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: 8789636
• 负责人: THILO STEHLE
• 金额: $ 25.32万
• 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/8789636
• 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感染引起致命的中枢神经系统疾病，进行性多灶性白质脑病， 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有效治疗PML的关键， 未来我们建议使用结构-功能方法来鉴定可以结合蛋白的分子。 重组产生的具有高亲和力的JCPyV衣壳蛋白VP 1（特异性目标1）。因为最初的袭击很可能 将不是很具体，我们将通过进行结构相关的结合试验来优化它们。 化合物，并通过晶体学以及生物测定验证结合， 与Atwood和DiMaio小组合作（项目2和3）。此外，我们将在结构上 表征JCPyV VP 1五聚体的受体转换突变体及其配体结合特性（特异性 目标2）。我们的初步结果表明，JCPyV可以参与一系列唾液酸化聚糖，但不是所有这些 相互作用导致感染。我们将设计JCPyV VP 1突变体，选择性地使用单一聚糖， 受体，我们将结晶与受体复合的VP 1蛋白，以确定特异性， 与项目#2和核心B合作分析相应的假病毒，用于细胞结合和进入。 博士DiMaio（项目#3）将确定针对不同受体的病毒是否显示不同的贩运 模式.在这个目标的第二部分，我们将描述JCPyV的配体结合特性和特异性， 使用聚糖微阵列筛选以及晶体学从PML患者分离。最后我们将 对整个JCPyV假病毒进行结构-功能分析（具体目标3）。一些结合位点 受体可能位于病毒体表面上的VP 1五聚体之间，并且这些位点将不存在于 重组表达的五聚体单独的背景。JCPyV假病毒最近已经产生 由阿特伍德小组（项目#2），他们忠实地复制病毒的基本功能， 入境将在结构上分析JCPyV假病毒颗粒的配体结合 性质以及聚糖微阵列。