Functions of the reovirus capsid

拟议的研究使用哺乳动物呼肠孤病毒（MRV）来定义单个衣壳蛋白μ 1， 影响病毒衣壳的不同功能。MRV颗粒由两个同心的蛋白质组成， 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，μ 1至少有三种功能。第一、 即使这两种蛋白质没有物理接触，μ 1的性质也会影响病毒在细胞中的组装。 这是一种改变病毒体上sigma1附着蛋白呈递的方式。第二，两个μ 1 在病毒分解过程中产生的肽与宿主脂膜合作， 进一步使病毒去包被并透化宿主膜。第三，μ 1影响颗粒间 相互作用以形成多病毒体感染单位。在以下三个目标的帮助下， 研究试图提供对μ 1功能的深入了解。在目标1中，μ 1在维持 将确定装配保真度。sigma1在粒子上的位置将由下式确定： 生化研究和冷冻电子显微镜。通过改变 μ 1和相邻衣壳蛋白之间的相互作用对σ 1的呈递将使用 遗传分析sigma1突变和sigma1构象的变化如何影响病毒在大肠杆菌中的复制 将使用病毒性疾病的小鼠模型来确定体内。在目标2中，μ 1在 将核心颗粒递送到宿主细胞质中。μ 1肽如何招募进入 将通过生物化学和遗传学研究确定膜的中间体。最小 成功募集病毒进入中间体所需的μ 1肽的数量， 形成和成功感染将被量化。病毒进入中间体的结构 将通过冷冻电子显微镜测定与膜结合的分子量。宿主蛋白质， 与衣壳结合后拆卸和影响感染的效率将被确定 通过亲和纯化和质谱分析。在目标3中，μ 1对MVIU形成的贡献将 被识别。对颗粒间相互作用重要的区域将通过有限的蛋白水解来确定 和质谱分析。MVIU形成、共感染效率和 将确定重组频率。MVIU的形成是否也决定了 体内，如果MVIU形成的决定因素和那些影响恢复的抑制剂 子子孙孙，将被确定。完成这项工作将提供全面的见解， μ 1如何完成这些功能，并定义影响容量的μ 1属性 MRV在细胞培养物中复制，产生抗性后代，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.