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

基本信息

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
项目类别：
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

项目摘要

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、进行性多灶性白质脑病、 PML，对此没有有效的治疗方法。对结构功能的透彻理解。这种人类病原体之间的关系是开发针对 PML 的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。重组产生的 JCPyV 衣壳蛋白 VP1 具有高亲和力（具体目标 1）。不会非常具体，我们将通过与结构相关的结合测定来优化它们使用核磁共振波谱分析化合物，并通过晶体学以及生物测定验证结合我们将进一步与 Atwood 和 DiMaio 小组合作（项目#2 和#3）。表征 JCPyV VP1 五聚体的受体转换突变体及其配体结合特性（具体目标 2)。我们的初步结果表明，JCPyV 可以结合一系列唾液酸化聚糖，但不是所有这些聚糖。我们将设计 JCPyV VP1 突变体，选择性地仅使用单个聚糖。受体，我们将结晶与受体复合的 VP1 蛋白，以确定特异性，并且与项目 #2 和核心 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）将确定针对不同受体的病毒是否表现出不同的贩运在该目标的第二部分中，我们将表征 JCPyV 的配体结合特性和特异性。最后，我们将使用聚糖微阵列筛选和晶体学从 PML 患者中分离。对整个 JCPyV 假病毒进行结构功能分析（特定目标 3）。受体可能位于病毒颗粒表面的 VP1 五聚体之间，并且此类位点不会存在于病毒粒子中最近仅产生了重组表达的五聚体 JCPyV 假病毒。由阿特伍德小组（项目＃2）开发，它们忠实地复制了病毒在附件和 JCPyV 假病毒颗粒将根据其配体结合进行结构分析。特性以及聚糖微阵列。