Molecular Basis of Reovirus Pathogenesis

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一个成熟的实验模型。新生小鼠肠道原发感染后，3型呼肠孤病毒通过神经传播，感染神经元，导致致死性脑炎。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蛋白是一种具有头部和尾部形态的丝状三聚体。Sigma1尾巴结合唾液酸(SA)，而Sigma1头部结合连接黏附分子A(JAM-A)，JAM-A是细胞间紧密连接的一种成分。这项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。提出了研究Sigma1-受体相互作用的结构和功能特性的四个综合的特定目标。在具体目标1中，将确定sigma1-JAM-A相互作用的序列决定因素。结构导向突变将被引入到每个分子的假定结合表面，纯化的野生型和突变蛋白的亲和力将通过表面等离子共振来确定。在补充实验中，将测试表达突变形式的Sigma1的腺病毒感染Jam-A表达细胞的能力。在具体目标2中，将确定呼肠孤病毒感染极化细胞的生理后果。在呼肠孤病毒内化过程中，将监测JAM-A和JAM-A相关蛋白在细胞内的分布以及紧密连接的完整性。JAM-A磷酸化在呼肠孤病毒附着和感染中的意义将被确定。在具体目标3中，将明确SA在呼肠孤病毒致病中的作用。由于sigma1的单一氨基酸多态，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趋向性。SA在神经元呼肠孤病毒感染中的重要性将通过测试不同的呼肠孤病毒株在原代神经元培养中与SA结合生长的能力来确定。在特定的目标4中，将确定JAM-A在呼肠孤病毒致病中的作用。JAM-A表达缺陷的小鼠将被接种呼肠孤病毒，病毒的生长和组织病理学将在复制的主要和次要位置进行评估。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。

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.