Vaccinia virus biochemical genetics

描述（由申请人提供）：本项目的目的是了解牛痘病毒感染过程中复制后基因转录的后起始事件的分子机制。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，包括原核生物和真核生物中基因表达调控的重要控制点。我们最近的工作表明，在牛痘病毒感染过程中，复制后mRNA 3'末端的形成是一个协调的过程，涉及转录终止和RNA切割，它与病毒DNA复制偶联，并且它是由病毒和宿主因子以动态复合体介导的。所述因子包括病毒RNA释放因子（A18）、病毒RNA切割/转录/DNA复制因子（H5）、两种病毒正转录延伸因子（G2和J3）以及A18 RNA释放活性和RNA切割所需的至少两种宿主因子。本项目包括两个目标，重点是了解转录终止和内切核糖核酸裂解RNA的机制。终止研究（目的1）涉及A18及其所需宿主因子的机制研究，mRNA切割研究（目的2）涉及由病毒H5蛋白和宿主蛋白组成的核糖核酸内切酶的机制研究。研究中的病毒基因产物在脊椎动物痘病毒中是高度保守的，它们对于病毒复制是必不可少的，它们毫无疑问都在介导复制后基因转录延长和终止中协作，然而它们在这些过程中的确切作用仅被部分理解。本文提出的研究将显著改善我们对这些必需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。结果将提供关键的洞察牛痘病毒基因表达的基本机制，特别是，该系统可能被证明是一个重要的模型，在真核生物中的转录延长的调节研究。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，它推进了我们对真核生物基因表达转录调控的基本机制的理解，这反过来又是我们对疾病理解的正常框架的核心部分。其次，它推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，这对治疗病毒诱导的疾病具有普遍意义。第三，该项目提供了深入了解痘病毒的复制，这是特别感兴趣的公共卫生作为研究工具，作为治疗性蛋白质的来源，作为溶瘤载体，并作为潜在的生物恐怖武器。

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.