Reovirus Attachment Mechanisms

 描述（由申请人提供）：病毒-受体相互作用通常由多功能病毒附着蛋白介导，该蛋白结合受体并指导附着后细胞进入事件。关于这些分子的知识的关键差距包括受体结合促进病毒向性的机制和在单个蛋白质中协调多种活性的构象变化。拟议的研究使用呼肠孤病毒，一种遗传上易于处理的dsRNA病毒，显示出溶瘤和疫苗应用的前景，以剖析特异性和成功的病毒受体接合过程。将进行实验以确定聚糖结合在病毒嗜性中的作用，确定受体识别后病毒附着蛋白的功能，并阐明不同衣壳组分附着于独特受体的机制。在小鼠肠道中的原发感染后，呼肠孤病毒传播到中枢神经系统（CNS），在中枢神经系统中，它表现出嗜性的类型特异性差异。呼肠孤病毒附着通过与唾液酸化聚糖的低亲和力结合，随后与连接粘附分子-A（JAM-A）或Nogo受体-1（NgR 1）的高亲和力结合来启动。呼肠孤病毒血清型1（T1）和血清型3（T3）毒株与JAM-A和NgR 1结合，但它们的聚糖利用不同。β 1纤维蛋白结合聚糖和JAM-A，而β 3衣壳表面蛋白结合NgR 1。提出了三个综合的具体目标，以提高呼肠孤病毒附着机制的知识。在具体目标1中，将确定聚糖结合对呼肠孤病毒神经嗜性的贡献。T1和T3 β 1蛋白的最小碳水化合物结合区域将使用通过反向遗传学工程化的嵌合病毒来定义。将用嵌合病毒感染小鼠，以阐明聚糖结合特异性如何将呼肠孤病毒靶向至离散的CNS位点。将鉴定呼肠孤病毒在神经元上结合的特异性聚糖。在特定目标2中，将通过检测对不同BMP 1构象具有特异性的中和性单克隆抗体（mAb）阻断病毒附着、内化和分解的能力来定义BMP 1的附着后功能和相关构象变化。将确定与阻止病毒进入的不同步骤的mAb复合的RISK 1的晶体结构，以建立mAb介导的感染阻断的生物物理基础，并鉴定新的RISK 1功能结构域。将使用具有工程化二硫键的病毒来定义需要α 1构象迁移率的病毒进入步骤，以将α 1锁定在不同的构象状态。在具体目标3中， 将阐明呼肠孤病毒与NgR 1的相互作用。将定义结合和感染所需的NGR 3和NgR 1中的序列。将使用X射线晶体学和低温电子显微镜来确定NgR 1与β 3复合物的结构。这些研究将加深对病毒参与细胞受体的机制的理解，提供有关多功能病毒附着蛋白的新信息，并加速用于临床应用的病毒载体的合理设计。

 DESCRIPTION (provided by applicant): Virus-receptor interactions are often mediated by multifunctional viral attachment proteins that bind receptors and guide post-attachment cell-entry events. Key gaps in knowledge about these molecules include mechanisms by which receptor binding facilitates viral tropism and the conformational changes that orchestrate multiple activities in a single protein. The proposed research uses reovirus, a genetically tractable dsRNA virus that shows promise for oncolytic and vaccine applications, to dissect the process of specific and successful viral receptor engagement. Experiments will be performed to determine the role of glycan binding in viral tropism, define functions of viral attachment protein subsequent to receptor recognition, and elucidate mechanisms by which different capsid components attach to unique receptors. Following primary infection in the murine intestine, reovirus disseminates to the central nervous system (CNS), where it exhibits serotype-specific differences in tropism. Reovirus attachment is initiated by low-affinity binding to sialylated glycans followed by high-affinity binding to either junctional adhesion molecule-A (JAM-A) or Nogo receptor-1 (NgR1). Reovirus serotype 1 (T1) and serotype 3 (T3) strains bind to JAM-A and NgR1, but they vary in glycan utilization. The 1 fiber protein binds glycan and JAM-A, whereas the 3 capsid-surface protein binds NgR1. Three integrated specific aims are proposed to enhance knowledge of reovirus attachment mechanisms. In Specific Aim 1, the contribution of glycan engagement to reovirus neural tropism will be determined. Minimal carbohydrate-binding regions of T1 and T3 1 proteins will be defined using chimeric viruses engineered by reverse genetics. Mice will be infected with chimeric viruses to elucidate how glycan-binding specificity targets reovirus to discrete CNS sites. The specific glycan bound by reovirus on neurons will be identified. In Specific Aim 2, post-attachment functions and associated conformational changes in 1 will be defined by testing neutralizing monoclonal antibodies (mAbs) specific for different 1 conformations for the capacity to block viral attachment, internalization, and disassembly. Crystal structures of 1 in complex with mAbs that impede distinct steps in viral entry will be determined to establish a biophysical basis for mAb-mediated infection blockade and identify new 1 functional domains. Viral entry steps requiring 1 conformational mobility will be defined using viruses with engineered disulfide bridges to lock 1 in different conformational states. In Specific Aim 3, the structural basis of reovirus interactions with NgR1 will be elucidated. Sequences in 3 and NgR1 required for binding and infection will be defined. The structure of NgR1 in complex with 3 will be determined using X-ray crystallography and cryo-electron microscopy. These studies will enhance an understanding of mechanisms by which viruses engage cellular receptors, contribute new information about multifunctional viral attachment proteins, and accelerate the rational design of viral vectors for clinical applications.