These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


PUBMED FOR HANDHELDS

Search MEDLINE/PubMed


  • Title: Sindbis virus RNA-negative mutants that fail to convert from minus-strand to plus-strand synthesis: role of the nsP2 protein.
    Author: Dé I, Sawicki SG, Sawicki DL.
    Journal: J Virol; 1996 May; 70(5):2706-19. PubMed ID: 8627744.
    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.
    [Abstract] [Full Text] [Related] [New Search]