Mansell E A, Patton J T
Department of Microbiology and Immunology, University of Miami School of Medicine, Florida 33101.
J Virol. 1990 Oct;64(10):4988-96. doi: 10.1128/JVI.64.10.4988-4996.1990.
Temperature-sensitive mutants of simian rotavirus SA11 were previously developed and organized into 10 of a possible 11 recombination groups on the basis of genome reassortment studies. Two of these mutants, tsF and tsG, map to genes encoding VP2 (segment 2) and VP6 (segment 6), respectively. To gain insight into the role of these proteins in genome replication, MA104 cells were infected with tsF or tsG and then maintained at permissive temperature (31 degrees C) until 9 h postinfection, when some cells were shifted to nonpermissive temperature (39 degrees C). Subviral particles (SVPs) were recovered from the infected cells at 10.5 and 12 h postinfection and assayed for associated replicase activity in a cell-free system shown previously to support rotavirus genome replication in vitro. The results showed that the level of replicase activity associated with tsF SVPs from cells shifted to nonpermissive temperature was ca. 20-fold less than that associated with tsF SVPs from cells maintained at permissive temperature. In contrast, the level of replicase activity associated with tsG SVPs from cells maintained at nonpermissive temperature was only slightly less (twofold or less) than that associated with tsG SVPs from cells maintained at permissive temperature. Analysis of the structure of replicase particles from tsG-infected cells shifted to nonpermissive temperature showed that they were similar in size and density to virion-derived core particles and contained the major core protein VP2 but lacked the major inner shell protein VP6. Taken together, these data indicate that VP2, but not VP6, is an essential component of enzymatically active replicase particles.
猿猴轮状病毒SA11的温度敏感突变体先前已被培育出来，并根据基因组重配研究，在可能的11个重组组中被归为10个组。其中两个突变体，tsF和tsG，分别定位于编码VP2（第2节段）和VP6（第6节段）的基因上。为深入了解这些蛋白质在基因组复制中的作用，用tsF或tsG感染MA104细胞，然后在允许温度（31摄氏度）下培养至感染后9小时，此时将一些细胞转移至非允许温度（39摄氏度）。在感染后10.5小时和12小时从感染细胞中回收亚病毒颗粒（SVP），并在先前已证明能支持轮状病毒基因组体外复制的无细胞系统中检测其相关的复制酶活性。结果显示，从转移至非允许温度的细胞中获得的tsF SVP的复制酶活性水平，比从在允许温度下培养的细胞中获得的tsF SVP的复制酶活性水平低约20倍。相比之下，从在非允许温度下培养的细胞中获得的tsG SVP的复制酶活性水平，仅比从在允许温度下培养的细胞中获得的tsG SVP的复制酶活性水平略低（两倍或更低）。对转移至非允许温度的tsG感染细胞的复制酶颗粒结构分析表明，它们在大小和密度上与病毒体衍生的核心颗粒相似，含有主要核心蛋白VP2，但缺乏主要内壳蛋白VP6。综上所述，这些数据表明VP2而非VP6是具有酶活性的复制酶颗粒的必需成分。
