimulated adenosine triphosphatases of human red cell membranes. Proceedings of the National Academy of Sciences, U. S. A 71:3367–3371
    [Google Scholar]
  16. Harmon S. A., Marnell L. L., Summers D. F. 1983; The major ribonucleoprotein-associated protein kinase of vesicular stomatitis virus is a host cell protein. Journal of Biological Chemistry 258:15283–15290
    [Google Scholar]
  17. Hatanaka M., Twiddy E., Gilden R. V. 1972; Protein kinase associated with RNA tumor viruses and other budding RNA viruses. Virology 47:536–538
    [Google Scholar]
  18. Hsu E. -H., Morgan E. M., Kingsbury D. W. 1982; Site-specific phosphorylation regulates the transcriptase activity of vesicular stomatitis virus NS protein. Journal of Virology 43:104–112
    [Google Scholar]
  19. Hunter T., Sefton B. M. 1980; Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proceedings of the National Academy of Sciences, U. S. A 77:1311–1315
    [Google Scholar]
  20. Imblum R. L., Wagner R. R. 1974; Protein kinase and phosphorylation of vesicular stomatitis virus. Journal of Virology 13:113–124
    [Google Scholar]
  21. Kingsford L., Emerson S. U. 1980; Transcriptional activities of different phosphorylated species of NS protein purified from vesicular stomatitis virions and cytoplasm of infected cells. Journal of Virology 33:1097–1105
    [Google Scholar]
  22. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
    [Google Scholar]
  23. Lemaster S., Roizman B. 1980; Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virus. Journal of Virology 35:798–8
    [Google Scholar]
  24. Moyer S. A., Summers D. F. 1974; Phosphorylation of vesicular stomatitis virus in vivo and in vitro . Journal of Virology 13:455–465
    [Google Scholar]
  25. Naito S., Ishihama A. 1976; Function and structure of RNA polymerase from vesicular stomatitis virus. Journal of Biological Chemistry 251:4307–4314
    [Google Scholar]
  26. Randall C. C., Rogers H. W., Downer D. N., Gentry G. A. 1972; Protein kinase activity in equine herpes virus. Journal of Virology 9:216–222
    [Google Scholar]
  27. Schaffausen B. S., Benjamin T. L. 1979; Phosphorylation of polyoma T antigens. Cell 18:935–946
    [Google Scholar]
  28. Scheidtmann K. -H., Echle B., Walter G. 1982; Simian virus 40 large T antigen is phosphorylated at multiple sites clustered in two separate regions. Journal of Virology 44:116–133
    [Google Scholar]
  29. Silberstein H., August J. T. 1973; Phosphorylation of animal virus proteins by a virion protein kinase. Journal of Virology 21:511–522
    [Google Scholar]
  30. Sinacore M. S., Lucas-Lenard J. 1982; The effect of the vesicular stomatitis virus-associated protein kinase on viral mRNA transcription in vivo . Virology 121:404–413
    [Google Scholar]
  31. Smith A. E., Smith R., Griffin G., Fried M. 1979; Protein kinase activity associated with polyoma virus middle T antigen in vivo . Cell 18:915–924
    [Google Scholar]
  32. Snyder H. W. Jr 1982; Biochemical characterization of protein kinase activities associated with transforming gene products of the Snyder-Theilen and Gardner-Arnstein strains of feline sarcoma virus. Virology 117:165–172
    [Google Scholar]
  33. Sokol F., Clark H. F. 1973; Phosphoproteins, structural components of rhabdovirus. Virology 52:246–263
    [Google Scholar]
  34. Strand M., August J. T. 1971; Protein kinase and phosphate acceptor proteins in Rauscher murine leukemia virus. Nature New Biology 233:137–140
    [Google Scholar]
  35. Tan K. B. 1975; Comparative study of the protein kinase associated with animal viruses. Virology 64:566–570
    [Google Scholar]
  36. Tan K. B., Sokol F. 1972; Structural proteins of simian virus 40: phosphoproteins. Journal of Virology 10:985–994
    [Google Scholar]
  37. Testa D., Banerjee A. K. 1979; Initiation of RNA synthesis in vitro: role of ATP. Journal of Biological Chemistry 254:2053–2058
    [Google Scholar]
  38. Testa D., Chanda P. K., Banerjee A. K. 1980; In vitro synthesis of the full-length complement of the negative strand genome RNA of vesicular stomatitis virus. Proceedings of the National Academy of Sciences, U. S. A 77:294–298
    [Google Scholar]
  39. Watanabe Y., Sakuma S., Tanaka S. 1974; A possible biological function of the protein kinase associated with vaccinia and vesicular stomatitis virions. FEBS Letters 41:331–341
    [Google Scholar]
  40. Witt D. J., Summers D. F. 1980; Relationship between virion-associated kinase-effected phosphorylation and transcription activity of vesicular stomatitis virus. Virology 107:34–49
    [Google Scholar]
  41. Witte O. N., Dasgupta A., Baltimore D. 1980; Abelson murine leukaemia virus protein is phosphorylated in vitro to form phosphotyrosine. Nature, London 283:826–831
    [Google Scholar]
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