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. 1990 Jun;64(6):2948–2957. doi: 10.1128/jvi.64.6.2948-2957.1990

Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs.

A K Pattnaik 1, G W Wertz 1
PMCID: PMC249479  PMID: 2159555

Abstract

Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have until now been attained only in cells coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle RNA replication. VSV proteins were synthesized soon after transfection in amounts that depended on the amount of input plasmid DNA and at rates that remained constant for at least 16 h after transfection. When cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with the DI particles, rapid and efficient replication and amplification of DI particle RNA was observed. Omission of any one of the three viral proteins abrogated the replication. The maximum levels of DI particle RNA replication that were achieved in the system exceeded those seen with wild-type helper VSV by 8- to 10-fold and were observed at molar L:NS:N protein ratios of approximately 1:200:200. This replication system can be used for analysis of structure-function relationships of VSV proteins that are involved in RNA replication and has potential for use in the identification of RNA sequences in the viral genome that control transcription and replication of VSV RNA.

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Selected References

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

  1. Abraham G., Banerjee A. K. Sequential transcription of the genes of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1976 May;73(5):1504–1508. doi: 10.1073/pnas.73.5.1504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arnheiter H., Davis N. L., Wertz G., Schubert M., Lazzarini R. A. Role of the nucleocapsid protein in regulating vesicular stomatitis virus RNA synthesis. Cell. 1985 May;41(1):259–267. doi: 10.1016/0092-8674(85)90079-0. [DOI] [PubMed] [Google Scholar]
  3. Ball L. A., White C. N. Order of transcription of genes of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1976 Feb;73(2):442–446. doi: 10.1073/pnas.73.2.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Banerjee A. K. The transcription complex of vesicular stomatitis virus. Cell. 1987 Feb 13;48(3):363–364. doi: 10.1016/0092-8674(87)90184-x. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  8. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [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. Davis N. L., Arnheiter H., Wertz G. W. Vesicular stomatitis virus N and NS proteins form multiple complexes. J Virol. 1986 Sep;59(3):751–754. doi: 10.1128/jvi.59.3.751-754.1986. [DOI] [PMC free article] [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. 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]
  13. Fuerst T. R., Earl P. L., Moss B. Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. Mol Cell Biol. 1987 Jul;7(7):2538–2544. doi: 10.1128/mcb.7.7.2538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Halonen P. E., Murphy F. A., Fields B. N., Reese D. R. Hemagglutinin of rabies and some other bullet-shaped viruses. Proc Soc Exp Biol Med. 1968 Apr;127(4):1037–1042. doi: 10.3181/00379727-127-32864. [DOI] [PubMed] [Google Scholar]
  16. Harmon S. A., Robinson E. N., Jr, Summers D. F. Ultrastructural localization of L and NS enzyme subunits on vesicular stomatitis virus RNPs using gold sphere-staphylococcal protein A-monospecific IgG conjugates. Virology. 1985 Apr 30;142(2):406–410. doi: 10.1016/0042-6822(85)90348-4. [DOI] [PubMed] [Google Scholar]
  17. Helfman W. B., Perrault J. Altered ATP utilization by the poIR mutants of vesicular stomatitis virus maps to the N-RNA template. Virology. 1988 Nov;167(1):311–313. doi: 10.1016/0042-6822(88)90087-6. [DOI] [PubMed] [Google Scholar]
  18. Helfman W. B., Perrault J. Redistributive properties of the vesicular stomatitis virus polymerase. Virology. 1989 Aug;171(2):319–330. doi: 10.1016/0042-6822(89)90599-0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Hudson L. D., Condra C., Lazzarini R. A. Cloning and expression of a viral phosphoprotein: structure suggests vesicular stomatitis virus NS may function by mimicking an RNA template. J Gen Virol. 1986 Aug;67(Pt 8):1571–1579. doi: 10.1099/0022-1317-67-8-1571. [DOI] [PubMed] [Google Scholar]
  22. Iverson L. E., Rose J. K. Localized attenuation and discontinuous synthesis during vesicular stomatitis virus transcription. Cell. 1981 Feb;23(2):477–484. doi: 10.1016/0092-8674(81)90143-4. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Laskey R. A. The use of intensifying screens or organic scintillators for visualizing radioactive molecules resolved by gel electrophoresis. Methods Enzymol. 1980;65(1):363–371. doi: 10.1016/s0076-6879(80)65047-2. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Li Y., Luo L. Z., Snyder R. M., Wagner R. R. Expression of the M gene of vesicular stomatitis virus cloned in various vaccinia virus vectors. J Virol. 1988 Mar;62(3):776–782. doi: 10.1128/jvi.62.3.776-782.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Masters P. S., Banerjee A. K. Complex formation with vesicular stomatitis virus phosphoprotein NS prevents binding of nucleocapsid protein N to nonspecific RNA. J Virol. 1988 Aug;62(8):2658–2664. doi: 10.1128/jvi.62.8.2658-2664.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Masters P. S., Banerjee A. K. Resolution of multiple complexes of phosphoprotein NS with nucleocapsid protein N of vesicular stomatitis virus. J Virol. 1988 Aug;62(8):2651–2657. doi: 10.1128/jvi.62.8.2651-2657.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meier E., Harmison G. G., Schubert M. Homotypic and heterotypic exclusion of vesicular stomatitis virus replication by high levels of recombinant polymerase protein L. J Virol. 1987 Oct;61(10):3133–3142. doi: 10.1128/jvi.61.10.3133-3142.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mirakhur B., Peluso R. W. In vitro assembly of a functional nucleocapsid from the negative-stranded genome RNA of a defective interfering particle of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7511–7515. doi: 10.1073/pnas.85.20.7511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Naito S., Ishihama A. Function and structure of RNA polymerase from vesicular stomatitis virus. J Biol Chem. 1976 Jul 25;251(14):4307–4314. [PubMed] [Google Scholar]
  32. Pattnaik A. K., Brown D. J., Nayak D. P. Formation of influenza virus particles lacking hemagglutinin on the viral envelope. J Virol. 1986 Dec;60(3):994–1001. doi: 10.1128/jvi.60.3.994-1001.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. Perrault J., Clinton G. M., McClure M. A. RNP template of vesicular stomatitis virus regulates transcription and replication functions. Cell. 1983 Nov;35(1):175–185. doi: 10.1016/0092-8674(83)90220-9. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. 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]
  44. Wertz G. W., Davis N. Characterization and mapping of RNase III cleavage sites in VSV genome RNA. Nucleic Acids Res. 1981 Dec 11;9(23):6487–6503. doi: 10.1093/nar/9.23.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Whitt M. A., Chong L., Rose J. K. Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant. J Virol. 1989 Sep;63(9):3569–3578. doi: 10.1128/jvi.63.9.3569-3578.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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