title: Sindbis virus RNA-negative mutants that fail to convert from minus-strand to plus-strand synthesis: role of the nsP2 protein.
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.
authors.affiliation: Department of Microbiology, Medical College of Ohio, Toledo 43699, USA.
authors.forename: I
authors.initials: I
authors.lastname: Dé
authors.forename: S G
authors.initials: SG
authors.lastname: Sawicki
authors.forename: D L
authors.initials: DL
authors.lastname: Sawicki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005347:Fibroblasts; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D017354:Point Mutation; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic; D012845:Sindbis Virus / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase