Host factors in the alphavirus replication complex

描述(由申请人提供)：由Togaviridae内的甲型病毒属成员感染人类和其他哺乳动物可导致发烧、皮疹、关节炎、脑炎和死亡。几种甲型病毒已被疾病控制和预防中心指定为可能用于生物恐怖主义的B类令人担忧的制剂，并被NIAID指定为优先病原体。目前还没有针对这些病原体引起的疾病的特效药。 我们正在研究甲型病毒的复制，长期目标是确定潜在的治疗靶点。复制传入的正义基因组RNA，发生在含有病毒非结构蛋白(NSP)的膜相关复合体中，首先产生负链拷贝，然后产生新的后代正链RNA。我们推测，除了负链和正链复制酶病毒NSP组分的已知差异外，不同宿主因子的招募促进了它们不同的功能。我们还假设甲型病毒利用宿主因子招募的共同策略来复制它们各自的基因组。 利用表达标记NSP3蛋白的突变体，我们建议使用免疫学技术从三种典型的甲型病毒，辛德比斯病毒，罗斯河病毒和委内瑞拉马脑炎病毒中分离正链和负链复制酶。复制复合体中存在的宿主因子将通过蛋白质组学技术进行鉴定。聚焦于甲型病毒正链和/或负链复制酶复合体中常见的因子，我们将使用共聚焦荧光显微镜验证每个因子与完整细胞中复制酶的关联。使用分子和生化方法，我们将评估相关宿主成分对病毒复制的影响，并绘制宿主因子的蛋白质-蛋白质(或蛋白质-RNA)相互作用图。了解甲型病毒正链和负链复制酶的组成和分子相互作用将有助于未来探索这些重要病原体的抑制剂。

DESCRIPTION (provided by applicant): Infection of humans and other mammals by members of the Alphavirus genus within the Togaviridae can result in fever, rash, arthritis, encephalitis and death. Several alphaviruses have been designated by the Centers for Disease Control and Prevention as Category B agents of concern for possible use in bioterrorism, and as Priority Pathogens by the NIAID. There is currently no specific treatment available for diseases caused by these pathogens. We are studying Alphavirus replication with the long-term goal of identifying potential therapeutic targets. Replication of incoming positive-sense genomic RNA, which occurs in membrane-associated complexes containing the viral nonstructural proteins (nsPs), first generates a minus strand copy, followed by new progeny plus strand RNAs. We hypothesize that in addition to known differences in the viral nsP components of the minus and plus strand replicases, that recruitment of different host factors facilitates their disparate functions. We also hypothesize that alphaviruses utilize a common strategy of host factor recruitment to replicate their respective genomes. Using mutants expressing a tagged nsP3 protein, we propose to use immunological techniques to isolate the plus and minus strand replicases from three representative alphaviruses, Sindbis virus, Ross River virus, and Venezuelan equine encephalitis virus. The host factors present in the replication complexes will be identified by proteomic techniques. Focusing on factors commonly present in the Alphavirus plus and/or minus strand replicase complexes, we will verify each factor's association with the replicase in intact cells using confocal fluorescence microscopy. Using molecular and biochemical approaches, we will assess the effects of associated host components on viral replication and map the protein-protein (or protein-RNA) interactions of the host factors. Understanding the components of and molecular interactions within the Alphavirus minus and plus strand replicases will facilitate future exploration for inhibitors of these important pathogens.