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. 1996 Apr;70(4):2252–2259. doi: 10.1128/jvi.70.4.2252-2259.1996

Expression of L protein of vesicular stomatitis virus Indiana serotype from recombinant baculovirus in insect cells: requirement of a host factor(s) for its biological activity in vitro.

M Mathur 1, T Das 1, A K Banerjee 1
PMCID: PMC190066  PMID: 8642651

Abstract

The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) Indiana serotype, a multifunctional catalytic subunit of the viral RNA polymerase, has been expressed in Spodoptera frugiperda cells infected with recombinant baculovirus BacPAK6-L containing the L gene under the control of a polyhedrin promoter. The recombinant L protein was biologically active and supported viral mRNA synthesis in vitro. When the expressed L protein was purified by phosphocellulose column chromatography, it eluted in two peaks, one at 0.4 M NaCl (peak I) and the second at 0.75 M NaCl (peak II). The L protein in peak I showed significant transcriptional activity in an in vitro transcription reconstitution experiment, whereas the L protein in peak II was inactive. Interestingly, the addition of cytoplasmic extract from uninfected Sf21 cells to peak II completely restored transcription in vitro, indicating the requirement of a host factor(s) for the activity of the L protein. This factor is relatively heat stable and is dissociable from the recombinant L protein. It is also present in BHK, COS, and HeLa cells in detectable levels. The role of the putative host protein(s) in the activation of the L protein is discussed.

