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: 7955643
• 负责人: PHOEBE L STEWART
• 金额: $ 1.36万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955643
• 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; Modeling; 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; computational anatomy; 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 该项目的长期目标是了解分子水平上腺病毒（AD）细胞进入的事件。下一个资金期间的具体目标是进行AD和AD/整合素复合物的高分辨率结构研究，以研究AD与其宿主细胞受体之间相互作用的几何形状，并通过与单价和多价配体结合来定义Alpha-V Beta-5整合素中诱导的构象变化。拟议的研究将确定测试AD已发展有效途径的范式，以感染特定的细胞类型和诱导整联蛋白细胞信号传导事件。结果将弥合我们对AD分子生物学的理解与基于AD媒介基因疗法的快速扩展领域之间的知识差距。在上一个资金期间，我们做出了令人兴奋的新发现，从而更好地表征了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，该病毒及其相关受体的精确三维取向是导致病毒性向流的一个因素。拟议的高分辨率研究将使我们能够表征Ad Penton碱基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整合蛋白相互作用，以及当由多价AD Penton碱基蛋白绑定并聚集时alpha-V整合蛋白的构象。具体目的旨在解决两个基本问题：1）AD的哪些结构特征对于有效地与宿主细胞受体结合至关重要？ 2）AD在Alpha-V Beta-5整合素中诱导什么构象变化来启动信号通路？冷冻电子显微镜（Cryo-EM）的进展使确定了二十面体病毒的高分辨率结构和AD/受体囊泡复合物的低温电子层析成分可行。这些进步包括开发自动数据采集软件，计算机控制的断层扫描软件，并行的图像处理软件以及具有液态冷却标本阶段的显微镜。最近还将冷冻EM方法扩展到洗涤剂溶解的膜蛋白，我们将采用这种方法来确定alpha-V beta-5整合素和AD/Alpha-V beta-5整合蛋白复合物的结构。对AD细胞进入过程的了解增加可能为开发阻断病毒细胞进入的抗病毒药提供机会，并将促进靶向AD载体的合理设计。