Regulation of Sindbis Virus Subgenomic RNA Synthesis

描述（由申请人提供）：Sindbis病毒（SV）是最简单的包膜RNA病毒之一，是托加病毒科甲病毒属的原型病毒。它通过蚊子传播给脊椎动物宿主。甲病毒属的许多病毒是人类疾病（如脑炎）的重要病因。其中一些也可能被认为是可能的生物恐怖主义制剂。Sindbis病毒复制的一个重要步骤是合成亚基因组（SG） RNA。这种RNA为病毒的三种结构蛋白提供信息。本研究的总体目标是研究SG RNA的合成是如何被调控的，以及SG RNA的合成是如何与基因组(G) RNA的合成协调的。这两种正链RNA都是由相同的负链RNA模板合成的。迄今为止，几乎所有关于SG RNA合成的研究都是在全细胞中完成的，并且主要集中在SG启动子的定义上。我们提出的研究，我们已经开发了三个新的体外系统，和一个独特的病毒突变体，SV-pzf，缺乏SG RNA的合成。体外系统是1)SV转录酶与SG启动子结合的实验，2)依赖外源启动子模板（PT）合成SG RNA的无细胞系统，以及3)SG和G RNA由相同的负链PT合成的无细胞系统。这些系统为研究SG RNA合成提供了全新的方法。利用这些系统，我们不仅将研究SG启动子，还将研究nsP4（病毒RNA依赖的RNA聚合酶）的作用，以及NTP浓度对SG RNA合成的影响。我们的第一个目标是在体外重现SV-pzf在SG RNA合成中的缺陷。我们的实验将使我们能够确定1)nsP4和SG启动子中的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最大活性G启动子所需的茎环结构来表征RNA（-）链RNA的3'端序列，并确定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 rna合成的协调性，了解这两种rna的合成在感染细胞中是如何平衡的。我们会问SG启动子和G启动子是否在无细胞系统中相互竞争，从而产生这两种rna。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。我们对SV - SG RNA合成的了解应该可以应用于其他制造SG RNA的病毒。此外，这一新的信息可以促进SG RNA合成作为抗病毒治疗靶点的发展。

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.