Functions of the reovirus capsid

这项拟议的研究使用哺乳动物呼肠孤病毒来定义单个衣壳蛋白，μ1， 影响病毒衣壳的不同功能。MRV颗粒由两个同心蛋白质组成 外壳、外壳和内核。μ-1蛋白是外衣壳的主要成分。 已发表的证据和我们的初步数据表明，μ1至少具有三种功能。第一, 尽管这两种蛋白质没有物理接触，但μ1的性质会影响病毒在 一种改变Sigma1附着蛋白在病毒粒子上呈现的方式。第二，两个μ1 在病毒分解过程中产生的多肽，与宿主脂膜合作，以促进 进一步揭开病毒的外衣，使宿主膜通透。第三，μ1影响粒子间 相互作用形成多病毒感染单位。在以下三个目标的帮助下，拟议 研究试图提供对μ1功能的洞察。在目标1中，μ1在维持 装配的保真度将被确定。粒子上的sigma1的位置将由下式确定 生化研究和冷冻电子显微镜。改变力量的贡献 μ-1与相邻衣壳蛋白之间的相互作用对Sigma1呈递的影响将用 基因分析。Sigma1封装和Sigma1构象的变化如何影响病毒复制 活体将使用病毒性疾病的小鼠模型来确定。在目标2中，μ1在 将核心颗粒输送到宿主细胞质将被定义。μ-1多肽如何招募入门 膜的中间体将通过生化和遗传学研究来确定。最小的 成功招募病毒进入中间体所需的μ1多肽数，用于毛孔 将对病毒的形成和成功感染进行量化。病毒进入中间体的结构 将通过低温电子显微镜来确定与膜相关的物质。宿主蛋白 将确定拆解后与衣壳相关并影响感染效率的因素 经亲和纯化和质谱分析。在目标3中，μ1对MVIU形成的贡献将 被指认出来。对颗粒间相互作用重要的区域将通过有限的蛋白质分解来确定 和质谱学。MVIU的形成与混合感染效率的关系 重新分类的频率将被确定。MVIU的形成是否也决定了 体内和IF形成MVIU的决定因素和影响重配子回收的那些因素 后代的关联，将被确定。这项工作的完成将提供对 μ1如何完成这些功能并定义影响容量的μ1属性 在细胞培养中复制，产生重组后代，并引发疾病。

The proposed research uses mammalian reovirus (MRV) to define how a single capsid protein, μ1, influences distinct functions of the viral capsid. MRV particles are comprised of two concentric protein shells, the outer capsid and the inner core. The μ1 protein is a major component of the outer capsid. Published evidence and our preliminary data indicate that μ1 performs at least three functions. First, even though the two proteins are not in physical contact, properties of μ1 influence virus assembly in a way that alters the presentation of the sigma1 attachment protein on the virion. Second, two μ1 peptides, generated during virus disassembly, cooperate with host lipid membranes to facilitate further uncoating of the virus and permeabilize host membranes. Third, μ1 influences inter-particle interactions to form multivirion infectious units. With the aid of the following three Aims, the proposed research seeks to provide insight into the functions of μ1. In Aim 1, the role of μ1 in maintaining assembly fidelity will be determined. The position of sigma1 on the particle will be determined by biochemical studies and cryoelectron microscopy. The contribution of altering the strength of interaction between μ1 and adjacent capsid proteins on the presentation of sigma1 will be evaluated using genetic analysis. How changes to sigma1 encapsidation and sigma1 conformation influence virus replication in vivo will be determined using a mouse model of viral disease. In Aim 2, the function of μ1 in delivering core particles into the host cytoplasm will be defined. How μ1 peptides recruit entry intermediates to membranes will be determined by biochemical and genetic studies. The minimal number of μ1 peptides needed for successful recruitment of a virus entry intermediate, for pore formation and for successful infection will be quantified. The structure of the virus entry intermediate associated with the membrane will be determined by cryoelectron microscopy. Host proteins that associate with capsids following disassembly and influence the efficiency of infection will be identified by affinity purification and mass spectrometry. In Aim 3, the contribution of μ1 to MVIU formation will be identified. Regions important for inter-particle interactions will be determined by limited proteolysis and mass spectrometry. The relationship between MVIU formation, coinfection efficiency, and reassortment frequency will be determined. Whether MVIU formation also determines reassortment in vivo and if the determinants of MVIU formation and those that influence the recovery of reassortant progeny correlate, will be determined. Completion of this work will provide comprehensive insight into how μ1 completes each of these functions and define the properties of μ1 that influence the capacity of MRV to replicate in cell culture, produce reassortant progeny, and elicit disease.