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Selected References

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  1. Baltimore D., Huang A. S., Stampfer M. Ribonucleic acid synthesis of vesicular stomatitis virus, II. An RNA polymerase in the virion. Proc Natl Acad Sci U S A. 1970 Jun;66(2):572–576. doi: 10.1073/pnas.66.2.572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Barik S., Banerjee A. K. Sequential phosphorylation of the phosphoprotein of vesicular stomatitis virus by cellular and viral protein kinases is essential for transcription activation. J Virol. 1992 Feb;66(2):1109–1118. doi: 10.1128/jvi.66.2.1109-1118.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barik S. Transcription of human respiratory syncytial virus genome RNA in vitro: requirement of cellular factor(s). J Virol. 1992 Nov;66(11):6813–6818. doi: 10.1128/jvi.66.11.6813-6818.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Beckes J. D., Perrault J. Stepwise phosphorylation of vesicular stomatitis virus P protein by virion-associated kinases and uncoupling of second step from in vitro transcription. Virology. 1992 Jun;188(2):606–617. doi: 10.1016/0042-6822(92)90515-q. [DOI] [PubMed] [Google Scholar]
  7. Canter D. M., Jackson R. L., Perrault J. Faithful and efficient in vitro reconstitution of vesicular stomatitis virus transcription using plasmid-encoded L and P proteins. Virology. 1993 Jun;194(2):518–529. doi: 10.1006/viro.1993.1290. [DOI] [PubMed] [Google Scholar]
  8. Dasgupta A. Purification of host factor required for in vitro transcription of poliovirus RNA. Virology. 1983 Jul 15;128(1):245–251. doi: 10.1016/0042-6822(83)90335-5. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. De B. P., Banerjee A. K. Specific interactions of vesicular stomatitis virus L and NS proteins with heterologous genome ribonucleoprotein template lead to mRNA synthesis in vitro. J Virol. 1984 Sep;51(3):628–634. doi: 10.1128/jvi.51.3.628-634.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. De B. P., Lesoon A., Banerjee A. K. Human parainfluenza virus type 3 transcription in vitro: role of cellular actin in mRNA synthesis. J Virol. 1991 Jun;65(6):3268–3275. doi: 10.1128/jvi.65.6.3268-3275.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Emerson S. U., Wagner R. R. L protein requirement for in vitro RNA synthesis by vesicular stomatitis virus. J Virol. 1973 Dec;12(6):1325–1335. doi: 10.1128/jvi.12.6.1325-1335.1973. [DOI] [PMC free article] [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. 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]
  15. Hayes R. J., Buck K. W. Complete replication of a eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase. Cell. 1990 Oct 19;63(2):363–368. doi: 10.1016/0092-8674(90)90169-f. [DOI] [PubMed] [Google Scholar]
  16. Hill V. M., Summers D. F. A minor microtubule-associated protein is responsible for the stimulation of vesicular stomatitis virus transcription in vitro. J Gen Virol. 1990 Feb;71(Pt 2):289–298. doi: 10.1099/0022-1317-71-2-289. [DOI] [PubMed] [Google Scholar]
  17. Horikami S. M., Moyer S. A. Host range mutants of vesicular stomatitis virus defective in in vitro RNA methylation. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7694–7698. doi: 10.1073/pnas.79.24.7694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huang Y. T., Romito R. R., De B. P., Banerjee A. K. Characterization of the in vitro system for the synthesis of mRNA from human respiratory syncytial virus. Virology. 1993 Apr;193(2):862–867. doi: 10.1006/viro.1993.1195. [DOI] [PubMed] [Google Scholar]
  19. Hunt D. M., Smith E. F., Buckley D. W. Aberrant polyadenylation by a vesicular stomatitis virus mutant is due to an altered L protein. J Virol. 1984 Nov;52(2):515–521. doi: 10.1128/jvi.52.2.515-521.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kamen R., Kondo M., Römer W., Weissmann C. Reconstitution of Q replicase lacking subunit with protein-synthesis-interference factor i. Eur J Biochem. 1972 Nov 21;31(1):44–51. doi: 10.1111/j.1432-1033.1972.tb02498.x. [DOI] [PubMed] [Google Scholar]
  21. Kitts P. A., Possee R. D. A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques. 1993 May;14(5):810–817. [PubMed] [Google Scholar]
  22. 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]
  23. Livingstone C., Jones I. Baculovirus expression vectors with single strand capability. Nucleic Acids Res. 1989 Mar 25;17(6):2366–2366. doi: 10.1093/nar/17.6.2366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Massey D. M., Deans N., Lenard J. Phosphorylation of NS protein by vesicular stomatitis virus nucleocapsids: lack of effect during RNA synthesis and separation of kinase from L protein. J Virol. 1990 Jul;64(7):3259–3264. doi: 10.1128/jvi.64.7.3259-3264.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Matsuura Y., Possee R. D., Overton H. A., Bishop D. H. Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. J Gen Virol. 1987 May;68(Pt 5):1233–1250. doi: 10.1099/0022-1317-68-5-1233. [DOI] [PubMed] [Google Scholar]
  26. Moyer S. A., Baker S. C., Horikami S. M. Host cell proteins required for measles virus reproduction. J Gen Virol. 1990 Apr;71(Pt 4):775–783. doi: 10.1099/0022-1317-71-4-775. [DOI] [PubMed] [Google Scholar]
  27. Moyer S. A., Baker S. C., Lessard J. L. Tubulin: a factor necessary for the synthesis of both Sendai virus and vesicular stomatitis virus RNAs. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5405–5409. doi: 10.1073/pnas.83.15.5405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Quadt R., Kao C. C., Browning K. S., Hershberger R. P., Ahlquist P. Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1498–1502. doi: 10.1073/pnas.90.4.1498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rigaut K. D., Gao Y., Lenard J. Effects of staurosporine on transcription by vesicular stomatitis virus. Virology. 1993 Jun;194(2):433–440. doi: 10.1006/viro.1993.1282. [DOI] [PubMed] [Google Scholar]
  30. Rose J. K., Lodish H. F., Brock M. L. Giant heterogeneous polyadenylic acid on vesicular stomatitis virus mRNA synthesized in vitro in the presence of S-adenosylhomocysteine. J Virol. 1977 Feb;21(2):683–693. doi: 10.1128/jvi.21.2.683-693.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Schubert M., Harmison G. G., Richardson C. D., Meier E. Expression of a cDNA encoding a functional 241-kilodalton vesicular stomatitis virus RNA polymerase. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7984–7988. doi: 10.1073/pnas.82.23.7984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sleat D. E., Banerjee A. K. Transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus RNA polymerase. J Virol. 1993 Mar;67(3):1334–1339. doi: 10.1128/jvi.67.3.1334-1339.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sleat D. E., Chikkala N. F., Gautam S., Banerjee A. K. Restricted replication of vesicular stomatitis virus in T lymphocytes is coincident with a deficiency in a cellular protein kinase required for viral transcription. J Gen Virol. 1992 Dec;73(Pt 12):3125–3132. doi: 10.1099/0022-1317-73-12-3125. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Sánchez A., De B. P., Banerjee A. K. In vitro phosphorylation of NS protein by the L protein of vesicular stomatitis virus. J Gen Virol. 1985 May;66(Pt 5):1025–1036. doi: 10.1099/0022-1317-66-5-1025. [DOI] [PubMed] [Google Scholar]
  37. Thomas D., Newcomb W. W., Brown J. C., Wall J. S., Hainfeld J. F., Trus B. L., Steven A. C. Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis. J Virol. 1985 May;54(2):598–607. doi: 10.1128/jvi.54.2.598-607.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

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