PROJECT 1: STRUCTURAL BIOLOGY OF JCPYV HOST CELL RECOGNITION

项目1：JCPYV宿主细胞识别的结构生物学

• 批准号: 8881340
• 负责人: THILO STEHLE
• 金额: $ 22.17万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8881340
• 关键词: Adenoviruses; Affinity; Antiviral Agents; B-Lymphocytes; Binding; Binding Sites; Biological Assay; Capsid; Capsid Proteins; Carbohydrates; Cells; Central Nervous System Diseases; Clathrin; Clinical; Collaborations; Complement; Complex; Crystallography; Development; Disease; Endocytosis; Engineering; Funding; Future; Ganglioside GM1; Human; Infection; Lead; Libraries; Ligand Binding; Ligands; Link; Merkel Cells; Methods; Modification; Molecular; Molecular Bank; Mutation; NMR Spectroscopy; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pattern; Play; Polyomavirus; Polysaccharides; Population; Progressive Multifocal Leukoencephalopathy; Property; Proteins; Role; Sialic Acids; Site; Specificity; Structure; Structure-Activity Relationship; Surface; Techniques; Viral; Virion; Virus; arm; base; design; effective therapy; follow-up; inhibitor/antagonist; monomer; mutant; particle; pathogen; receptor; receptor binding; research study; screening; structural biology; tissue tropism; trafficking

PROJECT SUMMARY – PROJECT 1 JCPyV infection causes a fatal central nervous system disease, progressive multifocal leukoencephalopathy, PML, for which there are no effective treatments. A thorough understanding of the structure-function relationships of this human pathogen is key for the development of effective therapies against PML in the future. We propose to use structure-function approaches to identify molecules that can bind to the recombinantly produced JCPyV capsid protein VP1 with high affinity (Specific Aim 1). Since the initial hits likely will not be very specific, we will optimize them by conducting binding assays with structurally related compounds using NMR spectroscopy, and verify binding by crystallography as well as in biological assays in collaboration with the Atwood and DiMaio groups (projects #2 and #3). We will furthermore structurally characterize receptor-switching mutants of JCPyV VP1 pentamers and their ligand-binding properties (Specific Aim 2). Our preliminary results show that JCPyV can engage a range of sialylated glycans, but not all of these interactions lead to an infection. We will engineer JCPyV VP1 mutants that selectively use only a single glycan receptor, and we will crystallize the VP1 proteins in complex with the receptor to define specificities and also analyze the corresponding pseudoviruses in collaboration with project #2 and core B for cell binding and entry. Dr. DiMaio (project #3) will determine if viruses targeted to different receptors display different trafficking patterns. In a second part of this aim, we will characterize ligand-binding properties and specificities of JCPyV isolates from PML patients using glycan microarray screening as well as crystallography. Finally, we will perform structure-function analyses of entire JCPyV pseudoviruses (Specific Aim 3). Binding sites for some receptors may lie between VP1 pentamers on the virion surface, and such sites will not be present in the context of the recombinantly expressed pentamers alone. JCPyV pseudoviruses have recently been generated by the Atwood group (project #2), and they faithfully replicate essential functions of the virus in attachment and entry. The JCPyV pseudovirus particles will be analyzed structurally with respect to their ligand binding properties as well as in glycan microarrays.

项目摘要 - 项目1 JCPYV感染会导致致命的中枢神经系统疾病，进行性多灶性白细胞症状， PML，没有有效的治疗方法。对结构功能的透彻理解 这种人类病原体的关系是开发针对PML有效疗法的关键 未来。我们建议使用结构功能方法来识别可以结合的分子 重组产生具有高亲和力的JCPYV衣壳蛋白VP1（特定AIM 1）。由于最初的命中可能 不会很具体，我们将通过进行结构相关的结合测定来优化它们 使用NMR光谱法，并通过晶体学以及在生物学测定中验证结合 与Atwood和Dimaio团体合作（项目＃2和＃3）。我们将在结构上 表征JCPYV VP1 pentamers的接收器开关突变体及其配体结合特性（特定 目标2）。我们的初步结果表明，JCPYV可以参与一系列垂直的聚糖，但并非所有这些 相互作用导致感染。我们将设计仅选择性使用单个聚糖的JCPYV VP1突变体 受体，我们将与受体中的复合物中的VP1蛋白结晶以定义规格，也是 与项目＃2和Core B合作分析相应的假病毒，以进行细胞结合和进入。 Dimaio博士（项目＃3）将确定针对不同接收者的病毒是否显示不同的贩运 模式。在此目标的第二部分中，我们将表征JCPYV的配体结合属性和规格 使用聚糖微阵列筛查以及晶体学分离的PML患者分离。最后，我们会的 对整个JCPYV假病毒进行结构功能分析（特定AIM 3）。有些人的绑定位点 接收器可能位于病毒体表面上的VP1五聚体之间，此类位点不会存在于 重组表达的五聚会的背景。 JCPYV伪病毒最近已经生成 由Atwood Group（项目＃2），他们忠实地复制了病毒的基本功能， 入口。 JCPYV假病毒颗粒将在结构上相对于其配体结合进行分析 属性以及聚糖微阵列。