Structural Analysis of Reovirus Attachment Mechanisms

描述(由申请人提供)：受体识别是病毒感染的第一步，在受感染宿主的靶细胞选择中起着至关重要的作用。许多病毒使用细胞黏附分子或细胞表面碳水化合物作为受体。然而，在原子水平上管理受体识别的一般规则还没有建立，并且多个受体对病毒附着和细胞进入的贡献还知之甚少。这项拟议的研究使用呼肠孤病毒，这是一种高度可处理的实验模型，显示出在溶瘤和疫苗应用方面的前景，以原子分辨率定义病毒-受体相互作用的结构基础。呼肠孤病毒在小鼠肠道初次感染后扩散到中枢神经系统(CNS)，在中枢神经系统(CNS)表现出不同血清型特异性的趋向性和致病机制的差异，可归因于病毒附着蛋白？1。？1蛋白是一个丝状三聚体，由N-末端和C-末端头部组成。T3D呼肠孤病毒株的？1尾部与唾液酸(SA)结合，3种呼肠孤病毒血清型的？1头部与免疫球蛋白超家族受体结合黏附分子-A(JAM-A)结合。提出了三个综合的特定目标来定义？1与其受体相互作用的结构和功能基础。在具体目标1中，将使用X射线结晶学来确定与JAM-A形成的复合体中的三种血清型？1的结构。JAM-A结合所需的每种血清型中的残基将通过使用新开发的基于质粒的反向遗传学系统对完整病毒进行结构引导突变来鉴定。JAM-A结合在小鼠中枢神经系统的呼肠孤病毒嗜性中的作用将通过JAM-A利用和室管膜细胞和神经元的原代培养中改变的突变来确定。在具体目标2中，将用X射线结晶学确定T3D？1与SA形成的络合物的结构。三种血清型的碳水化合物配体将通过糖链阵列筛选和功能分析进行鉴定。菌株T1L和T3D？1中的碳水化合物结合所需的最小序列单位将使用嵌合病毒以及病毒结合和传染性的分析来确定。在具体目标3中，将使用具有改变的？1灵活性和长度的突变病毒来阐明呼肠孤病毒附着中的？1受体结合域与细胞进入之间的功能关系。SA结合区的粘附性及其与JAM-A结合的相互作用将通过将额外的SA结合位点工程到？1尾部来确定。？1头将用腺病毒纤维旋钮取代，以确定受体特异性在呼肠孤病毒结合、内化和拆解中的功能。这些研究将加强对致病病毒与细胞受体结合的机制的基本了解，并加速用于治疗目的的病毒载体的合理设计。公共卫生相关性：病毒与受体的相互作用在病毒性疾病中起着关键作用。这项拟议的研究使用呼肠孤病毒，这是一个研究病毒附着和发病机制的强大实验系统，以确定病毒与细胞受体结合的一般机制。这项工作将提供有关病毒如何选择细胞靶标的重要新信息，并有助于开发新的抗病毒疫苗和治疗药物。

DESCRIPTION (provided by applicant): Receptor recognition is the first step in viral infection and plays an essential role in target-cell selection in the infected host. Many viruses use cell-adhesion molecules or cell-surface carbohydrates as receptors. However, general rules governing receptor recognition at an atomic level have not been established, and contributions of multiple receptors to viral attachment and cell entry are poorly understood. The proposed research uses reovirus, a highly tractable experimental model that shows promise for oncolytic and vaccine applications, to define the structural basis of virus-receptor interactions at atomic resolution. Following primary infection in the murine intestine, reovirus disseminates to the central nervous system (CNS), where it exhibits serotype-specific differences in tropism and pathogenesis attributable to viral attachment protein ?1. The ?1 protein is a filamentous trimer consisting of an N-terminal tail and a C-terminal head. The ?1 tail of strain T3D reovirus binds sialic acid (SA), and the ?1 head of all three reovirus serotypes binds immunoglobulin superfamily receptor junctional adhesion molecule-A (JAM-A). Three integrated specific aims are proposed to define the structural and functional basis of ?1 interactions with its receptors. In Specific Aim 1, structures of the three serotypes of ?1 in complex with JAM-A will be determined using X-ray crystallography. Residues in each serotype required for JAM-A binding will be identified by structure-guided mutagenesis of intact virus using a newly developed plasmid-based reverse genetics system. The role of JAM-A binding in reovirus tropism in the murine CNS will be defined using mutants altered in JAM-A utilization and primary cultures of ependymal cells and neurons. In Specific Aim 2, the structure of T3D ?1 in complex with SA will be determined using X-ray crystallography. Carbohydrate ligands of the three serotypes will be identified using glycan array screening and functional assays. Minimum sequence units required for carbohydrate binding in strains T1L and T3D ?1 will be defined using chimeric viruses and assays of viral binding and infectivity. In Specific Aim 3, functional relationships between the ?1 receptor-binding domains in reovirus attachment and cell entry will be elucidated using mutant viruses with alterations in ?1 flexibility and length. Adhesive properties of the SA-binding region and its interaction with JAM-A binding will be determined by engineering additional SA-binding sites into the ?1 tail. The ?1 head will be replaced with the adenovirus fiber knob to define the function of receptor specificity in reovirus binding, internalization, and disassembly. These studies will enhance a basic understanding of mechanisms by which pathogenic viruses engage cellular receptors and accelerate the rational design of viral vectors for therapeutic purposes. PUBLIC HEALTH RELEVANCE: Virus-receptor interactions serve a pivotal function in viral disease. The proposed research uses reovirus, a powerful experimental system for studies of viral attachment and pathogenesis, to define general mechanisms by which viruses bind to cellular receptors. This work will contribute important new information about how viruses select cellular targets and aid in the development of new antiviral vaccines and therapeutics.