Requirement for the Amino-Terminal Domain of Sindbis Virus nsP4 during Virus Infection
Requirement for the Amino-Terminal Domain of Sindbis Virus nsP4 during Virus Infection
复制标题
DOI：
10.1128/jvi.02058-10
复制
发表时间：
2011-04-01
影响因子：
5.4
通讯作者：
Hardy, Richard W.
中科院分区：
文献类型：
--
作者：
Rupp, Jonathan C.;Jundt, Natasha;Hardy, Richard W.
信德比斯病毒依赖RNA的RNA聚合酶nsP4具有甲病毒特有的氨基末端区域，预计该区域是紊乱的。为了确定该区域在甲病毒复制过程中的重要性，引入了29个突变，并评估了由此产生的病毒的生长缺陷。三个小斑块突变体D41A、G83L和三突变体GPG((8-10)) VAV分别在亚基因组合成、负链合成和病毒RNA合成的总体水平上存在缺陷。选择在BHK-21细胞中传代的大斑块病毒，并对其基因组进行测序。在nsP1、nsP2和nsP3中发现了恢复病毒RNA合成的抑制突变。nsP2从M282变为L， nsP3从H99变为N，纠正了d41a诱导的亚基因组RNA合成缺陷。nsP1， I351至V， I388至V，或N374至H （C. L. Fata， S. G. Sawicki， D. L. Sawicki， J. Virol. 76: 8641-8649, 2002）的三个变化抑制了负链合成缺陷。直接还原到8位G降低了GPG(8-10) VAV病毒的RNA合成缺陷。这些结果表明，在不同功能的RNA合成复合物中，nsP4的氨基末端结构域参与与其他nsp的不同相互作用，并且该结构域的灵活性对病毒RNA合成很重要。此外，突变病毒无法有效抑制宿主蛋白合成表明nsP4在调控宿主细胞基因表达中起作用。
The Sindbis virus RNA-dependent RNA polymerase nsP4 possesses an amino-terminal region that is unique to alphaviruses and is predicted to be disordered. To determine the importance of this region during alphavirus replication, 29 mutations were introduced, and resultant viruses were assessed for growth defects. Three small plaque mutants, D41A, G83L, and the triple mutant GPG((8-10)) VAV, had defects in subgenome synthesis, minus-strand synthesis, and overall levels of viral RNA synthesis, respectively. Large plaque viruses were selected following passage in BHK-21 cells, and the genomes of these were sequenced. Suppressor mutations in nsP1, nsP2, and nsP3 that restored viral RNA synthesis were identified. An nsP2 change from M282 to L and an nsP3 change from H99 to N corrected the D41A-induced defect in subgenomic RNA synthesis. Three changes in nsP1, I351 to V, I388 to V, or the previously identified change, N374 to H (C. L. Fata, S. G. Sawicki, and D. L. Sawicki, J. Virol. 76: 8641-8649, 2002), suppressed the minus-strand synthetic defect. A direct reversion back to G at position 8 reduced the RNA synthesis defect of the GPG(8-10) VAV virus. These results imply that nsP4's amino-terminal domain participates in distinct interactions with other nsPs in the context of differentially functioning RNA synthetic complexes, and flexibility in this domain is important for viral RNA synthesis. Additionally, the inability of the mutant viruses to efficiently inhibit host protein synthesis suggests a role for nsP4 in the regulation of host cell gene expression.