A cis-acting mutation in the Sindbis virus junction region which affects subgenomic RNA synthesis (original) (raw)

Abstract

The synthesis of Sindbis virus minus-strand and genomic and subgenomic RNAs is believed to require specific cis-acting sequences or structures in the template RNAs and a combination of virus-specific proteins and host components which act in trans. A conserved sequence of about 21 nucleotides in the junction region and encompassing the start site for the subgenomic RNA has been proposed to function as the promoter on the minus-strand template for synthesis of the subgenomic RNA (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982). We introduced a three-base insertion in this sequence, which also inserts a single amino acid near the COOH terminus of nsP4, in a cDNA clone of Sindbis virus from which infectious RNA transcripts can be generated. The phenotype of this mutant, called Toto1100CR4.1, was studied after RNA transfection of chicken embryo fibroblasts or BHK cells. The mutation leads to a drastic reduction in the level of the subgenomic RNA but does not alter the start site of the RNA. Probably as a consequence of depressed structural-protein synthesis, very few progeny virions are released and the mutant makes tiny or indistinct plaques even after prolonged incubation. The cis-acting effect of this mutation was demonstrated by incorporating either a wild-type or mutant junction region into a defective-interfering RNA and examining the relative synthesis of defective-interfering RNA-derived subgenomic RNA in vivo in the presence of wild-type helper virus. These results show that the junction region is recognized by yet unidentified viral trans-acting components for subgenomic RNA synthesis. When the Toto1100CR4.1 mutant was passaged in culture, plaque morphology variants readily arose. A total of 24 independent revertants were isolated, and 16 were characterized in detail. All revertants analyzed showed an increase in the level of subgenomic RNA synthesis. Sequence analysis of the junction region showed that all were pseudorevertants, with only two containing potentially compensating changes in the junction region. An assay was developed to identify revertants with second-site changes in trans-acting viral components involved in subgenomic RNA synthesis. At least two such revertants were identified. Mapping of these and other second-site compensating mutations may provide genetic clues as to which virus-specific protein(s) is responsible for interaction with the conserved junction region to promote subgenomic RNA synthesis.

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  1. BORNSTEIN M. B. Reconstituted rattail collagen used as substrate for tissue cultures on coverslips in Maximow slides and roller tubes. Lab Invest. 1958 Mar-Apr;7(2):134–137. [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. Boros I., Pósfai G., Venetianer P. High-copy-number derivatives of the plasmid cloning vector pBR322. Gene. 1984 Oct;30(1-3):257–260. doi: 10.1016/0378-1119(84)90130-6. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Burge B. W., Pfefferkorn E. R. Functional defects of temperature-sensitive mutants of Sindbis virus. J Mol Biol. 1968 Jul 14;35(1):193–205. doi: 10.1016/s0022-2836(68)80047-6. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Chang G. J., Trent D. W. Nucleotide sequence of the genome region encoding the 26S mRNA of eastern equine encephalomyelitis virus and the deduced amino acid sequence of the viral structural proteins. J Gen Virol. 1987 Aug;68(Pt 8):2129–2142. doi: 10.1099/0022-1317-68-8-2129. [DOI] [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. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hahn C. S., Rice C. M., Strauss E. G., Lenches E. M., Strauss J. H. Sindbis virus ts103 has a mutation in glycoprotein E2 that leads to defective assembly of virions. J Virol. 1989 Aug;63(8):3459–3465. doi: 10.1128/jvi.63.8.3459-3465.1989. [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., 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]
  14. 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]
  15. Kinney R. M., Johnson B. J., Brown V. L., Trent D. W. Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins. Virology. 1986 Jul 30;152(2):400–413. doi: 10.1016/0042-6822(86)90142-x. [DOI] [PubMed] [Google Scholar]
  16. Lehtovaara P., Söderlund H., Keränen S., Pettersson R. F., Käriäinen L. 18S defective interfering RNA of Semliki Forest virus contains a triplicated linear repeat. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5353–5357. doi: 10.1073/pnas.78.9.5353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Lustig S., Jackson A. C., Hahn C. S., Griffin D. E., Strauss E. G., Strauss J. H. Molecular basis of Sindbis virus neurovirulence in mice. J Virol. 1988 Jul;62(7):2329–2336. doi: 10.1128/jvi.62.7.2329-2336.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lutz C. T., Hollifield W. C., Seed B., Davie J. M., Huang H. V. Syrinx 2A: an improved lambda phage vector designed for screening DNA libraries by recombination in vivo. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4379–4383. doi: 10.1073/pnas.84.13.4379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Monroe S. S., Schlesinger S. Common and distinct regions of defective-interfering RNAs of Sindbis virus. J Virol. 1984 Mar;49(3):865–872. doi: 10.1128/jvi.49.3.865-872.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ou J. H., Rice C. M., Dalgarno L., Strauss E. G., Strauss J. H. Sequence studies of several alphavirus genomic RNAs in the region containing the start of the subgenomic RNA. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5235–5239. doi: 10.1073/pnas.79.17.5235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ou J. H., Strauss E. G., Strauss J. H. Comparative studies of the 3'-terminal sequences of several alpha virus RNAs. Virology. 1981 Mar;109(2):281–289. doi: 10.1016/0042-6822(81)90499-2. [DOI] [PubMed] [Google Scholar]
  26. Ou J. H., Strauss E. G., Strauss J. H. The 5'-terminal sequences of the genomic RNAs of several alphaviruses. J Mol Biol. 1983 Jul 25;168(1):1–15. doi: 10.1016/s0022-2836(83)80319-2. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Polo J. M., Davis N. L., Rice C. M., Huang H. V., Johnston R. E. Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro. J Virol. 1988 Jun;62(6):2124–2133. doi: 10.1128/jvi.62.6.2124-2133.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Rice C. M., Strauss J. H. Synthesis, cleavage and sequence analysis of DNA complementary to the 26 S messenger RNA of Sindbis virus. J Mol Biol. 1981 Aug 15;150(3):315–340. doi: 10.1016/0022-2836(81)90550-7. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. 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]
  39. Wieslander L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem. 1979 Oct 1;98(2):305–309. doi: 10.1016/0003-2697(79)90145-3. [DOI] [PubMed] [Google Scholar]
  40. 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]