Effect of Viral Capsid Stability and Flexibility on Viral Pathogenesis

描述（由申请方提供）：拟定研究的主要目的是确定病毒衣壳的保护和基因组递送功能之间的平衡如何影响病毒发病机制。这一目标将通过使用易处理的哺乳动物呼肠孤病毒系统作为实验模型来实现。在其天然状态下，呼肠孤病毒外衣壳的μ1蛋白赋予病毒颗粒稳定性。在进入细胞期间，μ1暴露并裂解以产生μ 1 N、δ和μ 1 N片段。这些μ1衍生的肽被掩埋并保持与病毒颗粒结合。在与宿主细胞膜相互作用时，μ1蛋白发生剧烈的构象重排，暴露并释放μ 1 N和μ 1 N。释放的肽在靶膜中形成孔，允许约70 nm的病毒内衣壳（核心）穿过膜递送。提出了三个综合目标来定义μ1的相互拮抗、保护和基因组递送功能如何平衡，以及这种平衡如何调节病毒疾病。在目的1中，将确定呼肠孤病毒衣壳如何去稳定以促进细胞进入所需的构象变化。将鉴定调节衣壳稳定性和构象灵活性的病毒决定簇。的机制 将确定哪些宿主蛋白酶促进病毒衣壳中的构象变化。构象改变的呼肠孤病毒颗粒的亚纳米结构将通过冷冻电子显微镜（cryo-EM）解析。在目标2中，将阐明μ1如何破坏膜。将确定启动感染所需的孔形成肽的最小数量。将定义μ 1 N和μ 1 N内允许这些肽形成孔的特征。将鉴定衣壳内控制呼肠孤病毒进入细胞期间形成的孔的形状和大小的决定因素。将通过cryo-EM测定与模型膜相关的病毒进入中间体的结构。在目标3中，将确定μ1在维持衣壳稳定性和允许基因组递送中的功能如何影响病毒发病机制。将在新生小鼠模型中评价具有改变的稳定性、构象灵活性或基因组递送效率的μ1突变病毒在初始感染位点复制、传播至继发感染位点以及在继发位点复制的能力。将比较μ1突变病毒的毒力。还将确定衣壳稳定性对动物间传播的影响。这些目标的成功完成将定义病毒衣壳的不同功能是如何调节的，提供病毒衣壳在其跨膜转运期间发生的变化的前所未有的快照，并确定病毒衣壳特性，组织嗜性和病毒疾病之间的关系。这项工作将有助于确定无包膜病毒保守的细胞进入途径中的关键控制点，这些关键控制点可以作为抗病毒治疗的潜在靶点。此外，这些研究的结果可以促进更有效的呼肠孤病毒溶瘤剂的开发。

DESCRIPTION (provided by applicant): The primary objective of the proposed research is to determine how the balance between the protective and genome delivery functions of the viral capsid influences viral pathogenesis. The goal will be attained using the tractable mammalian reovirus system as an experimental model. In its native state, the μ1 protein of the reovirus outer capsid confers stability to the viral particle. During entry into cells, μ1 is exposed and cleaved to generate μ1N, δ, and ɸ fragments. These μ1-derived peptides are buried and remain associated with the virus particle. Upon interaction with host membranes, the μ1 protein undergoes a dramatic conformational rearrangement to expose and release μ1N and ɸ. The released peptides form pores in target membranes that allow delivery of the ~70 nm viral inner capsid (core) across membranes. Three integrated aims are proposed to define how the mutually antagonistic, protective and genome delivery functions of μ1 are balanced, and how this balance modulates viral disease. In Aim 1, how the reovirus capsid is destabilized to promote conformational changes required for cell entry will be determined. Viral determinants that modulate capsid stability and conformational flexibility will be identified. The mechanisms by which host proteases promote conformational changes in the viral capsid will be defined. A subnanometer structure of the conformationally-altered reovirus particle will be solved by cryo-electron microscopy (cryo-EM). In Aim 2, how membranes are breached by μ1 will be elucidated. The minimal number of pore-forming peptides needed to initiate infection will be determined. Features within μ1N and ɸ that allow these peptides to form pores will be defined. Determinants within the capsid that control the shape and size of the pore formed by reovirus during cell entry will be identified. Structures of viral entry intermediates in association with model membranes will be determined by cryo-EM. In Aim 3, how functions of μ1 in maintaining capsid stability and allowing genome delivery influence viral pathogenesis will be determined. The capacity of μ1 mutant viruses with altered stability, conformational flexibility, or genome delivery efficiency to replicate at initial sites of infection, disseminate to secondary sites of infection, and replicate at secondary sites will be evaluated in a newborn mouse model. The virulence of μ1 mutant viruses will be compared. The effect of capsid stability on transmission between animals will also be determined. Successful completion of these goals will define how different functions of the viral capsid are regulated, provide unprecedented snapshots of changes occurring in the viral capsid during its transit across the membrane, and identify a relationship between viral capsid properties, tissue tropism, and viral disease. This work will help identify critical control points in the conserved cell entry pathway of nonenveloped viruses that could serve as potential targets for antiviral therapeutics. In addition, results of these studies could foster the development of a more efficacious reovirus oncolytic.