1887

Abstract

Semliki Forest virus (SFV) mutant ts4 has a reversible temperature-sensitive defect in the synthesis the subgenomic 26S mRNA. The viral nonstructural protein nsP2 was identified as a regulator of 26S synthesis by transferring nsP2 coding sequences from ts4 into the infectious SFV cDNA clone (SFoto) to create SFots4. Sequencing identified the causal mutation as C4038U, predicting the amino acid change M781T in nsP2. A revertant was isolated in which a back mutation of U to C restored the wild-type phenotype. Compared to Sindbis virus nsP2 mutants ts15, ts17, ts18, ts24 and ts133, which also exhibit temperature-sensitive 26S RNA synthesis, ts4 and SFots4 reduced 26S RNA synthesis faster and to lower levels after temperature shift. Under these conditions, ts4 and SFots4 also displayed complete conversion of RFII RFIII into RFI and reactivated minus-strand synthesis. After shift to 39 °C, ts4 nsP2 was released from a crude RNA polymerase preparation consisting of membranes sedimenting at 15000 (P15) and the remaining, unreleased nsP2 was capable of being cross-linked in almost equimolar ratio with nsP1 and nsP3. This supports the hypothesis that nsP2 binds directly or undirectly to the promoter for 26S RNA and that it is also an essential component of the viral replicase synthesizing 42S RNA plus strands. Only the former activity is temperature-sensitive in ts4 mutant.

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1998-02-01
2022-05-21
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References

