HOST FACTORS ASSOCIATED WITH THE SINDBIS VIRUS RNA-DEPENDENT RNA POLYMERASE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 辛德比斯病毒(SINV)是甲型病毒属的原型成员，其成员会导致严重的人类疾病，目前尚无特效治疗方法。为了确定在SINV RNA基因组复制中重要的宿主因素，我们构建了一个表达NSP4的SINV，NSP4是一种依赖于病毒RNA的RNA聚合酶，带有框内3xFlag表位标签。对从感染标记病毒的细胞中分离的含有NSP4的复合体进行蛋白质组学分析，发现29个相关的宿主蛋白。其中，10个蛋白仅在感染后期(12h)分离，14个蛋白在感染早期和晚期都有关联，5个蛋白仅在早期(感染后6h)分离到。这些结果证明了在感染过程中发生的病毒-宿主相互作用的动态性质，并表明NSP4执行的多种功能可能需要不同的宿主蛋白。在两次感染中都发现了与NSP4相关的两种蛋白，GTPase激活蛋白(SH3结构域)结合蛋白1(G3BP1)和G3BP2，这两种蛋白也被鉴定为与SINV nsP2和NSP3相关。我们展示了这些宿主因素在限制SINV复制事件中可能的重叠作用。本研究还确定了感染后6小时与NSP4相关的10个宿主因素，但未发现与NSP2或NSP3相关。这些因素是在RNA复制过程中发挥重要作用的候选因素。确定复制所必需的主机因素应该会导致中断甲型病毒复制的新策略。 克里斯蒂亚IM，罗兹雅贝克H，莫洛伊KR，卡尔基S，怀特LL，赖斯CM，Rrout MP，Chait BT，Macdonald MR。与Sindbis病毒依赖RNA聚合酶相关的宿主因素：G3BP1和G3BP2在病毒复制中的作用，病毒学杂志。84(2010)6720-32

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3xFlag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host proteins. Of these, 10 proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host proteins may be required for the multiple functions carried out by nsP4. Two related proteins found in association with nsP4 at both times of infection, GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication. Cristea IM, Rozjabek H, Molloy KR, Karki S, White LL, Rice CM, Rout MP, Chait BT, Macdonald MR.Host factors associated with the Sindbis virus RNA-dependent RNA polymerase: a role for G3BP1 and G3BP2 in virus replication, Journal of Virology. 84(2010)6720-32