Dé I, Sawicki S G, Sawicki D L
Department of Microbiology, Medical College of Ohio, Toledo 43699, USA.
J Virol. 1996 May;70(5):2706-19. doi: 10.1128/JVI.70.5.2706-2719.1996.
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.
我们在辛德毕斯病毒（一种甲病毒）的非结构蛋白nsP2基因中鉴定出了突变，这些突变似乎会阻止初始的、寿命短暂的负链复制酶复合体（RCinitial）转变为成熟、稳定的复制酶和转录酶复合体（RCstable），而后者可产生49S基因组或26S mRNA。nsP2 N结构域中核苷酸（nt）2166（G→A，预测Glu-163→Lys的变化）、nt 2502（G→A，预测Val-275→Ile的变化）和nt 2926（C→U，预测Leu-416→Ser的变化）处的碱基变化导致了辛德毕斯病毒RNA阴性突变体A互补组第11亚组（D.L. Sawicki和S.G. Sawicki，《病毒学》44:20 - 34，1985）中ts14、ts16和ts19成员的表型。与第I亚组突变体不同，在30℃形成的RCstable能正常转录26S mRNA，并且在温度转换后，在无蛋白质合成的情况下不合成负链。N结构域的替换并未使nsP2 C结构域中的巯基蛋白酶失活，也未阻止包含非结构蛋白的多蛋白的蛋白水解加工。第I亚组和第11亚组A互补组突变体的不同表型证明，nsP2的这两个结构域是必需的且功能不同。对ts14的详细分析发现，其nsP在40℃合成、加工、运输并组装成具有RCinitial特性的复合体，并在转换到40℃后短时间内合成负链。在负链复制酶P123或P23多蛋白切割成成熟的nsP1、nsP2、nsP3和nsP4之后，通往RCstable形成的途径出现了阻断，这表明在40℃形成了类似RCstable的结构。然而，这些RCstable或前RCstable结构在30℃时无法恢复活性。因此，转换到40℃后正链合成速率未能增加似乎是由于未能将RCinitial转变为RCstable。我们得出结论，RCstable是通过一个转换过程由RCinitial衍生而来，并且ts14是一个转换突变体。根据它们相似的表型，我们预测其他nsP2 N结构域突变体在RCinitial向RCstable的转换过程中也会被阻断。因此，nsP2的N结构域在nsP的折叠途径中起着至关重要的作用，该途径负责初始负链复制酶的形成及其向稳定的正链RNA合成酶的转变。
