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. 1997 Jun;71(6):4264–4271. doi: 10.1128/jvi.71.6.4264-4271.1997

Expression and purification of vesicular stomatitis virus N-P complex from Escherichia coli: role in genome RNA transcription and replication in vitro.

A K Gupta 1, A K Banerjee 1
PMCID: PMC191641  PMID: 9151813

Abstract

The nucleocapsid protein (N) and phosphoprotein (P) genes of vesicular stomatitis virus (VSV), Indiana serotype, were coexpressed in Escherichia coli BL21(DE3) by using the expression vector pET-3a. The coexpression resulted in the formation of N-P complex. The purified N-P complex was found to inhibit transcription in vitro mediated by viral ribonucleoprotein (RNP) complex in a dose-dependent manner. However, addition of uninfected mammalian cell extracts together with the N-P complex to the transcribing RNP resulted in the synthesis of full-length negative-strand genome RNA. These results indicate that the N-P complex regulated transcription and a cellular factor(s) in combination with the N-P complex may switch the RNA polymerase from transcription to replication mode.

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Selected References

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  1. Banerjee A. D., Abraham G., Colonno R. J. Vesicular stomatitis virus: mode of transcription. J Gen Virol. 1977 Jan;34(1):1–8. doi: 10.1099/0022-1317-34-1-1. [DOI] [PubMed] [Google Scholar]
  2. Banerjee A. K., Barik S., De B. P. Gene expression of nonsegmented negative strand RNA viruses. Pharmacol Ther. 1991;51(1):47–70. doi: 10.1016/0163-7258(91)90041-j. [DOI] [PubMed] [Google Scholar]
  3. Banerjee A. K. Transcription and replication of rhabdoviruses. Microbiol Rev. 1987 Mar;51(1):66–87. doi: 10.1128/mr.51.1.66-87.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barik S., Banerjee A. K. Cloning and expression of the vesicular stomatitis virus phosphoprotein gene in Escherichia coli: analysis of phosphorylation status versus transcriptional activity. J Virol. 1991 Apr;65(4):1719–1726. doi: 10.1128/jvi.65.4.1719-1726.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bell J. C., Brown E. G., Takayesu D., Prevec L. Protein kinase activity associated with immunoprecipitates of the vesicular stomatitis virus phosphoprotein NS. Virology. 1984 Jan 30;132(2):229–238. doi: 10.1016/0042-6822(84)90030-8. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Blumberg B. M., Giorgi C., Rose K., Kolakofsky D. Preparation and analysis of the nucleocapsid proteins of vesicular stomatitis virus and sendai virus, and analysis of the sendai virus leader-NP gene region. J Gen Virol. 1984 Apr;65(Pt 4):769–779. doi: 10.1099/0022-1317-65-4-769. [DOI] [PubMed] [Google Scholar]
  8. Cazenave C., Uhlenbeck O. C. RNA template-directed RNA synthesis by T7 RNA polymerase. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6972–6976. doi: 10.1073/pnas.91.15.6972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Condra J. H., Lazzarini R. A. Replicative RNA synthesis and nucleocapsid assembly in vesicular stomatitis virus-infected permeable cells. J Virol. 1980 Dec;36(3):796–804. doi: 10.1128/jvi.36.3.796-804.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Das T., Banerjee A. K. Expression of the vesicular stomatitis virus nucleocapsid protein gene in Escherichia coli: analysis of its biological activity in vitro. Virology. 1993 Mar;193(1):340–347. doi: 10.1006/viro.1993.1130. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. De B. P., Banerjee A. K. Requirements and functions of vesicular stomatitis virus L and NS proteins in the transcription process in vitro. Biochem Biophys Res Commun. 1985 Jan 16;126(1):40–49. doi: 10.1016/0006-291x(85)90568-6. [DOI] [PubMed] [Google Scholar]
  13. Emerson S. U., Yu Y. Both NS and L proteins are required for in vitro RNA synthesis by vesicular stomatitis virus. J Virol. 1975 Jun;15(6):1348–1356. doi: 10.1128/jvi.15.6.1348-1356.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Frankel S., Condeelis J., Leinwand L. Expression of actin in Escherichia coli. Aggregation, solubilization, and functional analysis. J Biol Chem. 1990 Oct 15;265(29):17980–17987. [PubMed] [Google Scholar]
  15. Ghosh K., Ghosh H. P. Synthesis in vitro of full length genomic RNA and assembly of the nucleocapsid of vesicular stomatitis virus in a coupled transcription-translation system. Nucleic Acids Res. 1982 Oct 25;10(20):6341–6351. doi: 10.1093/nar/10.20.6341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gupta A. K., Das T., Banerjee A. K. Casein kinase II is the P protein phosphorylating cellular kinase associated with the ribonucleoprotein complex of purified vesicular stomatitis virus. J Gen Virol. 1995 Feb;76(Pt 2):365–372. doi: 10.1099/0022-1317-76-2-365. [DOI] [PubMed] [Google Scholar]
  17. Hill V. M., Marnell L., Summers D. F. In vitro replication and assembly of vesicular stomatitis virus nucleocapsids. Virology. 1981 Aug;113(1):109–118. doi: 10.1016/0042-6822(81)90140-9. [DOI] [PubMed] [Google Scholar]
  18. Howard M., Wertz G. Vesicular stomatitis virus RNA replication: a role for the NS protein. J Gen Virol. 1989 Oct;70(Pt 10):2683–2694. doi: 10.1099/0022-1317-70-10-2683. [DOI] [PubMed] [Google Scholar]
  19. La Ferla F. M., Peluso R. W. The 1:1 N-NS protein complex of vesicular stomatitis virus is essential for efficient genome replication. J Virol. 1989 Sep;63(9):3852–3857. doi: 10.1128/jvi.63.9.3852-3857.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Patton J. T., Davis N. L., Wertz G. W. N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus. J Virol. 1984 Feb;49(2):303–309. doi: 10.1128/jvi.49.2.303-309.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Peluso R. W. Kinetic, quantitative, and functional analysis of multiple forms of the vesicular stomatitis virus nucleocapsid protein in infected cells. J Virol. 1988 Aug;62(8):2799–2807. doi: 10.1128/jvi.62.8.2799-2807.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Peluso R. W., Moyer S. A. Viral proteins required for the in vitro replication of vesicular stomatitis virus defective interfering particle genome RNA. Virology. 1988 Feb;162(2):369–376. doi: 10.1016/0042-6822(88)90477-1. [DOI] [PubMed] [Google Scholar]
  24. Schubert M., Harmison G. G., Meier E. Primary structure of the vesicular stomatitis virus polymerase (L) gene: evidence for a high frequency of mutations. J Virol. 1984 Aug;51(2):505–514. doi: 10.1128/jvi.51.2.505-514.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sprague J., Condra J. H., Arnheiter H., Lazzarini R. A. Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein. J Virol. 1983 Feb;45(2):773–781. doi: 10.1128/jvi.45.2.773-781.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  27. Takacs A. M., Banerjee A. K. Efficient interaction of the vesicular stomatitis virus P protein with the L protein or the N protein in cells expressing the recombinant proteins. Virology. 1995 Apr 20;208(2):821–826. doi: 10.1006/viro.1995.1219. [DOI] [PubMed] [Google Scholar]
  28. Takacs A. M., Das T., Banerjee A. K. Mapping of interacting domains between the nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10375–10379. doi: 10.1073/pnas.90.21.10375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]

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