Effect of Viral Capsid Stability and Flexibility on Viral Pathogenesis

描述（由申请人提供）：拟议研究的主要目的是确定病毒衣壳的保护功能和基因组传递功能之间的平衡如何影响病毒的发病机制。这一目标将通过使用可处理的哺乳动物呼肠孤病毒系统作为实验模型来实现。在原生状态下，呼肠孤病毒外衣壳的¿1蛋白赋予病毒颗粒稳定性。在进入细胞的过程中，¿1被暴露并裂解以产生¿1 n，¿1和？碎片。这些衍生的多肽被埋藏并与病毒颗粒保持联系。在与宿主膜相互作用后，¿1蛋白经历了一个戏剧性的构象重排，以暴露和释放¿1 n和？释放的多肽在靶膜上形成孔，允许将~70 nm的病毒内衣壳（核心）跨膜递送。提出了三个综合目标，以确定¿1的相互拮抗、保护和基因组传递功能如何平衡，以及这种平衡如何调节病毒疾病。在Aim 1中，将确定呼肠孤病毒衣壳如何不稳定以促进进入细胞所需的构象变化。将确定调节衣壳稳定性和构象灵活性的病毒决定因素。这些机制

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 ¿1 protein of the reovirus outer capsid confers stability to the viral particle. During entry into cells, ¿1 is exposed and cleaved to generate ¿1N, ¿, and ? fragments. These ¿1-derived peptides are buried and remain associated with the virus particle. Upon interaction with host membranes, the ¿1 protein undergoes a dramatic conformational rearrangement to expose and release ¿1N and ?. 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 ¿1 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 ¿1 will be elucidated. The minimal number of pore-forming peptides needed to initiate infection will be determined. Features within ¿1N and ? 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 ¿1 in maintaining capsid stability and allowing genome delivery influence viral pathogenesis will be determined. The capacity of ¿1 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 ¿1 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.