Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses
Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses
复制标题
辛德比斯和塞姆利基森林病毒RNA阴性温度敏感突变体的功能缺陷
作者：
Sirkka;Keranen;Leevi Kaariainen
研究了7个辛德比斯病毒和7个森姆利基森林病毒RNA阴性、对温度敏感的突变体在转变到生长周期中期的限制性温度(39摄氏度)后，RNA和蛋白质合成的缺陷。只有一个突变体，Sindbis病毒的ts-6，互补基团F的代表，显然无法在39摄氏度继续合成RNA，显然是由于温度敏感的聚合酶。该缺陷是可逆的，对42S和26S RNA的合成都有同样的影响，表明这两种RNA的合成都需要相同的聚合酶成分(S)。Sindbis病毒A组的3个突变体之一ts-4和塞姆利基森林病毒的1个RNA+/-突变体ts-10即使在允许的温度下也显示出聚合酶缺陷。在14个RNA阴性突变体中，有7个突变体的26S RNA合成优先减少。Sindbis病毒26S RNA缺陷突变体来自A和G两个不同的互补基团，这表明病毒的两个非结构蛋白(A和G)在调节26S RNA的合成中起着重要作用。由于42S RNA的合成仍在继续，因此在感染后期的RNA聚合中，蛋白A和G的这些功能是不需要的。26S RNA缺失突变体的RNA阴性表型意味着，在感染早期或在功能性聚合酶的组装中，调节这种亚基因组RNA合成的蛋白质必须具有另一种对RNA合成至关重要的功能。几个在26S RNA合成过程中存在特定缺陷的突变体显示出积累了一种大的非结构前体蛋白，其相对分子质量约为20万。在塞姆利基森林病毒和辛德比斯病毒感染的细胞中都发现了一种更大的蛋白质，它可能代表了整个非结构多蛋白。
Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.