Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1996 May;70(5):2706–2719. doi: 10.1128/jvi.70.5.2706-2719.1996

Sindbis virus RNA-negative mutants that fail to convert from minus-strand to plus-strand synthesis: role of the nsP2 protein.

I Dé 1, S G Sawicki 1, D L Sawicki 1
PMCID: PMC190127  PMID: 8627744

Abstract

We identified mutations in the gene for nsP2, a nonstructural protein of the alphavirus Sindbis virus, that appear to block the conversion of the initial, short-lived minus-strand replicase complex (RCinitial) into mature, stable forms that are replicase and transcriptase complexes (RCstable), producing 49S genome or 26S mRNA. Base changes at nucleotide (nt) 2166 (G-->A, predicting a change of Glu-163-->Lys), at nt 2502 (G-->A, predicting a change of Val-275-->Ile), and at nt 2926 (C-->U, predicting a change of Leu-416-->Ser) in the nsP2 N domain were responsible for the phenotypes of ts14, ts16, and ts19 members of subgroup 11 (D.L. Sawicki and S.G. Sawicki, Virology 44:20-34, 1985) of the A complementation group of Sindbis virus RNA-negative mutants. Unlike subgroup I mutants, the RCstable formed at 30 degrees C transcribed 26S mRNA normally and did not synthesize minus strands in the absence of protein synthesis after temperature shift. The N-domain substitutions did not inactivate the thiol protease in the C domain of nsP2 and did not stop the proteolytic processing of the polyprotein containing the nonstructural proteins. The distinct phenotypes of subgroup I and 11 A complementation group mutants are evidence that the two domains of nsP2 are essential and functionally distinct. A detailed analysis of ts14 found that its nsPs were synthesized, processed, transported, and assembled at 40 degrees C into complexes with the properties of RCinitial and synthesized minus strands for a short time after shift to 40 degrees C. The block in the pathway to the formation of RCstable occurred after cleavage of the minus-strand replicase P123 or P23 polyprotein into mature nsP1, nsP2, nsP3, and nsP4, indicating that structures resembling RCstable, were formed at 40 degrees C. However, these RCstable or pre-RCstable structures were not capable of recovering activity at 30 degrees C. Therefore, failure to increase the rate of plus-strand synthesis after shift to 40 degrees C appears to result from failure to convert RCinitial to RCstable. We conclude that RCstable is derived from RCinitial by a conversion process and that ts14 is a conversion mutant. From their similar phenotypes, we predict that other nsP2 N-domain mutants are blocked also in the conversion of RCinitial to RCstable. Thus, the N domain of nsP2 plays an essential role in a folding pathway of the nsPs responsible for formation of the initial minus-strand replicase and for its conversion into stable plus-strand RNA-synthesizing enzymes.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

