SINDBIS VIRUS RNA-NEGATIVE MUTANTS THAT FAIL TO CONVERT FROM MINUS-STRAND TO PLUS-STRAND SYNTHESIS - ROLE OF THE NSP2 PROTEIN

We identified mutations in the gene for nsP2, a nonstructural protein of the alphavirus Sindbis virus, that appear to block the conversion o f the initial, short-lived minus-strand replicase complex (RC,,,,,,,) into mature, stable forms that are replicase and transcriptase complex es (RC(stable)), producing 49S genome or 26S mRNA. Base changes at nuc leotide (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 292 6 (C --> U, predicting a change of Leu-416-->Ser) in the nsPZ N domain were responsible for the phenotypes of ts14, ts16, and ts19, members of subgroup II (D. L. Sawicki and S. G. Sawicki, Virology 44:20-34, 19 85) of the A complementation group of Sindbis virus RNA-negative mutan ts. Unlike subgroup I mutants, the RC(stable) formed at 30 degrees C t ranscribed 26S mRNA normally and did not synthesize minus strands in t he absence of protein synthesis after temperature shift. The N-domain substitutions did not inactivate the thiol protease in the C domain of nsPZ and did not stop the proteolytic processing of the polyprotein c ontaining the nonstructural proteins. The distinct phenotypes of subgr oup I and II A complementation group mutants are evidence that the two domains of nsPZ are essential and functionally distinct. A detailed a nalysis of ts14 found that its nsPs were synthesized, processed, trans ported, and assembled at 40 degrees C into complexes with the properti es of RC(initial) and synthesized minus strands for a short time after shift to 40 degrees C. The block in the pathway to the formation of R C(stable) occurred after cleavage of the minus-strand replicase P123 o r P23 polyprotein into mature nsP1, nsPZ, nsP3, and nsP4, indicating t hat structures resembling RC(stable) were formed at 40 degrees C. Howe ver, these RC(stable) or pre-RC(stable) structures were not capable of recovering activity at 30 degrees C. Therefore, failure to increase t he rate of plus-strand synthesis after shift to 40 degrees C appears t o result from failure to convert RC(initial) to RC(stable). We conclud e that RC(stable) is derived from RC(initial) by a conversion process and that ts14 is a conversion mutant. From their similar phenotypes, w e predict that other nsP2 N-domain mutants are blocked also in the con version of RC(initial) to RC(stable). Thus, the N domain of nsPZ plays an essential role in a folding pathway of the nsPs responsible for fo rmation of the initial minus-strand replicase and for its conversion i nto stable plus-strand RNA-synthesizing enzymes.