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

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

展开

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（由适用提供）：病毒受体相互作用通常是由结合受体和指导后连接后细胞入学事件的多功能病毒附着蛋白介导的。关于这些分子的知识的关键差距包括受体结合促进病毒tropism和构象变化的机制，这些变化在单个蛋白质中策划了多种活性。拟议中的研究使用依伏病毒，这是一种通用的dsRNA病毒，显示出对溶瘤和疫苗应用的有望，以剖析特定和成功的病毒受体参与过程。将进行实验，以确定聚糖结合在病毒性朝性中的作用，定义受体识别后的病毒附着蛋白的功能，并阐明机制，通过这些机制，不同的衣壳成分附着在独特的受体上。在鼠类肠道中产生原发性感染后，依默病毒将其传播到中枢神经系统（CNS），在中枢神经系统中表现出特定于血清型的特异性差异。小肠羊卷病毒的附着是通过低亲和力与硫化聚糖的结合，然后与连接胶分子-A（JAM-A）或Nogo受体1（NGR1）高亲和力结合。葡萄病毒血清型1（T1）和血清型3（T3）菌株与JAM-A和NGR1结合，但它们在聚糖利用率方面有所不同。 1纤维蛋白结合了聚糖和果酱-A，而3capsid-Surface蛋白结合NgR1。提出了三个集成的特定目的，以增强依孢病毒依恋机制的知识。在特定的目标1中，将确定聚糖参与对孢子病毒神经元热的贡献。 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异病毒靶向离散的中枢神经系统位点。将鉴定由e依病毒在神经元上绑定的特定聚糖。在特定的目标2中，将通过测试中和单克隆抗体（MAB）来定义1中的相关函数和相关的会议变化，该抗体（MAB）针对不同的1构型，以阻止病毒附着，内在化和拆卸的能力。将确定阻碍病毒进入中不同步骤的MAB复合物中的1的晶体结构，以建立MAB介导的感染阻滞的生物物理基础，并识别新的1功能域。需要使用二硫化桥的病毒来定义需要1会议活动的病毒进入步骤，以在不同的会议状态下锁定1。在特定目标3中，结构性基础将与NGR1相互作用将被阐明。将定义结合和感染所需的3和NGR1中的序列。使用X射线晶体学和冷冻电子显微镜确定与3复合物中NGR1的结构。这些研究将增强对病毒接触细胞受体的机制的理解，为多功能病毒附着蛋白提供新的信息，并加快病毒载体在临床应用中的合理设计。