Vaccinia virus genetics and morphogenesis

描述(由申请人提供)：本提案的目标是了解痘苗病毒核心的结构和组装。核心包含病毒DNA和至少50种病毒蛋白质。电子显微镜可区分核心亚结构，包括侧体、核心壁和核心腔内的管状结构，但对每个亚结构的确切蛋白质组成、核心区内单个蛋白质的功能或单个核蛋白在病毒形态发生过程中核心组装中的作用知之甚少。该项目将专注于九种核心蛋白的子集，分为四类：1)切割主要结构病毒粒子蛋白(I7L)所需的半胱氨酸蛋白酶，2)四种可能的核心壁蛋白(A3，A4，A10，F17)，3)两种可能的管蛋白(H4，L4)，以及两种可能的核心管状蛋白(E6和E8)。在一个目标中，将通过感染野生型病毒或携带特定核心结构蛋白突变的病毒，并通过电子显微镜和免疫荧光共聚焦显微镜分析感染，来研究痘病毒核心的组装。不仅将监测感染形成的特定结构，还将监测每种蛋白质和病毒DNA在病毒工厂内的分布。这些研究将揭示在形态形成过程中核心的各个组成部分是如何被运输的，以及每个组成部分如何对最终核心结构的演变做出贡献。第二个目的是通过对野生型病毒粒子或携带特定核心结构蛋白突变的病毒粒子进行受控降解，并通过电子显微镜、凝胶电泳法和Western blotting分析病毒粒子及其降解产物，来研究痘病毒粒子的核心结构。病毒粒子亚结构将用一组针对假设包含在每个主要病毒粒子亚结构中的蛋白质的抗体来探测，包括侧体、核心壁、管子和管腔。以这种方式分析野生型病毒粒子将揭示特定蛋白质在特定核心亚结构中的定位。对突变病毒粒子的分析将揭示特定核心亚结构中的缺陷，从而加强对野生型病毒粒子的研究，并提供关于核心结构中每个蛋白质功能的信息。 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，它促进了我们对蛋白质运输和大分子结构组装的基本机制的理解，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 proposal is to understand the structure and assembly of the vaccinia virion core. The core contains the viral DNA and at least 50 viral proteins. Core substructures can be distinguished by electron microscopy, including lateral bodies, a core wall, and a tubular structure in the lumen of the core, however very little is known about the precise protein composition of each substructure, the function of the individual proteins within the core, or the role of individual core proteins in core assembly during virus morphogenesis. The project will focus on a subset of nine core proteins that break down into four categories: 1) a cysteine protease required for cleavage of the major structural virion proteins (I7L), 2) four putative core wall proteins (A3, A4, A10, F17), 3) two putative tube proteins (H4, L4), and two putative core lumenal proteins (E6 and E8). In one aim the assembly of the pox virion core will be investigated by performing infections with wild type virus or virus bearing mutations in specific core structural proteins, and analyzing the infections by electron microscopy and immunofluorescence confocal microscopy. Infections will be monitored not only for the specific structures formed but also for the distribution within virus factories of each of the proteins and the viral DNA. These studies will reveal how individual components of the core are trafficked during morphogenesis, and how each component contributes to the evolution of the final core structure. In a second aim the structure of the pox virion core will be investigated by performing controlled degradation of wild type virions or virions bearing mutations in specific core structural proteins, and analyzing the virions and their degradation products by electron microscopy, gel electrophoresis and western blotting. Virion substructures will be probed with a panel of antibodies specific for proteins hypothetically contained in each of the major virion substructures, including lateral bodies, core wall, tube and lumen. Analysis of wild type virions in this fashion will reveal the localization of specific proteins within specific core substructures. Analysis of the mutant virions will reveal defects in specific core substructures, thus reinforcing assignments made from studies of wild type virions, and providing information on the function of each protein within the core structure. PUBLIC HEALTH RELEVANCE: This project is relevant to public health on three levels. First, it advances our understanding of the basic mechanisms of protein trafficking and assembly of macromolecular structures, 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.