Structural Analysis of Reovirus Attachment Mechanisms

呼肠孤病毒附着机制的结构分析

• 批准号: 6769334
• 负责人: THILO STEHLE
• 金额: $ 35.33万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6769334
• 关键词: Reoviridae; X ray crystallography; aspartate; binding sites; biological signal transduction; cell adhesion molecules; circular dichroism; molecular dynamics; protein protein interaction; protein structure function; receptor binding; receptor expression; sialate; site directed mutagenesis; virus infection mechanism; virus receptors

DESCRIPTION (provided by applicant): Reoviruses provide a well-established experimental system for studies of viral neuropathogenesis. Following primary infection in the intestine of newborn mice, type 3 reoviruses spread through nerves and infect neurons, causing encephalitis. Viral attachment protein sigma1 plays a crucial role in each of these progressive pathologic events. The sigma1 protein is a fibrous protein consisting of an N-terminal tail and a C-terminal head. The sigma1 tail contains a domain that binds sialic acid; the sigma1 head binds junctional adhesion molecule-1 (JAM-1). Recent high-resolution structural studies indicate that the JAM-1-binding domain of sigma1 is a homotrimer of loosely associated eight-stranded beta-barrels. However, the mechanism of dual receptor binding by sigma1 is not known, nor is it understood how sigma1-receptor interactions dictate viral tropism, cell entry, or disassembly. Four integrated specific aims are proposed to study structural and functional properties of sigma1, with the goal of defining the structural basis for the interaction of sigma1 with its receptors. In Specific Aim 1, the structural basis of sigma1 interactions with JAM-1 and sialic acid will be determined using X-ray crystallography. Truncated fragments of sigma1 that comprise the receptor-binding domains and the extracellular region of JAM-1 will be used for these analyses. In Specific Aim 2, local determinants of sigma1 receptor binding and the global relationships between the sigma1 receptor-binding domains will be defined. The length of a linker sequence between the receptor-binding domains will be altered, and modified sigma1 protein will be tested for viral attachment. In Specific Aim 3, determinants of conformational changes in sigma1 will be identified using X-ray crystallography and circular dichroism. The role of conserved aspartic acid residues at the head trimer interface will be defined using directed mutagenesis and assays of viral attachment and disassembly. In Specific Aim 4, dynamic regions of sigma1 structure will be defined using a very high-resolution structure of the G1 C-terminal domain and molecular dynamics simulation. These studies will yield a precise understanding of reovirus cell-attachment and provide insights into mechanisms by which reovirus selects cellular targets and produces neurologic disease. Moreover, since the atomic structures of so few virus-receptor complexes are known, this work may serve as a general model for ligand-receptor binding and aid in the rational design of antiviral therapeutics based on interference with specific pathogen-receptor interactions.

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完善的实验系统。新生小鼠肠道初次感染后，3型呼肠孤病毒通过神经传播并感染神经元，引起脑炎。病毒附着蛋白sigma1在这些进行性病理事件中起着至关重要的作用。sigma1蛋白是一种纤维蛋白，由n端尾部和c端头部组成。sigma1尾部包含一个结合唾液酸的结构域；sigma1头与连接粘附分子-1 （JAM-1）结合。最近的高分辨率结构研究表明，sigma1的jam -1结合域是一个松散关联的八链β -桶的同源三聚体。然而，sigma1结合双受体的机制尚不清楚，也不清楚sigma1受体的相互作用如何决定病毒的趋向性、细胞进入或分解。我们提出了四个综合的具体目标来研究sigma1的结构和功能特性，目的是确定sigma1与其受体相互作用的结构基础。在Specific Aim 1中，将使用x射线晶体学确定sigma1与JAM-1和唾液酸相互作用的结构基础。包括受体结合域和JAM-1细胞外区域的sigma1的截短片段将用于这些分析。在Specific Aim 2中，将定义sigma1受体结合的局部决定因素和sigma1受体结合域之间的全局关系。受体结合域之间的连接体序列的长度将被改变，并且修饰的sigma1蛋白将被测试是否有病毒附着。在具体目标3中，将使用x射线晶体学和圆二色性来确定sigma1构象变化的决定因素。保守的天冬氨酸残基在头部三聚体界面的作用将通过定向诱变和病毒附着和拆卸的测定来确定。在Specific Aim 4中，将使用G1 c端结构域的高分辨率结构和分子动力学模拟来定义sigma1结构的动态区域。这些研究将产生呼肠孤病毒细胞附着的精确理解，并提供对呼肠孤病毒选择细胞靶点和产生神经系统疾病的机制的见解。此外，由于已知的病毒-受体复合物的原子结构如此之少，这项工作可以作为配体-受体结合的一般模型，并有助于基于干扰特定病原体-受体相互作用的抗病毒治疗的合理设计。