Structural Analysis of Reovirus Attachment Mechanisms

呼肠孤病毒附着机制的结构分析

• 批准号: 8206800
• 负责人: TERENCE S. DERMODY
• 金额: $ 31.6万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-16 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8206800
• 关键词: Adenoviruses; Adhesives; Affinity; Antiviral Agents; Binding; Binding Sites; Biological Assay; C-terminal; Carbohydrates; Cell Adhesion Molecules; Cell surface; Cell-Matrix Junction; Cells; Complex; Coupled; Coxsackie Viruses; Development; Disease; Engineering; Ependymal Cell; Exhibits; Experimental Models; Fiber; Foundations; Head; Human; Immunoglobulin A; Immunoglobulins; Infection; Intestines; Knowledge; Length; Ligand Binding; Ligands; Link; Location; Mediating; Microbe; Morphology; Mus; Mutagenesis; N-terminal; Neuraxis; Neurons; Neurotropism; Oligosaccharides; Oncolytic; Organ; Pathogenesis; Plasmids; Play; Polysaccharides; Process; Property; Proteins; Reoviridae Infections; Reovirus; Research; Resolution; Role; Screening procedure; Serotyping; Sialic Acids; Site; Specificity; Structural Models; Structure; System; Tail; Therapeutic; Tropism; Vaccines; Viral; Viral Vector; Virus; Virus Diseases; Virus Receptors; Work; X-Ray Crystallography; activator 1 protein; adenovirus receptor; base; carbohydrate structure; cellular targeting; design; flexibility; junctional adhesion molecule; member; microbial; mutant; positional cloning; programs; prototype; receptor; receptor binding; receptor function; research study; therapeutic vaccine; tissue tropism

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.

受体识别是病毒感染的第一步，在目标细胞中起着至关重要的作用 在受感染的主机中进行选择。许多病毒使用细胞粘附分子或细胞表面碳氢化物 作为受体。但是，在原子水平上控制受体识别的一般规则尚未 已建立，多个接收器对病毒附着和细胞进入的贡献很差 理解。拟议的研究使用依默病毒，这是一个高度可触犯的实验模型，显示 溶瘤和疫苗应用的承诺，定义病毒受体的结构基础 原子分辨率的相互作用。在鼠肠中的原发性感染后，埃洛谢德病毒 传播到中枢神经系统（CNS），在该系统中表现出血清型特异性差异 归因于病毒附着蛋白的向流和发病机理»1。1蛋白是一种丝状蛋白 三聚体由N末端尾部和C末端头组成。 «菌株T3D静脉病毒的1个尾巴结合 唾液酸（SA）和所有三种依肠病毒血清型的1头结合免疫球蛋白超家族 受体连接粘附分子-A（JAM-A）。提出了三个集成的特定目标 定义与接收器的1个相互作用的结构和功能基础。在特定的目标1中， 将使用X射线确定与JAM-A复合体中三种血清型的结构 晶体学。 JAM-A结合所需的每种血清型中的住宅将通过结构确定 使用新开发的基于质粒的反向遗传学系统的完整病毒的诱变。 JAM-A结合在鼠CN中的葡萄病毒tropism中的作用将使用变化的突变体定义 在jam-a利用率和室心室细胞和神经元的原发性培养物中。在特定的目标2中 T3D»与SA复合体中的结构将使用X射线晶体学确定。糖类 将使用聚糖阵列筛选和功能测定法确定这三种血清型的配体。 菌株T1L和T3D•1将定义的最小序列单元将定义 使用病毒结合和感染的嵌合病毒和刺客。在特定目标3中，功能 e; evirus附着和细胞进入中的1个受体结合域之间的关系将是 使用突变病毒阐明，其柔性和长度改变。粘附特性 SA结合区域及其与JAM-A绑定的相互作用将通过工程额外确定 SA结合位点进入1尾。 1头将被腺病毒纤维旋钮代替以定义 受体特异性在葡萄病毒结合，内在化和拆卸中的功能。这些研究 将增强对病原病毒接纳细胞的机制的基本理解 受体并加速了用于治疗目的的病毒载体的合理设计。