  1. Ahola T., Kääriäinen L. 1995; Reaction in alphavirus mRNA capping : formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP. Proceedings of the National Academy of Sciences, USA 92:507–511
    [Google Scholar]
  2. Ahola T., Laakkonen P., Vihinen H., Kääriäinen L. 1997; Critical residues of Semliki Forest virus RNA capping enzyme involved in methyltransferase and guanylyltransferase-like activities. Journal of Virology 71:392–397
    [Google Scholar]
  3. Barton D. J., Sawicki S. G., Sawicki D. L. 1988; Demonstration in vitro of temperature-sensitive elongation of RNA in Sindbis virus mutant ts6 . Journal of Virology 62:3597–3602
    [Google Scholar]
  4. Birney E., Kumar S., Krainer A. R. 1993; Analysis of the RNA- recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Research 21:5803–5816
    [Google Scholar]
  5. de Groot R. H., Hardy W. R., Shirako Y., Strauss J. H. 1990; Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase : evidence for temporal regulation of polyprotein processing in vivo. EMBO Journal 9:2631–2638
    [Google Scholar]
  6. I., Sawicki S. G., Sawicki D. L. 1996; Sindbis virus RNA- negative mutants that fail to convert from minus-strand to plus-strand synthesis: role of the nsP2 protein. Journal of Virology 70:2706–2719
    [Google Scholar]
  7. Ding M., Schlesinger M. J. 1989; Evidence that Sindbis virus nsP2 is an autoprotease which processes the virus nonstructural polyprotein. Virology 171:280–284
    [Google Scholar]
  8. Grakoui A., Levis R., Raju R., Huang H. V., Rice C. M. 1989; A cis- acting mutation in the Sindbis virus junction region which affects subgenomic RNA synthesis. Journal of Virology 63:5216–5227
    [Google Scholar]
  9. Hahn Y. S., Grakoui A., Rice C. M., Strauss E. G., Strauss J. H. 1989a; Mapping of RNA- temperature-sensitive mutants of Sindbis virus : complementation group F mutants have lesions in nsP4. Journal of Virology 63:1194–1202
    [Google Scholar]
  10. Hahn Y. S., Strauss E. G., Strauss J. H. 1989b; Mapping of RNA temperature-sensitive mutants of Sindbis virus : assignment of complementation groups A, B, and G to nonstructural proteins. Journal of Virology 63:3142–3150
    [Google Scholar]
  11. Kääriäinen L., Söderlund H. 1978; Structure and replication of alphaviruses. Current Topics in Microbiology and Immunology 82:15–69
    [Google Scholar]
  12. Keränen S., Kääriäinen L. 1974; Isolation and basic characterization of temperature-sensitive mutants from Semliki Forest virus. Acta Pathologica et Microbiologica Scandinavica Section B Microbiology 82:810–820
    [Google Scholar]
  13. Keränen S., Kääriäinen L. 1979; Functional defects of RNA- negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses. Journal of Virology 32:19–29
    [Google Scholar]
  14. Laakkonen P., Hyvönen M., Peränen J., Kääriäinen L. 1994; Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli . Journal of Virology 68:7418–7425
    [Google Scholar]
  15. Laakkonen P., Ahola T., Kääriäinen L. 1996; The effects of palmitoylation on membrane association of Semliki Forest virus RNA capping enzyme. Journal of Biological Chemistry 271:28567–28571
    [Google Scholar]
  16. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  17. LaStarza M. W., Lemm J. A., Rice C. M. 1994; Genetic analysis of the nsP3 region of Sindbis virus : evidence for roles in minus-strand and subgenomic RNA synthesis. Journal of Virology 68:5781–5791
    [Google Scholar]
  18. Lemm J. A., Rümenapf T., Strauss E. G., Strauss J. H., Rice C. M. 1994; Polypeptide requirements for assembly of functional Sindbis virus replication complexes : a model for the temporal regulation of minus- and plus-strand RNA synthesis. EMBO Journal 13:2925–2934
    [Google Scholar]
  19. Levis R., Schlesinger S., Huang H. V. 1990; Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription. Journal ofVirology 64:1726–1733
    [Google Scholar]
  20. Li G., LaStarza M. W., Hardy W. R., Strauss J. H., Rice C. M. 1990; Phosphorylation of Sindbis virus nsP3 in vivo and in vitro . Virology 179:416–427
    [Google Scholar]
  21. Liljeström P., Lusa S., Huylebroeck D., Garoff H. 1991; In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6000-molecular-weight membrane protein modulates virus release. Journal of Virology 65:4107–4113
    [Google Scholar]
  22. Mi S., Stollar V. 1991; Expression of Sindbis virus nsPl and methyltransferase activity in Escherichia coli . Virology 184:423–427
    [Google Scholar]
  23. Peränen J., Takkinen K., Kalkkinen N., Kääriäinen L. 1988; Semliki Forest virus-specific non-structural protein nsP3 is a phospho- protein. Journal of General Virology 69:2165–2178
    [Google Scholar]
  24. Pettersson R. F., Söderlund H., Kääriäinen L. 1980; The nucleotide sequences of 57-terminal T1 oligonucleotides of Semliki- Forest-virus 42S and 26S RNAs are different. European Journal of Biochemistry 105:435–443
    [Google Scholar]
  25. Raju R., Huang H. V. 1991; Analysis of Sindbis virus promoter recognition in vivo, using novel vectors with two subgenomic mRNA promoters. Journal of Virology 65:2501–2510
    [Google Scholar]
  26. Ranki M., Ulmanen I., Kääriäinen L. 1979; Semliki Forest virus- specific nonstructural protein is associated with ribosomes. FEBS Letters 108:299–302
    [Google Scholar]
  27. Rikkonen M. 1996; Functional significance of the nuclear-targeting and NTP-binding motifs of Semliki Forest virus nonstructural protein nsP2. Virology 218:352–361
    [Google Scholar]
  28. Rikkonen M., Peränen J., Kääriäinen L. 1992; Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2. Virology 189:462–473
    [Google Scholar]
  29. Rikkonen M., Peränen J., Kääriäinen L. 1994; ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2. Journal of Virology 68:5804–5810
    [Google Scholar]
  30. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  31. Saraste J., Kääriäinen L., Söderlund H., Keränen S. 1977; RNA synthesis directed by a temperature-sensitive mutant of Semliki Forest virus. Journal of General Virology 37:399–406
    [Google Scholar]
  32. Sawicki D. L., Gomatos P. J. 1978; Cleavage defect in the nonstructural polyprotein of Semliki Forest virus has two separate effects on virus RNA synthesis. Journal of General Virology 39:463–473
    [Google Scholar]
  33. Sawicki D. L., Sawicki S. G. 1980; Short-lived minus-strand polymerase for Semliki Forest virus. Journal of Virology 34:108–118
    [Google Scholar]
  34. Sawicki D. L., Sawicki S. G. 1985; Functional analysis of the A complementation group mutants of Sindbis HR virus. Virology 144:20–34
    [Google Scholar]
  35. Sawicki S. G., Sawicki D. L. 1986; The effect of loss regulation of minus-strand RNA synthesis on Sindbis virus replication. Virology 151:339–349
    [Google Scholar]
  36. Sawicki D. L., Sawicki S. G. 1993; A second nonstructural protein functions in the regulation of alphavirus negative-strand RNA synthesis. Journal of Virology 67:3605–3610
    [Google Scholar]
  37. Sawicki D. L., Kääriäinen L., Lambek C., Gomatos P. J. 1978; Mechanism for control of synthesis of Semliki Forest virus 26S and 42S RNA. Journal of Virology 25:19–27
    [Google Scholar]
  38. Sawicki D. L., Sawicki S. G., Keränen S., Kääriäinen L. 1981a; Specific Sindbis virus-coded function for minus-strand RNA synthesis. Journal of Virology 39:348–358
    [Google Scholar]
  39. Sawicki S. G., Sawicki D. L., Kääriäinen L., Keränen S. 1981b; A Sindbis virus mutant temperature-sensitive in the regulation of minus- strand RNA synthesis. Virology 115:161–172
    [Google Scholar]
  40. Sawicki D. L., Barkhimer D. B., Sawicki S. G., Rice C. M., Schlesinger S. 1990; Temperature sensitive shut-off of alphavirus minus strand RNA synthesis maps to a nonstructural protein, nsP4. Virology 174:43–52
    [Google Scholar]
  41. Scheele C. M., Pfefferkorn E. R. 1969; Inhibition of intrajacent (26S) ribonucleic acid synthesis in cells infected with Sindbis virus. Journal of Virology 4:117–122
    [Google Scholar]
  42. Shirako Y., Strauss J. H. 1994; 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. Journal of Virology 68:1874–1885
    [Google Scholar]
  43. Simmons D. T., Strauss J. H. 1972; Replication of Sindbis virus. II. Multiple forms of double-stranded RNA isolated from infected cells. Journal of Molecular Biology 71:615–631
    [Google Scholar]
  44. Strauss J. H., Strauss E. G. 1994; The alphaviruses : gene expression, replication, and evolution. Microbiological Reviews 58:491–562
    [Google Scholar]
  45. Strauss E. G., deGroot R. J., Levinson R., Strauss J. H. 1992; Identification of the active site residues in the nsP2 proteinase of Sindbis virus. Virology 191:932–940
    [Google Scholar]
  46. Wang Y.-F., Sawicki S. G., Sawicki D. L. 1991; Sindbis virus nsP1 functions in negative-strand RNA synthesis. Journal of Virology 65:985–988
    [Google Scholar]
  47. Wang Y.-F., Sawicki S. G., Sawicki D. L. 1994; Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA. Journal of Virology 68:6466–6475
    [Google Scholar]
  48. Wang H.-L., O’Rear J., Stollar V. 1996; Mutagenesis of the Sindbis virus nsP1 protein: effects on methyltransferase activity and viral infectivity. Virology 217:527–531
    [Google Scholar]
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