Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1994 Sep;68(9):5781–5791. doi: 10.1128/jvi.68.9.5781-5791.1994

Genetic analysis of the nsP3 region of Sindbis virus: evidence for roles in minus-strand and subgenomic RNA synthesis.

M W LaStarza 1, J A Lemm 1, C M Rice 1
PMCID: PMC236982  PMID: 8057460

Abstract

Sindbis virus nonstructural polyproteins and their cleavage products are believed to be essential components of viral RNA replication and transcription complexes. Although numerous studies have investigated the effect of mutations in nsP1-, nsP2-, and nsP4-coding regions on Sindbis virus-specific RNA synthesis, relatively little is known about the function of the region encoding nsP3. nsP3 is a phosphoprotein comprising two regions: an N-terminal portion which is highly conserved among alphaviruses and a C-terminal portion which is not conserved, varying both in sequence and in length. We have constructed a library of random linker insertion mutations in the nsP3-coding region and characterized selected viable mutants. Initially, 126 mutants containing insertions in the conserved region and 23 with insertions in the nonconserved region were screened for temperature-sensitive (ts) plaque formation or for significant differences in plaque morphology. All nonconserved-region mutants were similar to the parental virus, whereas 13 of those in the conserved region were either ts or exhibited altered plaque phenotypes. Ten of these 13 mutants were ts for plaque formation as well as RNA accumulation at 40 degrees C. Highly ts mutants CR3.36 and CR3.39 were defective in their ability to synthesize minus-strand RNAs at the nonpermissive temperature. The CR3.36 and CR3.39 insertion mutations localized to different regions near nsP3 residues 58 and 226, respectively. CR3.39 was able to complement ts mutants from Sindbis virus complementation groups A, B, F, and G. Another mutant isolated from the library, CR3.34, while not ts for plaque formation or RNA synthesis, formed smaller plaques and was defective in subgenomic RNA synthesis at all temperatures examined. These results suggest a role for nsP3 or nsP3-containing polyproteins in the synthesis of viral minus-strand and subgenomic RNAs.

Full text

PDF
5785

Images in this article

5786Image
on p.5786
5788Image
on p.5788

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. Burge B. W., Pfefferkorn E. R. Complementation between temperature-sensitive mutants of Sindbis virus. Virology. 1966 Oct;30(2):214–223. doi: 10.1016/0042-6822(66)90097-3. [DOI] [PubMed] [Google Scholar]
  4. Burge B. W., Pfefferkorn E. R. Isolation and characterization of conditional-lethal mutants of Sindbis virus. Virology. 1966 Oct;30(2):204–213. doi: 10.1016/0042-6822(66)90096-1. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Dominguez G., Wang C. Y., Frey T. K. Sequence of the genome RNA of rubella virus: evidence for genetic rearrangement during togavirus evolution. Virology. 1990 Jul;177(1):225–238. doi: 10.1016/0042-6822(90)90476-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Frolov I., Schlesinger S. Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells. J Virol. 1994 Mar;68(3):1721–1727. doi: 10.1128/jvi.68.3.1721-1727.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Hahn C. S., Hahn Y. S., Braciale T. J., Rice C. M. Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2679–2683. doi: 10.1073/pnas.89.7.2679. [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. 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]
  18. Kinney R. M., Johnson B. J., Welch J. B., Tsuchiya K. R., Trent D. W. The full-length nucleotide sequences of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and its attenuated vaccine derivative, strain TC-83. Virology. 1989 May;170(1):19–30. doi: 10.1016/0042-6822(89)90347-4. [DOI] [PubMed] [Google Scholar]
  19. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  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. Lastarza M. W., Grakoui A., Rice C. M. Deletion and duplication mutations in the C-terminal nonconserved region of Sindbis virus nsP3: effects on phosphorylation and on virus replication in vertebrate and invertebrate cells. Virology. 1994 Jul;202(1):224–232. doi: 10.1006/viro.1994.1338. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  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. 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]
  28. 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]
  29. 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]
  30. 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]
  31. 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]
  32. 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]
  33. Pierce J. S., Strauss E. G., Strauss J. H. Effect of ionic strength on the binding of Sindbis virus to chick cells. J Virol. 1974 May;13(5):1030–1036. doi: 10.1128/jvi.13.5.1030-1036.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Raju R., Huang H. V. Analysis of Sindbis virus promoter recognition in vivo, using novel vectors with two subgenomic mRNA promoters. J Virol. 1991 May;65(5):2501–2510. doi: 10.1128/jvi.65.5.2501-2510.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sawicki D. L., Sawicki S. G. A second nonstructural protein functions in the regulation of alphavirus negative-strand RNA synthesis. J Virol. 1993 Jun;67(6):3605–3610. doi: 10.1128/jvi.67.6.3605-3610.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. Scheidel L. M., Stollar V. Mutations that confer resistance to mycophenolic acid and ribavirin on Sindbis virus map to the nonstructural protein nsP1. Virology. 1991 Apr;181(2):490–499. doi: 10.1016/0042-6822(91)90881-b. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Shirako Y., Strauss J. H. Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis. J Virol. 1994 Mar;68(3):1874–1885. doi: 10.1128/jvi.68.3.1874-1885.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Simmons D. T., Strauss J. H. Replication of Sindbis virus. II. Multiple forms of double-stranded RNA isolated from infected cells. J Mol Biol. 1972 Nov 28;71(3):615–631. doi: 10.1016/s0022-2836(72)80027-5. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Strauss E. G., Lenches E. M., Strauss J. H. Mutants of sindbis virus. I. Isolation and partial characterization of 89 new temperature-sensitive mutants. Virology. 1976 Oct 1;74(1):154–168. doi: 10.1016/0042-6822(76)90137-9. [DOI] [PubMed] [Google Scholar]
  49. Strauss E. G., Levinson R., Rice C. M., Dalrymple J., Strauss J. H. Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses. Virology. 1988 May;164(1):265–274. doi: 10.1016/0042-6822(88)90644-7. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. Takkinen K. Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus. Nucleic Acids Res. 1986 Jul 25;14(14):5667–5682. doi: 10.1093/nar/14.14.5667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. 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]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES