CRYO-EM STRUCTURAL STUDIES OF ADENOVIRUS CELL ENTRY

腺病毒细胞进入的冷冻电镜结构研究

• 批准号: 8171036
• 负责人: PHOEBE L STEWART
• 金额: $ 1.22万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171036
• 关键词: Address; Adenovirus Vector; Adenoviruses; Antiviral Agents; Binding; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer software; Cryoelectron Microscopy; Detergents; Development; Event; Funding; Goals; Grant; Helium; Institution; Integrin Binding; Integrins; Knowledge; Ligands; Liquid substance; Membrane Proteins; Methods; Microscope; Molecular; Molecular Biology; Molecular Conformation; Pathway interactions; Process; Receptor Cell; Research; Research Personnel; Resolution; Resources; Signal Pathway; Signal Transduction; Source; Specimen; Staging; Structure; Testing; Tropism; United States National Institutes of Health; Vesicle; Viral; Virus; adenovirus receptor; cell type; data acquisition; design; gene therapy; image processing; integrin beta5; penton base; protein folding; receptor; tomography

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term objective of this project is to understand the events involved in adenovirus (Ad) cell entry at the molecular level. The specific goals for the next funding period are to undertake high resolution structural studies of Ad and Ad/integrin complexes, to investigate the geometry of the interaction between Ad and its host cell receptors, and to define the conformational changes induced in alpha-v beta-5 integrin by binding to monovalent and multivalent ligands. The proposed research will definitively test the paradigm that Ad has evolved efficient pathways for infecting specific cell types and for inducing integrin cell signaling events. The results will bridge the knowledge gap between our understanding of Ad molecular biology and the rapidly expanding field of Ad vector based gene therapy. In the previous funding period, we have made exciting new discoveries that have provided a better characterization of Ad structure and its interaction with host cell receptors. An emerging concept is that the precise three-dimensional orientation of the virus with its associated receptors is a contributing factor to viral tropism. The proposed higher resolution studies will enable us to characterize the tertiary protein fold of the Ad penton base protein, which interacts with alpha-v integrins during viral cell entry, as well as the conformation of alpha-v integrin when bound and clustered by the multivalent Ad penton base protein. The specific aims are designed to address two fundamental questions: 1) What structural features of Ad are critical for efficient binding to host cell receptors? 2) What conformational changes does Ad induce in alpha-v beta-5 integrin to initiate signaling pathways? Advances in cryo-electron microscopy (cryo-EM) have made determining a high resolution structure of an icosahedral virus and cryoelectron tomography of Ad/receptor vesicle complexes feasible. These advances include the development of automated data acquisition software, computer-controlled tomography software, parallelized image processing software, and microscopes with liquid-helium-cooled specimen stages. Cryo-EM methods have also recently been extended to detergent solubilized membrane proteins and we will apply this approach to determine structures of alpha-v beta-5 integrin and an Ad/alpha-v beta-5 integrin complex. Increased knowledge of the Ad cell entry process may provide an opportunity to develop antivirals that block viral cell entry and will facilitate the rational design of targeted Ad vectors.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 该项目的长期目标是在分子水平上了解腺病毒（Ad）进入细胞所涉及的事件。下一个资助期的具体目标是对 Ad 和 Ad/整合素复合物进行高分辨率结构研究，研究 Ad 与其宿主细胞受体之间相互作用的几何形状，并确定通过与单价和多价配体结合而在 α-v beta-5 整合素中诱导的构象变化。拟议的研究将最终测试 Ad 已进化出用于感染特定细胞类型和诱导整合素细胞信号传导事件的有效途径的范例。这些结果将弥合我们对 Ad 分子生物学的理解和快速扩展的基于 Ad 载体的基因治疗领域之间的知识差距。在之前的资助期间，我们取得了令人兴奋的新发现，更好地表征了 Ad 结构及其与宿主细胞受体的相互作用。一个新兴的概念是，病毒及其相关受体的精确三维方向是病毒趋向性的一个影响因素。拟议的更高分辨率研究将使我们能够表征 Ad 五邻体基础蛋白的三级蛋白质折叠，该蛋白在病毒细胞进入过程中与 α-V 整合素相互作用，以及当多价 Ad 五邻体基础蛋白结合和聚集时 α-V 整合素的构象。具体目标旨在解决两个基本问题：1）Ad 的哪些结构特征对于有效结合宿主细胞受体至关重要？ 2) Ad 诱导 alpha-v beta-5 整合素发生哪些构象变化以启动信号通路？冷冻电子显微镜 (cryo-EM) 的进步使得确定二十面体病毒的高分辨率结构和 Ad/受体囊泡复合物的冷冻电子断层扫描变得可行。这些进步包括自动数据采集软件、计算机控制断层扫描软件、并行图像处理软件以及带有液氦冷却样品台的显微镜的开发。冷冻电镜方法最近也已扩展到去污剂溶解的膜蛋白，我们将应用这种方法来确定 α-v beta-5 整合素和 Ad/α-v beta-5 整合素复合物的结构。增加对 Ad 细胞进入过程的了解可能会为开发阻止病毒细胞进入的抗病毒药物提供机会，并将促进目标 Ad 载体的合理设计。