Molecular Basis of Reovirus Pathogenesis

呼肠孤病毒发病机制的分子基础

• 批准号: 7030402
• 负责人: TERENCE S. DERMODY
• 金额: $ 34.41万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7030402
• 关键词: Reoviridae; cell surface receptors; chimeric proteins; laboratory mouse; membrane proteins; molecular cloning; mutant; nervous system infection; protein structure function; receptor binding; tissue /cell culture; virus infection mechanism; virus protein; virus receptors

DESCRIPTION (provided by applicant): Reoviruses provide a well-established experimental model for studies of viral neuropathogenesis. Following primary infection in the intestine of newborn mice, serotype 3 reoviruses spread through nerves and infect neurons, causing lethal encephalitis. Viral attachment protein sigma1 plays a crucial role in the progression of these pathologic events. The sigma1 protein is a filamentous trimer with head and tail morphology. The sigma1 tail binds sialic acid (SA), whereas the sigma1 head binds junctional adhesion molecule A (JAM-A), a component of intercellular tight junctions. The major objective of the proposed research is to determine how interactions between sigma1 and cell-surface receptors lead to organ-specific disease. Four integrated specific aims are proposed to study structural and functional properties of sigma1-receptor interactions. In Specific Aim 1, sequence determinants of sigma1- JAM-A interactions will be identified. Structure-guided mutations will be introduced into putative binding surfaces of each molecule, and affinities of purified wild-type and mutant proteins will be determined by surface plasmon resonance. In complementary experiments, adenoviruses expressing mutant forms of sigma1 will be tested for the capacity to infect JAM-A-expressing cells. In Specific Aim 2, the physiological consequences of reovirus infection of polarized cells will be determined. Intracellular distribution of JAM-A and JAM-A-associated proteins and integrity of tight junctions will be monitored during reovirus internalization. The significance of JAM-A phosphorylation in reovirus attachment and infection will be determined. In Specific Aim 3, the role of SA in reovirus pathogenesis will be defined. Reovirus strains differing in the capacity to bind SA due to a single amino acid polymorphism in sigma1 will be studied for pathways of spread and neural tropism in mice. The importance of SA in reovirus infection of neurons will be defined by testing reovirus strains that differ in SA-binding capacity for growth in primary neuronal cultures. In Specific Aim 4, the role of JAM-A in reovirus pathogenesis will be determined. Mice deficient in expression of JAM-A will be inoculated with reovirus, and viral growth and histopathology will be assessed at primary and secondary sites of replication. These studies will yield a precise understanding of reovirus cell-attachment and provide novel insights into mechanisms by which reoviruses and other neurotropic viruses select cellular targets and produce disease.

描述（由申请人提供）：呼肠孤病毒为病毒神经发病机制的研究提供了完善的实验模型。新生小鼠肠道初次感染后，血清型 3 呼肠孤病毒通过神经传播并感染神经元，引起致命性脑炎。病毒附着蛋白 sigma1 在这些病理事件的进展中起着至关重要的作用。 sigma1 蛋白是一种具有头尾形态的丝状三聚体。 sigma1 尾部结合唾液酸 (SA)，而 sigma1 头结合连接粘附分子 A (JAM-A)，这是细胞间紧密连接的一个组成部分。拟议研究的主要目标是确定 sigma1 和细胞表面受体之间的相互作用如何导致器官特异性疾病。提出了四个综合的具体目标来研究 sigma1-受体相互作用的结构和功能特性。在具体目标 1 中，将鉴定 sigma1-JAM-A 相互作用的序列决定因素。结构引导的突变将被引入每个分子的假定结合表面，并且纯化的野生型和突变蛋白的亲和力将通过表面等离振子共振来确定。在补充实验中，将测试表达 sigma1 突变形式的腺病毒感染 JAM-A 表达细胞的能力。在具体目标 2 中，将确定极化细胞的呼肠孤病毒感染的生理后果。在呼肠孤病毒内化过程中，将监测 JAM-A 和 JAM-A 相关蛋白的细胞内分布以及紧密连接的完整性。将确定 JAM-A 磷酸化在呼肠孤病毒附着和感染中的重要性。在具体目标 3 中，将定义 SA 在呼肠孤病毒发病机制中的作用。由于 sigma1 中的单个氨基酸多态性，呼肠孤病毒株与 SA 的结合能力有所不同，我们将研究其在小鼠中的传播途径和神经向性。 SA 在神经元呼肠孤病毒感染中的重要性将通过测试在原代神经元培养物中与 SA 结合能力不同的呼肠孤病毒株来确定。在具体目标 4 中，将确定 JAM-A 在呼肠孤病毒发病机制中的作用。 JAM-A表达缺陷的小鼠将接种呼肠孤病毒，并在初级和次级复制位点评估病毒生长和组织病理学。这些研究将产生对呼肠孤病毒细胞附着的精确理解，并为呼肠孤病毒和其他嗜神经病毒选择细胞靶标和产生疾病的机制提供新的见解。