Regulation of Sindbis Virus Subgenomic RNA Synthesis

描述(由申请人提供)：辛德比斯病毒(Sindbis Virus，SV)是一种最简单的包膜RNA病毒，是甲病毒属Togaviridae科的原型病毒。它是通过蚊子传播给脊椎动物宿主的。甲型病毒属中的许多病毒是人类疾病的重要原因，如脑炎。几个也可能被认为是可能的生物恐怖主义代理人。复制Sindbis病毒的一个重要步骤是合成一个亚基因组(SG)RNA。这种RNA是病毒三种结构蛋白的信息。该建议的总体目标是研究调节SG RNA合成的方法，以及SG RNA的合成如何与基因组(G)RNA的合成相协调。这两个正链RNA都是由相同的负链RNA模板制成的。到目前为止，几乎所有关于SG RNA合成的研究都是在整个细胞中完成的，并且主要集中在SG启动子的定义上。在我们提出的研究中，我们带来了我们开发的三个新的体外系统，以及一个独特的病毒突变体SV-pzf，它在合成SG RNA方面存在缺陷。体外系统包括：1)将SV转录酶与SG启动子结合的方法；2)依赖于外源启动子-模板(PT)的无细胞体系；3)SG和G RNA均由同一负链PT合成的无细胞体系。这些系统为研究SG RNA合成提供了全新的途径。利用这些系统，我们将不仅检测SG启动子，而且还将检测病毒RNA依赖的RNA聚合酶NSP4的作用，以及NTP浓度对SG RNA合成的影响。我们的第一个目标将是在体外复制在SV-pzf中看到的SG RNA合成的缺陷。我们的实验将使我们能够确定1)NSP4和SG启动子中SV-pzf突变对SG RNA合成缺陷的相对贡献，2)这些突变是否影响转录酶与SG启动子的结合。我们的第二个目标是根据G启动子最大活性所需的茎环结构来表征RNA(-)链RNA的3‘末端序列，并鉴定NSP4中与该启动子结合的氨基酸序列。我们的第三个目标是研究SG和G RNAs合成的协调，并了解这两个RNAs的合成在感染细胞中是如何平衡的。我们将询问SG和G启动子是否在制造这两个RNA的无细胞系统中相互竞争。我们还将研究作为复制酶/转录酶底物的NTP的浓度如何影响SG和G RNA合成之间的平衡。我们所了解到的关于SV SG RNA合成的知识应该适用于其他制造SG RNA的病毒。此外，这一新信息可能有助于将SG RNA合成作为抗病毒治疗的靶点。

DESCRIPTION (provided by applicant): Sindbis virus, (SV) one of the simplest, enveloped RNA viruses, is the prototype virus of the family Togaviridae, genus alphavirus. It is transmitted to its vertebrate hosts by mosquitoes. Many viruses in the genus alphavirus are important causes of human disease, such as encephalitis. Several may also be considered possible bioterrorism agents. An essential step in the replication of Sindbis virus is the synthesis of a subgenomic (SG) RNA. This RNA serves as the message for the three structural proteins of the virus. The overall goal of this proposal is to study the means by which the synthesis of the SG RNA is regulated, and how the synthesis of the SG RNA is coordinated with synthesis of the genomic (G) RNA. Both of these plus-strand RNAs are made off the same minus strand RNA template. Virtually all studies of SG RNA synthesis to date have been done with whole cells, and have focused on definition of the SG promoter. We bring to our proposed studies, three new in vitro systems that we have developed, and a unique viral mutant, SV-pzf, that is deficient in the synthesis of SG RNA. The in vitro systems are 1) an assay for binding of the SV transcriptase to the SG promoter, 2) a cell-free system for making SG RNA which is dependent on addition of an exogenous promoter-template (PT) and 3) a cell-free system in which both SG and G RNA are made from the same minus-strand PT. These systems make possible completely new approaches for the study of SG RNA synthesis. Using these systems, we will examine not only the SG promoter, but also the role of nsP4, the viral RNA-dependent RNA polymerase, and the influence of NTP concentrations on the synthesis of SG RNA. Our first goal will be to reproduce in vitro the defect in SG RNA synthesis seen with SV-pzf. Our experiments will enable us to determine 1) the relative contributions of the SV-pzf mutations in nsP4, and in the SG promoter to the defect in SG RNA synthesis, 2) whether these mutations affect the binding of the transcriptase to the SG promoter. Our second goal is to characterize the 3' terminal sequence of the RNA (-) strand RNA in terms of the stem-loop structures needed for maximum activity of the G promoter, and to identify the amino acid sequence in nsP4 that binds to this promoter. Our third goal is to study the coordination of the synthesis of the SG and G RNAs, and to learn how the synthesis of these two RNAs is balanced in the infected cell. We will ask whether the SG and G promoters compete with each other in a cell-free system which makes both of these RNAs. We will also examine how the concentrations of the NTPs, which are the substrates of the replicase/transcriptase, influence the balance between synthesis of the SG and G RNAs. What we learn about the synthesis of SV SG RNA should have application to other viruses which make SG RNAs. As well, this new information could facilitate the development of SG RNA synthesis as a target for anti-viral therapy.