Reovirus Attachment Mechanisms

呼肠孤病毒附着机制

• 批准号: 9272356
• 负责人: TERENCE S. DERMODY
• 金额: $ 41.44万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-07 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9272356
• 关键词: Affinity; Binding; Binding Proteins; Biological Assay; Biophysics; Blocking Antibodies; Brain; Capsid; Carbohydrates; Cell surface; Cell-Matrix Junction; Cells; Chloride Ion; Complex; Cryoelectron Microscopy; Crystallization; Data; Disease; Disulfides; Double Stranded RNA Virus; Engineering; Ependymal Cell; Event; Exhibits; Fiber; Health; Human; Infection; Intestines; Knowledge; Laboratories; Link; Mediating; Membrane Proteins; Molecular Conformation; Monoclonal Antibodies; Mus; Nature; Neuraxis; Neurons; Neutralization Tests; Oligosaccharides; Oncolytic; Oncolytic viruses; Organ; Pathogenicity; Polysaccharides; Primary Infection; Process; Proteins; Reoviridae; Reovirus; Reovirus Type 1; Research; Role; Serotyping; Sialic Acids; Site; Specificity; Structure; Structure-Activity Relationship; System; Tail; Three-Dimensional Image; Tropism; Vaccines; Viral; Viral Physiology; Viral Vector; Virion; Virus; Virus Diseases; Virus Receptors; Virus Replication; Work; X-Ray Crystallography; biophysical properties; cell type; clinical application; defined contribution; design; experimental study; image reconstruction; junctional adhesion molecule; nervous system disorder; neutralizing monoclonal antibodies; public health relevance; receptor; receptor binding; relating to nervous system; reverse genetics; tissue tropism; vector vaccine; virus envelope

 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.

 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。关于这些分子的主要知识空白包括受体结合促进病毒趋向性的机制以及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（一种基因上可控制的 dsRNA 病毒）来剖析特定且成功的病毒受体结合的过程，该病毒在溶瘤和疫苗应用方面显示出前景。将进行实验以确定聚糖结合在病毒向性中的作用，定义受体识别后病毒附着蛋白的功能，并阐明不同衣壳成分附着到独特受体的机制。在小鼠肠道中初次感染后，呼肠孤病毒传播到中枢神经系统（CNS），在那里它表现出血清型特异性的趋向性差异。呼肠孤病毒的附着是通过与唾液酸化聚糖的低亲和力结合开始的，然后是与连接粘附分子-A (JAM-A) 或 Nogo 受体-1 (NgR1) 的高亲和力结合。呼肠孤病毒血清型 1 (T1) 和血清型 3 (T3) 菌株与 JAM-A 和 NgR1 结合，但它们的聚糖利用率有所不同。 1 纤维蛋白结合聚糖和 JAM-A，而 3 衣壳表面蛋白结合 NgR1。提出了三个综合的具体目标来增强对呼肠孤病毒附着机制的了解。在具体目标 1 中，将确定聚糖结合对呼肠孤病毒神经向性的贡献。 T1 和 T3 1 蛋白的最小碳水化合物结合区域将使用反向遗传学设计的嵌合病毒来定义。小鼠将被嵌合病毒感染，以阐明聚糖结合特异性如何将呼肠孤病毒靶向离散的中枢神经系统位点。将鉴定神经元上呼肠孤病毒结合的特定聚糖。在具体目标 2 中，将通过测试针对不同 1 构象的中和单克隆抗体 (mAb) 阻断病毒附着、内化和分解的能力来定义 1 中的附着后功能和相关构象变化。将确定 1 与 mAb 复合物的晶体结构，阻碍病毒进入的不同步骤，从而为 mAb 介导的感染阻断建立生物物理基础，并确定新的 1 功能域。需要 1 构象迁移性的病毒进入步骤将使用具有工程二硫键的病毒来定义，以将 1 锁定在不同的构象状态。在具体目标 3 中，结构基础 将阐明呼肠孤病毒与 NgR1 的相互作用。将定义结合和感染所需的3和NgR1中的序列。 NgR1 与 3 复合物的结构将使用 X 射线晶体学和冷冻电子显微镜确定。这些研究将加深对病毒与细胞受体结合机制的理解，提供有关多功能病毒附着蛋白的新信息，并加速临床应用病毒载体的合理设计。