A second nonstructural protein functions in the regulation of alphavirus negative-strand RNA synthesis.
Previous studies (D.L. Sawicki, D. B. Barkhimer, S. G. Sawicki, C. M. Rice, and S. Schlesinger, Virology 174:43-52, 1990) identified a temperature-sensitive (ts) defect in Sindbis virus nonstructural protein 4 (nsP4) that reactivated negative-strand synthesis after its normal cessation at the end of the early phase of replication. We now report identification of two different ts alterations in nsP2 of Ala-517 to Thr in ts17 or Asn-700 to Lys in ts133 that also reactivated negative-strand synthesis. These same mutations caused severely reduced protease processing by nsP2 and recognition of the internal promoter for subgenomic mRNA synthesis and were responsible for the conditional lethality and RNA negativity of these mutants. Reactivation of negative-strand synthesis by mutations in nsP2 resembled that in nsP4: it was a reversible property of stable replication complexes and did not require continuation of viral protein synthesis. Recombinant viruses expressing both mutant nsP2 and nsP4 reactivated negative-strand synthesis more efficiently than did either mutant protein alone, consistent with the hypothesis that both nsP2 and nsP4 participate in template recognition. We propose that these alterations cause nsP2 and nsP4 to switch from their normal preference to recognize negative strands as templates to recognize positive strands and thereby mimic the initial formation of a replication complex.
Department of Microbiology, Medical College of Ohio, Toledo 43699-0008.
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D000524:Alphavirus / Q000235:genetics* / Q000254:growth & development; D000595:Amino Acid Sequence; D002874:Chromosome Mapping; D003546:Cysteine Endopeptidases / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D008969:Molecular Sequence Data; D017354:Point Mutation; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis*; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D012845:Sindbis Virus / Q000235:genetics* / Q000254:growth & development; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D012333:RNA, Messenger; D012367:RNA, Viral; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; C499947:nsP4 protein, Sindbis virus; D012321:DNA-Directed RNA Polymerases; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase