Reovirus Attachment Mechanisms

呼肠孤病毒附着机制

基本信息

批准号：
9278506
负责人：
TERENCE S. DERMODY
金额：
$ 49.11万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2020-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9278506
关键词：
Affinity Binding Binding Proteins Biological Assay Brain Capsid Carbohydrates Cell surface Cell-Matrix Junction Cells Chloride Ion Complex Cryoelectron Microscopy 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 Neurologic Neurons Oligosaccharides Oncolytic Oncolytic viruses Organ Polysaccharides Primary Infection Process Proteins Reovirus Reovirus Type 1 Research Role Serotyping Sialic Acids Site Specificity Structure Structure-Activity Relationship System Tail Testing Three-Dimensional Image Tropism Vaccines Viral Viral Physiology Viral Vector Virion Virus Virus Diseases Virus Receptors Virus Replication Work X-Ray Crystallography base biophysical properties cell type clinical application defined contribution design junctional adhesion molecule neutralizing monoclonal antibodies receptor receptor binding reconstruction relating to nervous system research study reverse genetics tissue tropism vector vaccine

项目摘要

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蛋白附着介导，该蛋白附着结合受体并指导附着后细胞进入事件，有关这些分子的知识的主要空白包括受体结合促进病毒趋向性的机制和病毒趋向性。在单一蛋白质中协调多种活性的构象变化这项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）来剖析这一过程，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，并阐明初次感染后不同衣壳成分附着到独特受体的机制。在小鼠肠道中，呼肠孤病毒传播到中枢神经系统（CNS），在中枢神经系统中，呼肠孤病毒通过与唾液酸化物的低亲和力结合而表现出血清型特异性差异。聚糖，然后与连接粘附分子-A (JAM-A) 或 Nogo 受体-1 (NgR1) 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。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。提出了三个综合具体目标，以增强对呼肠孤病毒附着机制的了解。在具体目标 1 中，将使用 T1 和 T3 1 蛋白的最小碳水化合物结合区域来确定聚糖参与的贡献。通过反向遗传学设计的嵌合病毒将感染嵌合病毒，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。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、内化和病毒的能力来定义 1 的附着后功能和相关构象变化。将确定 1 与 mAb 复合物中阻止病毒进入的不同步骤的晶体结构，从而为 mAb 介导的感染阻断建立生物物理基础并识别新的病毒。需要 1 构象迁移性的 1 功能域将使用具有工程化二硫键的病毒来定义，以将 1 锁定在不同的构象状态中。将阐明呼肠孤病毒与 NgR1 的相互作用。将使用 X 射线晶体学和冷冻电子显微镜确定结合和感染所需的 NgR1 的序列。了解病毒与细胞受体结合的机制，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。