Molecular Basis of Reovirus Pathogenesis

描述（由申请方提供）：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。在新生小鼠的肠道中初次感染后，血清3型呼肠孤病毒通过神经传播并感染神经元，引起致命的脑炎。病毒附着蛋白sigma 1在这些病理事件的进展中起着至关重要的作用。sigma 1蛋白是一种具有头部和尾部形态的丝状三聚体。sigma 1尾部结合唾液酸（SA），而sigma 1头部结合连接粘附分子A（JAM-A），细胞间紧密连接的组成部分。这项研究的主要目的是确定sigma 1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，研究sigma 1受体相互作用的结构和功能特性。在具体目标1中，将确定sigma 1- JAM-A相互作用的序列决定因素。结构引导的突变将被引入到每个分子的推定结合表面中，并且纯化的野生型和突变蛋白的亲和力将通过表面等离子体共振来确定。在补充实验中，将测试表达sigma 1突变形式的腺病毒感染表达JAM-A的细胞的能力。在具体目标2中，将确定呼肠孤病毒感染极化细胞的生理后果。在呼肠孤病毒内化期间，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意义。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sigma 1中的单个氨基酸多态性，呼肠孤病毒株结合SA的能力不同，将研究其在小鼠中的传播途径和神经嗜性。SA在呼肠孤病毒感染神经元中的重要性将通过测试在原代神经元培养物中生长的SA结合能力不同的呼肠孤病毒株来定义。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。将用呼肠孤病毒接种JAM-A表达缺陷的小鼠，并在初级和次级复制位点评估病毒生长和组织病理学。这些研究将对呼肠孤病毒的细胞附着产生精确的理解，并为呼肠孤病毒和其他嗜神经病毒选择细胞靶点并产生疾病的机制提供新的见解。

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.