Structural Analysis of Reovirus Attachment Mechanisms

受体识别是病毒感染的第一步，在靶细胞中起着重要作用 在被感染的宿主中进行选择。许多病毒利用细胞粘附分子或细胞表面碳水化合物 作为受体。然而，在原子水平上控制受体识别的一般规则还没有被阐明。 已经建立，并且多种受体对病毒附着和细胞进入的贡献很差 明白这项拟议中的研究使用了呼肠孤病毒，这是一种非常容易处理的实验模型， 有望用于溶瘤和疫苗应用，以确定病毒受体的结构基础 原子分辨率的相互作用。在小鼠肠道中初次感染后，呼肠孤病毒 传播到中枢神经系统（CNS），在那里它表现出特定的类型差异， 嗜性和致病性归因于病毒附着蛋白1。1蛋白是一种丝状蛋白， 由N-末端尾部和C-末端头部组成的三聚体。T3 D呼肠孤病毒株的1号尾结合 唾液酸（SA），所有三种呼肠孤病毒血清型的1号头结合免疫球蛋白超家族 受体连接粘附分子-A（JAM-A）。提出了三个综合的具体目标， 定义与受体相互作用的结构和功能基础。具体目标1、 将使用X射线测定与JAM-A复合的3种血清型的结构 结晶学JAM-A结合所需的每种血清型中的残基将通过结构- 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引导诱变。 JAM-A结合在鼠CNS中呼肠孤病毒嗜性中的作用将使用改变的突变体来定义。 在JAM-A室管膜细胞和神经元的利用和原代培养中。具体目标2 与SA复合的T3 D 1的结构将使用X射线晶体学来确定。碳水化合 三种血清型的配体将使用聚糖阵列筛选和功能测定来鉴定。 将定义菌株T1 L和T3 D <$1中碳水化合物结合所需的最小序列单位 使用嵌合病毒和病毒结合和感染性的测定。在具体目标3中，功能性 呼肠孤病毒附着和进入细胞中的受体结合结构域之间的关系将是 使用变异病毒阐明了变异病毒的柔性和长度。粘合剂性能 SA结合区及其与JAM-A结合的相互作用将通过工程化另外的基因来确定。 SA结合位点进入到1尾。将用腺病毒纤维旋钮替换1号头， 受体特异性在呼肠孤病毒结合、内化和分解中的作用。这些研究 将增强对致病病毒与细胞结合的机制的基本理解， 受体，并加速用于治疗目的的病毒载体的合理设计。

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.