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. Ahola T., Käriäinen L. Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):507–511. doi: 10.1073/pnas.92.2.507. [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. 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. Dinant S., Janda M., Kroner P. A., Ahlquist P. Bromovirus RNA replication and transcription require compatibility between the polymerase- and helicase-like viral RNA synthesis proteins. J Virol. 1993 Dec;67(12):7181–7189. doi: 10.1128/jvi.67.12.7181-7189.1993. [DOI] [PMC free article] [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. 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]
  8. Franklin R. M. Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc Natl Acad Sci U S A. 1966 Jun;55(6):1504–1511. doi: 10.1073/pnas.55.6.1504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gorbalenya A. E., Koonin E. V., Lai M. M. Putative papain-related thiol proteases of positive-strand RNA viruses. Identification of rubi- and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha- and coronaviruses. FEBS Lett. 1991 Aug 19;288(1-2):201–205. doi: 10.1016/0014-5793(91)81034-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grady L. J., Campbell W. P. Amplification of large RNAs (greater than 1.5 kb) by polymerase chain reaction. Biotechniques. 1989 Sep;7(8):798–800. [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: 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]
  15. 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]
  16. Keränen S., Käriäinen L. Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses. J Virol. 1979 Oct;32(1):19–29. doi: 10.1128/jvi.32.1.19-29.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Keränen S., Ruohonen L. Nonstructural proteins of Semliki Forest virus: synthesis, processing, and stability in infected cells. J Virol. 1983 Sep;47(3):505–515. doi: 10.1128/jvi.47.3.505-515.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kroner P. A., Young B. M., Ahlquist P. Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis. J Virol. 1990 Dec;64(12):6110–6120. doi: 10.1128/jvi.64.12.6110-6120.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Laín S., Martín M. T., Riechmann J. L., García J. A. Novel catalytic activity associated with positive-strand RNA virus infection: nucleic acid-stimulated ATPase activity of the plum pox potyvirus helicaselike protein. J Virol. 1991 Jan;65(1):1–6. doi: 10.1128/jvi.65.1.1-6.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laín S., Riechmann J. L., García J. A. RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus. Nucleic Acids Res. 1990 Dec 11;18(23):7003–7006. doi: 10.1093/nar/18.23.7003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Lemm J. A., Rümenapf T., Strauss E. G., Strauss J. H., Rice C. M. Polypeptide requirements for assembly of functional Sindbis virus replication complexes: a model for the temporal regulation of minus- and plus-strand RNA synthesis. EMBO J. 1994 Jun 15;13(12):2925–2934. doi: 10.1002/j.1460-2075.1994.tb06587.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. McCauliffe D. P., Zappi E., Lieu T. S., Michalak M., Sontheimer R. D., Capra J. D. A human Ro/SS-A autoantigen is the homologue of calreticulin and is highly homologous with onchocercal RAL-1 antigen and an aplysia "memory molecule". J Clin Invest. 1990 Jul;86(1):332–335. doi: 10.1172/JCI114704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Nakhasi H. L., Cao X. Q., Rouault T. A., Liu T. Y. Specific binding of host cell proteins to the 3'-terminal stem-loop structure of rubella virus negative-strand RNA. J Virol. 1991 Nov;65(11):5961–5967. doi: 10.1128/jvi.65.11.5961-5967.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nakhasi H. L., Rouault T. A., Haile D. J., Liu T. Y., Klausner R. D. Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA. New Biol. 1990 Mar;2(3):255–264. [PubMed] [Google Scholar]
  30. Pardigon N., Strauss J. H. Cellular proteins bind to the 3' end of Sindbis virus minus-strand RNA. J Virol. 1992 Feb;66(2):1007–1015. doi: 10.1128/jvi.66.2.1007-1015.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pardigon N., Strauss J. H. Mosquito homolog of the La autoantigen binds to Sindbis virus RNA. J Virol. 1996 Feb;70(2):1173–1181. doi: 10.1128/jvi.70.2.1173-1181.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Peränen J. Localization and phosphorylation of Semliki Forest virus non-structural protein nsP3 expressed in COS cells from a cloned cDNA. J Gen Virol. 1991 Jan;72(Pt 1):195–199. doi: 10.1099/0022-1317-72-1-195. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Rikkonen M., Peränen J., Käriäinen L. ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2. J Virol. 1994 Sep;68(9):5804–5810. doi: 10.1128/jvi.68.9.5804-5810.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rikkonen M., Peränen J., Käriäinen L. Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2. Virology. 1992 Aug;189(2):462–473. doi: 10.1016/0042-6822(92)90570-f. [DOI] [PubMed] [Google Scholar]
  36. Rokeach L. A., Haselby J. A., Meilof J. F., Smeenk R. J., Unnasch T. R., Greene B. M., Hoch S. O. Characterization of the autoantigen calreticulin. J Immunol. 1991 Nov 1;147(9):3031–3039. [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. Alphavirus positive and negative strand RNA synthesis and the role of polyproteins in formation of viral replication complexes. Arch Virol Suppl. 1994;9:393–405. doi: 10.1007/978-3-7091-9326-6_39. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Sawicki D. L., Sawicki S. G. Short-lived minus-strand polymerase for Semliki Forest virus. J Virol. 1980 Apr;34(1):108–118. doi: 10.1128/jvi.34.1.108-118.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Sawicki S. G., Sawicki D. L. The effect of overproduction of nonstructural proteins on alphavirus plus-strand and minus-strand RNA synthesis. Virology. 1986 Jul 30;152(2):507–512. doi: 10.1016/0042-6822(86)90157-1. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. 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]
  49. 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]
  50. Strauss J. H., Strauss E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994 Sep;58(3):491–562. doi: 10.1128/mr.58.3.491-562.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wang Y. F., Sawicki S. G., Sawicki D. L. Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA. J Virol. 1994 Oct;68(10):6466–6475. doi: 10.1128/jvi.68.10.6466-6475.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Wengler G. Studies on the synthesis of viral RNA-polymerase-template complexes in BHK 21 cells infected with Semliki Forest virus. Virology. 1975 Jul;66(1):322–326. doi: 10.1016/0042-6822(75)90202-0. [DOI] [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