Structural Analysis of Reovirus Attachment Mechanisms

呼肠孤病毒附着机制的结构分析

• 批准号: 7759118
• 负责人: TERENCE S. DERMODY
• 金额: $ 31.92万
• 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/7759118
• 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; 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; adenovirus receptor; base; carbohydrate structure; cellular targeting; design; flexibility; junctional adhesion molecule; member; microbial; mutant; positional cloning; programs; prototype; public health relevance; receptor; receptor binding; receptor function; research study; therapeutic vaccine; tissue tropism

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.

描述（由申请人提供）：受体识别是病毒感染的第一步，在感染宿主中的目标细胞选择中起着至关重要的作用。许多病毒使用细胞粘附分子或细胞表面碳水化合物作为受体。但是，尚未建立控制受体识别的一般规则，并且对多种受体对病毒依恋和细胞进入的贡献的理解很少。拟议的研究使用依默病毒是一种高度可处理的实验模型，该模型显示出对溶瘤和疫苗应用的希望，以定义原子分辨率下病毒 - 受体相互作用的结构基础。在鼠肠中原发性感染后，葡萄病毒将其传播到中枢神经系统（CNS），在中枢神经系统（CNS）中表现出可归因于病毒附着蛋白的质和发病机理的血清型特异性差异？1。 1蛋白是由N末端尾部和C末端头组成的丝状三聚体。菌株T3D静脉病毒的1尾结合唾液酸（SA），并且所有三种孢子病毒血清型的头部结合了免疫球蛋白超家族受体联合粘附分子-A（JAM-A）。提出了三个集成的特定目标来定义与受体相互作用的结构和功能基础。在特定的目标1中，将使用X射线晶体学确定与JAM-A复合体中三种血清型的结构。 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的完整病毒的结构引导诱变来鉴定。 JAM-A结合在鼠CN中的葡萄病毒tropism中的作用将使用在JAM-A使用中改变的突变体和外膜细胞和神经元的原发性培养物来定义。在特定的目标2中，将使用X射线晶体学确定T3D？1的结构。三种血清型的碳水化合物配体将使用聚糖阵列筛选和功能分析来鉴定。 T1L和T3D？1中碳水化合物结合所需的最小序列单元将使用嵌合病毒和病毒结合和感染性的测定定义。在特定的目标3中，使用突变病毒在？1柔韧性和长度改变的突变病毒中，将阐明依孢病毒附着和细胞进入中的受体结合结构域之间的功能关系。 SA结合区域的粘合特性及其与JAM-A结合的相互作用将通过将其他SA结合位点工程化到？1尾部来确定。 1个头将被腺病毒纤维旋钮替换，以定义依孢病毒结合，内在化和拆卸中受体特异性的功能。这些研究将增强对致病性病毒参与细胞受体并加速病毒载体的理性设计的机制的基本理解。公共卫生相关性：病毒 - 受体相互作用在病毒疾病中起关键功能。拟议的研究使用依默病毒（用于病毒依恋和发病机理研究的强大实验系统）来定义病毒与细胞受体结合的一般机制。这项工作将贡献有关病毒如何选择细胞靶标并有助于开发新的抗病毒疫苗和治疗剂的重要新信息。