title: Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA.
abstract: The alphavirus mutant Sindbis virus HR ts4, which has been assigned to the A complementation group, possessed a selective defect in negative-strand synthesis that was similar although not identical to that observed for the B complementation group mutant ts11 (Y.-F. Wang, S. G. Sawicki, and D. L. Sawicki, J. Virol. 65:985-988, 1991). The causal mutation was identified as a change of a C to a U residue at nucleotide 4903 in the nsP3 open reading frame that predicted a change of Ala-268 to Val. Thus, both nsP3 and nsP1 play a role selectively in the transcription of negative strands early in infection. The assignment of the mutation carried by an A complementation group mutant of Sindbis virus HR to nsP3 was unexpected, as mutations in other A complementation group mutants studied to date mapped to nsP2. Another mutant with a conditionally lethal mutation, ts7 of the G complementation group, also possessed a causal mutation resulting from a single-residue change in nsP3. Negative-strand synthesis ceased more slowly after a shift to the nonpermissive temperature in ts7-than in ts4-infected cells, and ts7 complemented ts11, but ts4 did not. However, the nsP3 of both ts4 and ts7 allowed reactivation of negative-strand synthesis by stable replication complexes containing nsP4 from ts24. Therefore, mutations in nsP3 affected only early events in replication and probably prevent the formation and/or function of the initial replication complex that synthesizes its negative-strand template. Because neither ts4 nor ts7 complemented 10A complementation group mutants, the genes for nsP2 and nsP3 function initially as a single cistron. We interpret these findings and present a model to suggest that the initial alphavirus replication complex is formed from tightly associated nsP2 and nsP3, perhaps in the form of P23, and proteolytically processed and trans-active nsP4 and nsP1.
authors.affiliation: Department of Microbiology, Medical College of Ohio, Toledo 43699.
authors.forename: Y F
authors.initials: YF
authors.lastname: Wang
authors.forename: S G
authors.initials: SG
authors.lastname: Sawicki
authors.forename: D L
authors.initials: DL
authors.lastname: Sawicki
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis*; D005347:Fibroblasts; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D018096:Mononegavirales / Q000378:metabolism*; D016366:Open Reading Frames; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000096:biosynthesis*; D011995:Recombination, Genetic; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012845:Sindbis Virus / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014633:Valine; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C089383:nsp3 protein, alphavirus; D012321:DNA-Directed RNA Polymerases; D014633:Valine; D000409:Alanine