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. 1993 Apr;67(4):1905–1915. doi: 10.1128/jvi.67.4.1905-1915.1993

Assembly of functional Sindbis virus RNA replication complexes: requirement for coexpression of P123 and P34.

J A Lemm 1, C M Rice 1
PMCID: PMC240258  PMID: 8445716

Abstract

A vaccinia virus transient expression system was used to determine which of the Sindbis virus (SIN) proteins and/or polyproteins are necessary for the formation of active replication complexes and, in particular, to analyze the role of nsP4, the putative polymerase, versus P34 in RNA replication. We generated vaccinia virus recombinants in which the cDNA for the entire SIN nonstructural coding region as well as cDNA copies of the individual nonstructural proteins (nsPs) and several intermediate polyproteins were placed downstream of the promoter for T7 RNA polymerase and the encephalomyocarditis virus 5' untranslated region. The proteins expressed by the vaccinia virus recombinants comigrate with authentic proteins synthesized in SIN-infected cells, and the polyproteins appear to be processed to the individual proteins of the correct size. To examine the replication efficiencies of different protein combinations, a vaccinia virus recombinant was designed to express an engineered substrate RNA which could serve as a template for replication and subgenomic mRNA transcription by the SIN nsPs. Expression of the entire SIN nonstructural coding region resulted in the synthesis of high levels of both genomic and subgenomic RNAs derived from the engineered template. No RNA replication could be detected during coexpression of the four individual nsPs, although the proteins were indistinguishable, in terms of electrophoretic mobility, from those synthesized in SIN-infected cells. Coexpression of polyproteins P12, P23, and/or P34 with the individual nsPs also did not result in detectable levels of RNA replication. However, when P123 and P34 were coexpressed, efficient RNA replication and subgenomic mRNA transcription of the substrate RNA was observed. Coexpression of nsP4 with P123 resulted in the synthesis of only minus-strand RNAs. These studies show that expression of both P123 and P34 is necessary for establishment of functional RNA replication and transcription complexes and raise the possibility that the polyproteins themselves may be functional components of these complexes. In addition, these data indicate that an nsP4 moiety expressed independently with an additional N-terminal methionine is capable of functioning in minus-strand but not plus-strand RNA synthesis.

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Selected References

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  1. Bachmair A., Finley D., Varshavsky A. In vivo half-life of a protein is a function of its amino-terminal residue. Science. 1986 Oct 10;234(4773):179–186. doi: 10.1126/science.3018930. [DOI] [PubMed] [Google Scholar]
  2. Ball L. A. Cellular expression of a functional nodavirus RNA replicon from vaccinia virus vectors. J Virol. 1992 Apr;66(4):2335–2345. doi: 10.1128/jvi.66.4.2335-2345.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Clewley J. P., Kennedy S. I. Purification and polypeptide composition of Semliki Forest virus RNA polymerase. J Gen Virol. 1976 Sep;32(3):395–411. doi: 10.1099/0022-1317-32-3-395. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. Gomatos P. J., Käriäinen L., Keränen S., Ranki M., Sawicki D. L. Semliki Forest virus replication complex capable of synthesizing 42S and 26S nascent RNA chains. J Gen Virol. 1980 Jul;49(1):61–69. doi: 10.1099/0022-1317-49-1-61. [DOI] [PubMed] [Google Scholar]
  11. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gotoh H., Shioda T., Sakai Y., Mizumoto K., Shibuta H. Rescue of Sendai virus from viral ribonucleoprotein-transfected cells by infection with recombinant vaccinia viruses carrying Sendai virus L and P/C genes. Virology. 1989 Aug;171(2):434–443. doi: 10.1016/0042-6822(89)90612-0. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. Huang T. S., Palese P., Krystal M. Determination of influenza virus proteins required for genome replication. J Virol. 1990 Nov;64(11):5669–5673. doi: 10.1128/jvi.64.11.5669-5673.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Karacostas V., Nagashima K., Gonda M. A., Moss B. Human immunodeficiency virus-like particles produced by a vaccinia virus expression vector. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8964–8967. doi: 10.1073/pnas.86.22.8964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Lemm J. A., Rice C. M. Roles of nonstructural polyproteins and cleavage products in regulating Sindbis virus RNA replication and transcription. J Virol. 1993 Apr;67(4):1916–1926. doi: 10.1128/jvi.67.4.1916-1926.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Levis R., Schlesinger S., Huang H. V. Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription. J Virol. 1990 Apr;64(4):1726–1733. doi: 10.1128/jvi.64.4.1726-1733.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Levis R., Weiss B. G., Tsiang M., Huang H., Schlesinger S. Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging. Cell. 1986 Jan 17;44(1):137–145. doi: 10.1016/0092-8674(86)90492-7. [DOI] [PubMed] [Google Scholar]
  28. Li G. P., La Starza M. W., Hardy W. R., Strauss J. H., Rice C. M. Phosphorylation of Sindbis virus nsP3 in vivo and in vitro. Virology. 1990 Nov;179(1):416–427. doi: 10.1016/0042-6822(90)90310-n. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mi S., Durbin R., Huang H. V., Rice C. M., Stollar V. Association of the Sindbis virus RNA methyltransferase activity with the nonstructural protein nsP1. Virology. 1989 Jun;170(2):385–391. doi: 10.1016/0042-6822(89)90429-7. [DOI] [PubMed] [Google Scholar]
  34. Mi S., Stollar V. Expression of Sindbis virus nsP1 and methyltransferase activity in Escherichia coli. Virology. 1991 Sep;184(1):423–427. doi: 10.1016/0042-6822(91)90862-6. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Pattnaik A. K., Ball L. A., LeGrone A. W., Wertz G. W. Infectious defective interfering particles of VSV from transcripts of a cDNA clone. Cell. 1992 Jun 12;69(6):1011–1020. doi: 10.1016/0092-8674(92)90619-n. [DOI] [PubMed] [Google Scholar]
  38. Pattnaik A. K., Wertz G. W. Cells that express all five proteins of vesicular stomatitis virus from cloned cDNAs support replication, assembly, and budding of defective interfering particles. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1379–1383. doi: 10.1073/pnas.88.4.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pattnaik A. K., Wertz G. W. Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs. J Virol. 1990 Jun;64(6):2948–2957. doi: 10.1128/jvi.64.6.2948-2957.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ranki M., Käriäinen L. Solubilized RNA replication complex from Semliki Forest virus-infected cells. Virology. 1979 Oct 30;98(2):298–307. doi: 10.1016/0042-6822(79)90553-1. [DOI] [PubMed] [Google Scholar]
  41. Rice C. M., Levis R., Strauss J. H., Huang H. V. Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol. 1987 Dec;61(12):3809–3819. doi: 10.1128/jvi.61.12.3809-3819.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  43. Sawicki D. L., Sawicki S. G., Keränen S., Käriäinen L. Specific Sindbis virus-coded function for minus-strand RNA synthesis. J Virol. 1981 Aug;39(2):348–358. doi: 10.1128/jvi.39.2.348-358.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sawicki D., Barkhimer D. B., Sawicki S. G., Rice C. M., Schlesinger S. Temperature sensitive shut-off of alphavirus minus strand RNA synthesis maps to a nonstructural protein, nsP4. Virology. 1990 Jan;174(1):43–52. doi: 10.1016/0042-6822(90)90052-s. [DOI] [PubMed] [Google Scholar]
  45. Sawicki S. G., Sawicki D. L., Käriäinen L., Keränen S. A Sindbis virus mutant temperature-sensitive in the regulation of minus-strand RNA synthesis. Virology. 1981 Nov;115(1):161–172. doi: 10.1016/0042-6822(81)90098-2. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Wang Y. F., Sawicki S. G., Sawicki D. L. Sindbis virus nsP1 functions in negative-strand RNA synthesis. J Virol. 1991 Feb;65(2):985–988. doi: 10.1128/jvi.65.2.985-988.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Xiong C., Levis R., Shen P., Schlesinger S., Rice C. M., Huang H. V. Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. Science. 1989 Mar 3;243(4895):1188–1191. doi: 10.1126/science.2922607. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. 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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