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Journal of General Virology header logo Journal of General Virology

Volume 66, Issue 5

The Role of Polypeptides L and NS in the Transcription Process of Vesicular Stomatitis Virus New Jersey Using the Temperature-sensitive Mutant E1 No Access

Abstract

SUMMARY

The roles of the L and NS polypeptides in transcription by vesicular stomatitis virus New Jersey were studied using a mutant, E1, which contains a temperature-sensitive transcriptase and an altered NS polypeptide, both phenotypic changes being the consequence of the mutation. Mutant E1, its revertant (E1/R1) and the wild-type virus were dissociated into sub-viral fractions and, after reconstitution of these fractions in all combinations, the transcriptase was assayed at the permissive (31 °C) and restrictive (39 °C) temperatures. Reconstitution of the pellet fractions (containing polypeptide N complexed with the virion RNA) and the supernatant fractions (containing polypeptides L and NS) restored transcriptase activity at 31 °C in all combinations, but at 39 °C transcription was observed only in the presence of the supernatant fractions of wild-type and revertant viruses but not in the presence of the supernatant fractions of E1. When the pellet fractions and the L fractions were reconstituted, the transcriptase activity was restored in all combinations both at 31 °C and 39 °C. However, transcription at 39 °C by reconstituted pellet and L fractions was strongly inhibited when the NS fraction of E1 was also added, while addition of the NS fractions of wild-type and revertant viruses had no effect. Since only traces of polypeptide NS were present in the L fractions and none in the pellet fractions, the results strongly suggest that polypeptide L is the transcriptase itself while polypeptide NS exerts some control over transcription.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): dissociation-reconstitution , mutant tsE1 , transcription in vitro and VSV
© Journal of General Virology 1985
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1985-05-01
2025-05-12
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References

