Feller J A, Smallwood S, Skiadopoulos M H, Murphy B R, Moyer S A
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida, 32610, USA.
Virology. 2000 Oct 10;276(1):190-201. doi: 10.1006/viro.2000.0535.
The L subunit of the RNA-dependent RNA polymerase of negative strand RNA viruses is believed to possess all the enzymatic activities necessary for viral transcription and replication. Mutations in the L proteins of human parainfluenza virus type 3 (PIV3) and vesicular stomatitis virus (VSV) have been shown to confer temperature sensitivity to the viruses; however, their specific defects have not been determined. Mutant PIV3 L proteins expressed from plasmids were tested for temperature sensitivity in transcription and replication in a minigenome reporter system in cells and for in vitro transcription from purified PIV3 template. The single L mutants, Y942H and L992F, were temperature sensitive (ts) in both assays, although viral RNA synthesis was not completely abolished at the nonpermissive temperature. Surprisingly, the T1558I L mutant was not ts, although its cognate virus was ts. Thus the ts defect in this virus may be due to the abrogation of an essential interaction of the mutant polymerase with a host cell component, which is not measured by the RNA synthesis assays. Most of the combinations of the PIV3 L mutations were not additive and did not show temperature sensitivity in in vitro transcription. Since they were ts in the minigenome assay in vivo, replication appears to be specifically defective. The ts mutations in PIV3 and VSV L proteins were also substituted into the Sendai L protein to compare the defects in related systems. Only Sendai Y942H L was ts in both transcription and replication. One Sendai L mutant, L992F, gave much better replication than transcription. Several other mutants could transcribe but not replicate in vitro, while replication in vivo was normal.
负链RNA病毒的RNA依赖RNA聚合酶的L亚基被认为具有病毒转录和复制所需的所有酶活性。已证明人副流感病毒3型（PIV3）和水疱性口炎病毒（VSV）的L蛋白中的突变会使病毒具有温度敏感性；然而，它们的具体缺陷尚未确定。在细胞中的微型基因组报告系统中测试了从质粒表达的突变型PIV3 L蛋白在转录和复制中的温度敏感性，以及从纯化的PIV3模板进行的体外转录。单个L突变体Y942H和L992F在两种测定中均具有温度敏感性（ts），尽管在非允许温度下病毒RNA合成并未完全消除。令人惊讶的是，T1558I L突变体不具有温度敏感性，尽管其同源病毒具有温度敏感性。因此，该病毒中的温度敏感性缺陷可能是由于突变型聚合酶与宿主细胞成分的关键相互作用被消除，而RNA合成测定未检测到这种相互作用。PIV3 L突变的大多数组合不是累加的，并且在体外转录中未显示温度敏感性。由于它们在体内微型基因组测定中具有温度敏感性，因此复制似乎存在特异性缺陷。PIV3和VSV L蛋白中的温度敏感性突变也被替换到仙台L蛋白中，以比较相关系统中的缺陷。只有仙台Y942H L在转录和复制中均具有温度敏感性。一个仙台L突变体L992F的复制比转录要好得多。其他几个突变体在体外可以转录但不能复制，而在体内复制正常。
