Vaccinia virus biochemical genetics

描述(由申请人提供)：本项目的目标是了解在痘苗病毒感染过程中控制复制后基因转录后启动事件的分子机制。近年来，越来越清楚的是，转录起始后事件，包括转录延伸、终止和RNA切割，是原核生物和真核生物基因表达调控的重要控制点。我们最近的工作表明，痘苗病毒感染过程中复制后mRNA 3‘端的形成是一个涉及转录终止和RNA切割的协同过程，它与病毒DNA复制耦合，并由病毒和宿主因子以动态复合体的形式介导。这些因子包括病毒RNA释放因子(A18)、病毒RNA裂解/转录/DNA复制因子(H5)、两个病毒正转录延伸因子(G2和J3)以及至少两个A18 RNA释放活性和RNA切割所需的宿主因子。该项目包括两个目标，重点是了解转录终止和内源性核溶解RNA切割的机制。终止的研究(目标1)涉及对A18及其所需宿主因子的机制研究，而信使核糖核酸切割(目标2)的研究涉及由病毒H5蛋白和宿主蛋白组成的内切核酸酶的机制研究。研究中的病毒基因产物在脊椎动物痘病毒中高度保守，它们是病毒复制所必需的，它们无疑都在介导复制后基因转录延长和终止过程中协同工作，但它们在这些过程中的确切作用还只有部分了解。这项研究将大大加深我们对这些关键基因在痘病毒复制中的作用的理解，也将影响我们对病毒与宿主细胞之间相互作用的理解。这些结果将为深入了解痘苗病毒基因表达的基本机制提供关键的见解，该系统可能被证明是研究真核生物转录延伸调控的重要模型。公共卫生相关性：该项目在三个层面上与公共卫生相关。首先，它促进了我们对真核转录调控基因表达的基本机制的理解，而这反过来又是我们对疾病理解的正常框架的中心部分。第二，它促进了我们对病毒复制和病毒细胞相互作用的分子机制的理解，这对病毒引起的疾病的治疗具有普遍的指导意义。第三，该项目提供了对痘病毒复制的具体洞察，痘病毒作为研究工具、治疗性蛋白的来源、溶瘤载体和潜在的生物恐怖武器，对公共卫生特别感兴趣。

DESCRIPTION (provided by applicant): The goal of this project is to understand the molecular mechanisms governing post-initiation events in postreplicative gene transcription during vaccinia virus infection. In recent years it has become increasingly clear that post-initiation events in transcription, including transcription elongation, termination and RNA cleavage, comprise important control points for regulation of gene expression in both prokaryotes and eukaryotes. Our recent work suggests that postreplicative mRNA 3' end formation during vaccinia virus infection is a concerted process that involves both transcription termination and RNA cleavage, that it is coupled to viral DNA replication, and that it is mediated by both viral and host factors in a dynamic complex. The factors include a viral RNA release factor (A18), a viral RNA cleavage/transcription/DNA replication factor (H5), two viral positive transcription elongation factors (G2 and J3), and at least two host factors required for A18 RNA release activity and for RNA cleavage. This project comprises two aims, focusing on understanding the mechanism of transcription termination and endoribonucleolytic RNA cleavage. Studies of termination (Aim 1) involve mechanistic studies on A18 and its required host factor, and studies of mRNA cleavage (Aim 2) involve mechanistic studies of an endoribonuclease comprised of the viral H5 protein and host proteins. The viral gene products under study are highly conserved among vertebrate poxviruses, they are essential for virus replication, they all unquestionably collaborate in mediating postreplicative gene transcription elongation and termination, and yet their precise roles in these processes are only partially understood. The research proposed here will significantly refine our understanding of the roles of these essential genes in poxvirus replication, and it will also impact on our understanding of the interaction between the virus and the host cell. The results will provide critical insight into fundamental mechanisms of vaccinia virus gene expression in particular, and the system may prove to be an important model for study of regulation of transcription elongation in eukaryotes in general. PUBLIC HEALTH RELEVANCE: This project is relevant to public health on three levels. First, it advances our understanding of the basic mechanisms of eukaryotic transcriptional regulation of gene expression, which in turn is a central part of the normal framework on which our understanding of disease is built. Second, it advances our understanding of the molecular mechanisms of virus replication and virus cell interactions, which has implications in general for treatment of virus induced disease. Third, the project provides insight specifically into the replication of poxviruses, which are of particular interest to public health as research tools, as a source of therapeutic proteins, as oncolytic vectors, and as potential bioterrorist weapons.