  1. Baltimore D., Huang A. S., Stampfer M. 1970; Ribonucleic acid synthesis of vesicular stomatitis virus. II. An RNA polymerase in the virion. Proceedings of the National Academy of Sciences, U. S. A 66:572–576
     [Google Scholar]
  2. Bishop D. H. L., Roy P. 1971; Kinetics of RNA synthesis by vesicular stomatitis virus particles. Journal of Molecular Biology 57:513–527
     [Google Scholar]
  3. Bishop D. H. L., Roy P. 1972; Dissociation of vesicular stomatitis virus and relation of the virion proteins to the viral transcriptase. Journal of Virology 10:234–243
     [Google Scholar]
  4. Emerson S. U., Wagner R. R. 1972; Dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis B and T virions. Journal of Virology 10:297–309
     [Google Scholar]
  5. Emerson S. U., Wagner R. R. 1973; L protein requirement for in vitro RNA synthesis by vesicular stomatitis virus. Journal of Virology 12:1325–1335
     [Google Scholar]
  6. 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]
  7. Evans D., Pringle C. R., Szilagyi J. F. 1979; Temperature-sensitive mutants of complementation group E of vesicular stomatitis virus New Jersey serotype possess altered NS polypeptides. Journal of Virology 31:325–333
     [Google Scholar]
  8. Huang A. S., Wagner R. R. 1966; Comparative sedimentation coefficients of RNA extracted from plaque forming and defective particles of vesicular stomatitis virus. Journal of Molecular Biology 22:381–384
     [Google Scholar]
  9. Hunt D. M., Emerson S. U., Wagner R. R. 1976; RNA temperature-sensitive mutants of vesicular stomatitis virus: L-protein thermosensitivity accounts for transcriptase restriction of Group 1 mutants. Journal of Virology 18:596–603
     [Google Scholar]
  10. Kang C. Y., Prevec L. 1969; Proteins of vesicular stomatitis virus. I. Polyacrylamide gel analysis of viral antigens. Journal of Virology 3:404–413
     [Google Scholar]
  11. 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]
  12. Lesnaw J. A., Dickson L. R., Curry R. H. 1979; Proposed replicative role of the NS polypeptide of vesicular stomatitis virus: structural analysis of an electrophoretic variant. Virology 31:8–16
     [Google Scholar]
  13. Macpherson I. A., Stoker M. G. P. 1962; Polyoma transformation of hamster cell clones - an investigation of genetic factors affecting cell competence. Virology 16:147–151
     [Google Scholar]
  14. McSharry J. J., Wagner R. R. 1971; Lipid composition of purified vesicular stomatitis virus. Journal of Virology 7:59–70
     [Google Scholar]
  15. Mellon M. G., Emerson S. U. 1978; Binding of transcriptase components (L and NS proteins) to the nucleocapsid template of vesicular stomatitis virus. Journal of Virology 27:560–567
     [Google Scholar]
  16. Naito S., Ishihama A. 1970; Function and structure of RNA polymerase from vesicular stomatitis virus. Journal of Biological Chemistry 251:4307–4314
     [Google Scholar]
  17. Oakley B. R., Kirsch D. R., Morris N. R. 1980; A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry 105:361–363
     [Google Scholar]
  18. Ongrádi J., Szilagyi J. F. 1981; The role of the L and NS polypeptides in the transcription by vesicular stomatitis virus New Jersey serotype. In Replication of Negative Strand Viruses pp 837–844 Edited by Bishop D. H. L., Compans R. W. New York & Amsterdam: Elsevier/North-Holland;
     [Google Scholar]
  19. Preston C. M., Szilagyi J. F. 1977; Cell-free translation of RNA synthesized in vitro by a transcribing nucleoprotein complex prepared from purified vesicular stomatitis virus. Journal of Virology 21:1002–1009
     [Google Scholar]
  20. Pringle C. R., Szilagyi J. F. 1980; Gene assignment and complementation groups. In Rhahdoviruses vol. II pp 141–161 Edited by Bishop D. H. L. Florida: CRC Press;
     [Google Scholar]
  21. Pringle C. R., Duncan I. B., Stevenson M. 1971; Isolation and characterization of temperature-sensitive mutants of vesicular stomatitis virus, New Jersey serotype. Journal of Virology 8:836–841
     [Google Scholar]
  22. Pringle C. R., Devine V., Wilkie M., Preston C. M., Dolan A., Mcgeoch D. J. 1981; Enhanced mutability associated with a temperature-sensitive mutant of vesicular stomatitis virus. Journal of Virology 39:377–389
     [Google Scholar]
  23. Spath P. J., Koblet H. 1979; Properties of SDS-polyacrylamide gels highly cross-linked with N, N′-diallyltartardiamide and the rapid isolation of macromolecules from the gel matrix. Analytical Biochemistry 93:275–285
     [Google Scholar]
  24. Szilágyi J. F. 1975; Structure and functional activity of the RNA polymerase of vesicular stomatitis virus. In vitro Transcription and Translation of Viral Genomes pp 129–134 Edited by Haenni A. N., Beaud G. Paris: INSERM;
     [Google Scholar]
  25. Szilagyi J. F., Pringle C. R. 1972; Effect of temperature-sensitive mutations on the virion-associated RNA transcriptase of vesicular stomatitis virus. Journal of Molecular Biology 71:281–291
     [Google Scholar]
  26. Szilagyi J. F., Pringle C. R. 1975; Virion transcriptase activity differences in host range mutants of vesicular stomatitis virus. Journal of Virology 16:927–936
     [Google Scholar]
  27. Szilagyi J. F., Pringle C. R. 1978; A possible host factor requirement for the transcription of vesicular stomatitis virus RNA. In Negative Strand Viruses and the Host Cell pp 285–291 Edited by Mahy B. W. J., Barry R. D. London: Academic Press;
     [Google Scholar]
  28. Szilagyi J. F., Pringle C. R. 1979; Effect of temperature-sensitive mutation on activity of the RNA transcriptase of vesicular stomatitis virus New Jersey. Journal of Virology 30:692–700
     [Google Scholar]
  29. Szilagyi J. F., Uryvayev L. 1973; Isolation of an infectious ribonucleoprotein from vesicular stomatitis virus containing an active RNA transcriptase. Journal of Virology 11:279–286
     [Google Scholar]
  30. Szilagyi J. F., Pringle C. R., Macpherson T. M. 1977; Temperature-dependent host range mutation in vesicular stomatitis virus affecting polypeptide L. Journal of Virology 22:381–388
     [Google Scholar]
  31. Wagner R. R., Prevec L., Brown F., Summers D. F., Sokol F., Macleod R. 1972; Classification of rhabdovirus proteins: a proposal. Journal of Virology 10:1228–1230
     [Google Scholar]
/content/journal/jgv/10.1099/0022-1317-66-5-1011
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The Role of Polypeptides L and NS in the Transcription Process of Vesicular Stomatitis Virus New Jersey Using the Temperature-sensitive Mutant tsE1
J. Gen. Virol. 66, 1011 (1985); https://doi.org/10.1099/0022-1317-66-5-1011
/content/journal/jgv/10.1099/0022-1317-66-5-1011
/content/journal/jgv/10.1099/0022-1317-66-5-1011
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