Cryo-EM Structural Studies of Adenovirus Cell Entry

描述（由申请人提供）：本项目的长期目标是在分子水平上了解腺病毒（Ad）细胞进入所涉及的事件。下一个资助期的具体目标是进行Ad和Ad/整合素复合物的高分辨率结构研究，研究Ad与其宿主细胞受体之间相互作用的几何形状，并定义α -v β -5整合素通过与单价和多价配体结合而引起的构象变化。拟议的研究将明确地验证Ad已经进化出感染特定细胞类型和诱导整合素细胞信号事件的有效途径的范式。该结果将弥合我们对Ad分子生物学的理解与快速发展的Ad载体基因治疗领域之间的知识差距。在之前的资助期内，我们取得了令人兴奋的新发现，为Ad结构及其与宿主细胞受体的相互作用提供了更好的表征。一个新兴的概念是，病毒及其相关受体的精确三维定向是病毒趋向性的一个促成因素。提出的更高分辨率的研究将使我们能够表征在病毒进入细胞时与α -v整合素相互作用的Ad戊基蛋白的三级蛋白折叠，以及α -v整合素与多价Ad戊基蛋白结合和聚集时的构象。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α -v - β -5整合素的哪些构象变化启动信号通路？低温电子显微镜（cro - em）的进步使得确定二十面体病毒的高分辨率结构和Ad/受体囊泡复合物的低温电子断层扫描成为可能。这些进步包括自动数据采集软件、计算机控制的断层扫描软件、并行图像处理软件和液氦冷却标本级显微镜的发展。冷冻电镜方法最近也被扩展到洗涤剂溶解膜蛋白，我们将应用这种方法来确定α -v β -5整合素和Ad/ α -v β -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为开发阻断病毒进入细胞的抗病毒药物提供机会，并有助于合理设计靶向Ad载体。

DESCRIPTION (provided by applicant): 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.