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. 1993 Apr;67(4):1916–1926. doi: 10.1128/jvi.67.4.1916-1926.1993

Roles of nonstructural polyproteins and cleavage products in regulating Sindbis virus RNA replication and transcription.

J A Lemm 1, C M Rice 1
PMCID: PMC240259  PMID: 8445717

Abstract

Using vaccinia virus to express Sindbis virus (SIN) nonstructural proteins (nsPs) and template RNAs, we showed previously that synthesis of all three viral RNAs occurred only during expression of either the entire nonstructural coding region or the polyprotein precursors P123 and P34. In this report, the vaccinia virus system was used to express cleavage-defective polyproteins and nsP4 proteins containing various N-terminal extensions to directly examine the roles of the P123 and P34 polyproteins in RNA replication. Replication and subgenomic mRNA transcription occurred during coexpression of P34 and P123 polyproteins in which cleavage was blocked at either or both of the 1/2 and 2/3 sites. For all cleavage-defective P123 polyproteins, however, the ratio of subgenomic to genomic RNA was decreased, suggesting that both the 1/2 and 2/3 cleavages are required for efficient subgenomic RNA transcription. These studies indicate that the uncleaved P123 polyprotein can function as a component of the viral replicase capable of synthesizing both plus- and minus-strand RNAs. In contrast, cleavage-defective P34 was unable to function in RNA replication, even in complementation experiments in which minus-strand RNAs were provided by nsP4. A P34 polyprotein whose cleavage site was not altered could only function in RNA replication in the presence of an active nsP2 protease. Although nsP4, the putative RNA polymerase, was capable of synthesizing only minus-strand RNAs during coexpression with P123, the addition of only 22 upstream residues to nsP4 allowed both replication and transcription of subgenomic RNA to occur. These data show that the conserved domains of both nsP3 and the nsP4 polymerase do not need to be present in a P34 polyprotein to form a functional plus-strand replicase-transcriptase and suggest that the presence of an active nsP2 protease and a cleavable 3/4 site correlates with synthesis of all virus-specific RNA species.

