Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1984 Feb;49(2):303–309. doi: 10.1128/jvi.49.2.303-309.1984

N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.

J T Patton, N L Davis, G W Wertz
PMCID: PMC255465  PMID: 6319730

Abstract

Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent reticulocyte lysate to support protein synthesis. We report here an analysis of the requirement for individual viral proteins in VSV replication. Viral mRNAs purified by hybridization to cDNA clones were used to direct the synthesis of individual proteins in the in vitro system. By this method, it was demonstrated that the synthesis of the VSV nucleocapsid protein, N, alone, resulted in the replication of genome-length RNA by both defective interfering intracellular nucleocapsids and virion-derived nucleocapsids. Neither the viral phosphoprotein, NS, nor the matrix protein, M, supported RNA replication. The amount of RNA replication for a given amount of N protein was the same in reactions in which either all of the VSV proteins or only N protein were synthesized. In addition, RNA replication products synthesized in reactions containing only newly made N protein assembled with the N protein to form nucleocapsids. These results demonstrate that the major nucleocapsid protein (N) can by itself fulfill the requirement for protein synthesis in RNA replication and allow complete replication, i.e., initiation and elongation, as well as encapsidation of genome-length progeny RNA.

Full text

PDF
303

Images in this article

305Image
on p.305
306Image
on p.306
306Image
on p.306
306Image
on p.306
307Image
on p.307

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blumberg B. M., Giorgi C., Kolakofsky D. N protein of vesicular stomatitis virus selectively encapsidates leader RNA in vitro. Cell. 1983 Feb;32(2):559–567. doi: 10.1016/0092-8674(83)90475-0. [DOI] [PubMed] [Google Scholar]
  2. Blumberg B. M., Leppert M., Kolakofsky D. Interaction of VSV leader RNA and nucleocapsid protein may control VSV genome replication. Cell. 1981 Mar;23(3):837–845. doi: 10.1016/0092-8674(81)90448-7. [DOI] [PubMed] [Google Scholar]
  3. Chanda P. K., Banerjee A. K. Identification of promoter-proximal oligonucleotides and a unique dinucleotide, pppGpC, from in vitro transcription products of vesicular stomatitis virus. J Virol. 1981 Jul;39(1):93–103. doi: 10.1128/jvi.39.1.93-103.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Davis N. L., Wertz G. W. Synthesis of vesicular stomatitis virus negative-strand RNA in vitro: dependence on viral protein synthesis. J Virol. 1982 Mar;41(3):821–832. doi: 10.1128/jvi.41.3.821-832.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Emerson S. U., Dierks P. M., Parsons J. T. In vitro synthesis of a unique RNA species by a T particle of vesicular stomatitis virus. J Virol. 1977 Sep;23(3):708–716. doi: 10.1128/jvi.23.3.708-716.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Emerson S. U. Vesicular stomatitis virus: structure and function of virion components. Curr Top Microbiol Immunol. 1976;73:1–34. doi: 10.1007/978-3-642-66306-2_1. [DOI] [PubMed] [Google Scholar]
  7. Gallione C. J., Greene J. R., Iverson L. E., Rose J. K. Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus N and NS proteins. J Virol. 1981 Aug;39(2):529–535. doi: 10.1128/jvi.39.2.529-535.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kingsbury D. W. The molecular biology of paramyxoviruses. Med Microbiol Immunol. 1974;160(2-3):73–83. doi: 10.1007/BF02121714. [DOI] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Leamnson R. N., Reichmann M. E. The RNA of defective vesicular stomatitis virus particles in relation to viral cistrons. J Mol Biol. 1974 Jan 5;85(4):551–568. doi: 10.1016/0022-2836(74)90315-5. [DOI] [PubMed] [Google Scholar]
  11. Leppert M., Rittenhouse L., Perrault J., Summers D. F., Kolakofsky D. Plus and minus strand leader RNAs in negative strand virus-infected cells. Cell. 1979 Nov;18(3):735–747. doi: 10.1016/0092-8674(79)90127-2. [DOI] [PubMed] [Google Scholar]
  12. Patton J. T., Davis N. L., Wertz G. W. Cell-free synthesis and assembly of vesicular stomatitis virus nucleocapsids. J Virol. 1983 Jan;45(1):155–164. doi: 10.1128/jvi.45.1.155-164.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Peluso R. W., Moyer S. A. Initiation and replication of vesicular stomatitis virus genome RNA in a cell-free system. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3198–3202. doi: 10.1073/pnas.80.11.3198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Perlman S. M., Huang A. S. RNA synthesis of vesicular stomatitis virus. V. Interactions between transcription and replication. J Virol. 1973 Dec;12(6):1395–1400. doi: 10.1128/jvi.12.6.1395-1400.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ricciardi R. P., Miller J. S., Roberts B. E. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4927–4931. doi: 10.1073/pnas.76.10.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rose J. K., Gallione C. J. Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions. J Virol. 1981 Aug;39(2):519–528. doi: 10.1128/jvi.39.2.519-528.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schubert M., Keene J. D., Lazzarini R. A., Emerson S. U. The complete sequence of a unique RNA species synthesized by a DI particle of VSV. Cell. 1978 Sep;15(1):103–112. doi: 10.1016/0092-8674(78)90086-7. [DOI] [PubMed] [Google Scholar]
  18. Simonsen C. C., Hill V. M., Summers D. F. Further characterization of the replicative complex of vesicular stomatitis virus. J Virol. 1979 Aug;31(2):494–505. doi: 10.1128/jvi.31.2.494-505.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Soria M., Little S. P., Huang A. S. Characterization of vesicular stomatitis virus nucleocapsids. I. Complementary 40 S RNA molecules in nucleocapsids. Virology. 1974 Sep;61(1):270–280. doi: 10.1016/0042-6822(74)90261-x. [DOI] [PubMed] [Google Scholar]
  20. Stamminger G., Lazzarini R. A. Analysis of the RNA of defective VSV particles. Cell. 1974 Sep;3(1):85–93. doi: 10.1016/0092-8674(74)90044-0. [DOI] [PubMed] [Google Scholar]
  21. Wertz G. W., Davis N. L. RNase III cleaves vesicular stomatitis virus genome-length RNAs but fails to cleave viral mRNA's. J Virol. 1979 Apr;30(1):108–115. doi: 10.1128/jvi.30.1.108-115.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wertz G. W., Levine M. RNA synthesis by vesicular stomatitis virus and a small plaque mutant: effects of cycloheximide. J Virol. 1973 Aug;12(2):253–264. doi: 10.1128/jvi.12.2.253-264.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wertz G. W. Replication of vesicular stomatitis virus defective interfering particle RNA in vitro: transition from synthesis of defective interfering leader RNA to synthesis of full-length defective interfering RNA. J Virol. 1983 May;46(2):513–522. doi: 10.1128/jvi.46.2.513-522.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES