Effect of Viral Capsid Stability and Flexibility on Viral Pathogenesis

描述(由申请人提供)：拟议研究的主要目标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。这一目标将使用易于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，呼肠孤病毒外壳的1蛋白为病毒颗粒提供了稳定性。在进入细胞的过程中，？1暴露并被切割，生成？1N、？和？碎片。这些源自1的多肽被掩埋，并与病毒颗粒保持联系。当与宿主膜相互作用时，1蛋白经历戏剧性的构象重排，暴露并释放1N和？释放的多肽在靶膜上形成孔，使~70 nm的病毒内衣壳(核心)能够跨膜输送。提出了三个综合目标来定义病毒1的相互拮抗、保护和基因组传递功能是如何平衡的，以及这种平衡如何调节病毒疾病。在目标1中，将确定呼肠孤病毒衣壳如何不稳定以促进细胞进入所需的构象变化。将确定调节衣壳稳定性和构象灵活性的病毒决定因素。这些机制由 哪些宿主蛋白酶促进病毒衣壳的构象变化将被定义。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)得到解决。在目标2中，1将阐明膜是如何被破坏的。将确定启动感染所需的最小数量的造孔肽。第1N和？中的功能允许这些多肽形成毛孔的物质将被定义。衣壳内控制呼肠孤病毒进入细胞时形成的孔的形状和大小的决定因素将被识别。与模型膜相关的病毒进入中间体的结构将由低温电子显微镜确定。在目标3中，将确定1在维持衣壳稳定和允许基因组传递方面如何影响病毒的致病。具有改变的稳定性、构象灵活性或基因组传递效率的突变病毒在初始感染部位复制、传播到二级感染部位以及在二级感染部位复制的能力将在新生小鼠模型中进行评估。我们将比较1种变异病毒的毒力。衣壳的稳定性对动物间传播的影响也将被确定。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，提供前所未有的病毒衣壳在跨膜运输过程中发生的变化的快照，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，这些控制点可能成为抗病毒治疗的潜在靶点。此外，这些研究的结果可能会促进更有效的呼肠孤病毒溶瘤的发展。

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.