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Selected References

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

  1. Ahlquist P., Strauss E. G., Rice C. M., Strauss J. H., Haseloff J., Zimmern D. Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses. J Virol. 1985 Feb;53(2):536–542. doi: 10.1128/jvi.53.2.536-542.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barton D. J., Sawicki S. G., Sawicki D. L. Demonstration in vitro of temperature-sensitive elongation of RNA in Sindbis virus mutant ts6. J Virol. 1988 Oct;62(10):3597–3602. doi: 10.1128/jvi.62.10.3597-3602.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barton D. J., Sawicki S. G., Sawicki D. L. Solubilization and immunoprecipitation of alphavirus replication complexes. J Virol. 1991 Mar;65(3):1496–1506. doi: 10.1128/jvi.65.3.1496-1506.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ding M. X., Schlesinger M. J. Evidence that Sindbis virus NSP2 is an autoprotease which processes the virus nonstructural polyprotein. Virology. 1989 Jul;171(1):280–284. doi: 10.1016/0042-6822(89)90539-4. [DOI] [PubMed] [Google Scholar]
  5. Falkner F. G., Moss B. Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors. J Virol. 1988 Jun;62(6):1849–1854. doi: 10.1128/jvi.62.6.1849-1854.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. French R., Janda M., Ahlquist P. Bacterial gene inserted in an engineered RNA virus: efficient expression in monocotyledonous plant cells. Science. 1986 Mar 14;231(4743):1294–1297. doi: 10.1126/science.231.4743.1294. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Goelet P., Lomonossoff G. P., Butler P. J., Akam M. E., Gait M. J., Karn J. Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5818–5822. doi: 10.1073/pnas.79.19.5818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grakoui A., Levis R., Raju R., Huang H. V., Rice C. M. A cis-acting mutation in the Sindbis virus junction region which affects subgenomic RNA synthesis. J Virol. 1989 Dec;63(12):5216–5227. doi: 10.1128/jvi.63.12.5216-5227.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hahn Y. S., Grakoui A., Rice C. M., Strauss E. G., Strauss J. H. Mapping of RNA- temperature-sensitive mutants of Sindbis virus: complementation group F mutants have lesions in nsP4. J Virol. 1989 Mar;63(3):1194–1202. doi: 10.1128/jvi.63.3.1194-1202.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hahn Y. S., Strauss E. G., Strauss J. H. Mapping of RNA- temperature-sensitive mutants of Sindbis virus: assignment of complementation groups A, B, and G to nonstructural proteins. J Virol. 1989 Jul;63(7):3142–3150. doi: 10.1128/jvi.63.7.3142-3150.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hardy W. R., Hahn Y. S., de Groot R. J., Strauss E. G., Strauss J. H. Synthesis and processing of the nonstructural polyproteins of several temperature-sensitive mutants of Sindbis virus. Virology. 1990 Jul;177(1):199–208. doi: 10.1016/0042-6822(90)90473-5. [DOI] [PubMed] [Google Scholar]
  14. Hardy W. R., Strauss J. H. Processing the nonstructural polyproteins of Sindbis virus: study of the kinetics in vivo by using monospecific antibodies. J Virol. 1988 Mar;62(3):998–1007. doi: 10.1128/jvi.62.3.998-1007.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hardy W. R., Strauss J. H. Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans. J Virol. 1989 Nov;63(11):4653–4664. doi: 10.1128/jvi.63.11.4653-4664.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haseloff J., Goelet P., Zimmern D., Ahlquist P., Dasgupta R., Kaesberg P. Striking similarities in amino acid sequence among nonstructural proteins encoded by RNA viruses that have dissimilar genomic organization. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4358–4362. doi: 10.1073/pnas.81.14.4358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hirth L., Richards K. E. Tobacco mosaic virus: model for structure and function of a simple virus. Adv Virus Res. 1981;26:145–199. doi: 10.1016/s0065-3527(08)60423-6. [DOI] [PubMed] [Google Scholar]
  18. Hruby D. E., Guarino L. A., Kates J. R. Vaccinia virus replication. I. Requirement for the host-cell nucleus. J Virol. 1979 Feb;29(2):705–715. doi: 10.1128/jvi.29.2.705-715.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ishikawa M., Meshi T., Motoyoshi F., Takamatsu N., Okada Y. In vitro mutagenesis of the putative replicase genes of tobacco mosaic virus. Nucleic Acids Res. 1986 Nov 11;14(21):8291–8305. doi: 10.1093/nar/14.21.8291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kamer G., Argos P. Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res. 1984 Sep 25;12(18):7269–7282. doi: 10.1093/nar/12.18.7269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  22. Lemm J. A., Durbin R. K., Stollar V., Rice C. M. Mutations which alter the level or structure of nsP4 can affect the efficiency of Sindbis virus replication in a host-dependent manner. J Virol. 1990 Jun;64(6):3001–3011. doi: 10.1128/jvi.64.6.3001-3011.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lemm J. A., Rice C. M. Assembly of functional Sindbis virus RNA replication complexes: requirement for coexpression of P123 and P34. J Virol. 1993 Apr;67(4):1905–1915. doi: 10.1128/jvi.67.4.1905-1915.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li G. P., Prágai B. M., Rice C. M. Rescue of Sindbis virus-specific RNA replication and transcription by using a vaccinia virus recombinant. J Virol. 1991 Dec;65(12):6714–6723. doi: 10.1128/jvi.65.12.6714-6723.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li G. P., Rice C. M. Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication. J Virol. 1989 Mar;63(3):1326–1337. doi: 10.1128/jvi.63.3.1326-1337.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mackett M., Smith G. L. Vaccinia virus expression vectors. J Gen Virol. 1986 Oct;67(Pt 10):2067–2082. doi: 10.1099/0022-1317-67-10-2067. [DOI] [PubMed] [Google Scholar]
  27. Moss B., Elroy-Stein O., Mizukami T., Alexander W. A., Fuerst T. R. Product review. New mammalian expression vectors. Nature. 1990 Nov 1;348(6296):91–92. doi: 10.1038/348091a0. [DOI] [PubMed] [Google Scholar]
  28. Novak J. E., Kirkegaard K. Improved method for detecting poliovirus negative strands used to demonstrate specificity of positive-strand encapsidation and the ratio of positive to negative strands in infected cells. J Virol. 1991 Jun;65(6):3384–3387. doi: 10.1128/jvi.65.6.3384-3387.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Peränen J., Rikkonen M., Liljeström P., Käriäinen L. Nuclear localization of Semliki Forest virus-specific nonstructural protein nsP2. J Virol. 1990 May;64(5):1888–1896. doi: 10.1128/jvi.64.5.1888-1896.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rothstein M. A., Richards O. C., Amin C., Ehrenfeld E. Enzymatic activity of poliovirus RNA polymerase synthesized in Escherichia coli from viral cDNA. Virology. 1988 Jun;164(2):301–308. doi: 10.1016/0042-6822(88)90542-9. [DOI] [PubMed] [Google Scholar]
  31. Sawicki D. L., Sawicki S. G. Functional analysis of the A complementation group mutants of Sindbis HR virus. Virology. 1985 Jul 15;144(1):20–34. doi: 10.1016/0042-6822(85)90301-0. [DOI] [PubMed] [Google Scholar]
  32. Shirako Y., Strauss J. H. Cleavage between nsP1 and nsP2 initiates the processing pathway of Sindbis virus nonstructural polyprotein P123. Virology. 1990 Jul;177(1):54–64. doi: 10.1016/0042-6822(90)90459-5. [DOI] [PubMed] [Google Scholar]
  33. Strauss E. G., De Groot R. J., Levinson R., Strauss J. H. Identification of the active site residues in the nsP2 proteinase of Sindbis virus. Virology. 1992 Dec;191(2):932–940. doi: 10.1016/0042-6822(92)90268-T. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Strauss E. G., Rice C. M., Strauss J. H. Complete nucleotide sequence of the genomic RNA of Sindbis virus. Virology. 1984 Feb;133(1):92–110. doi: 10.1016/0042-6822(84)90428-8. [DOI] [PubMed] [Google Scholar]
  35. Strauss E. G., Rice C. M., Strauss J. H. Sequence coding for the alphavirus nonstructural proteins is interrupted by an opal termination codon. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5271–5275. doi: 10.1073/pnas.80.17.5271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. de Groot R. J., Hardy W. R., Shirako Y., Strauss J. H. Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo. EMBO J. 1990 Aug;9(8):2631–2638. doi: 10.1002/j.1460-2075.1990.tb07445.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. de Groot R. J., Rümenapf T., Kuhn R. J., Strauss E. G., Strauss J. H. Sindbis virus RNA polymerase is degraded by the N-end rule pathway. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8967–8971. doi: 10.1073/pnas.88.20.8967. [DOI] [PMC free article] [PubMed] [Google Scholar]

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