CRYO-EM STRUCTURAL STUDIES OF ADENOVIRUS CELL ENTRY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 本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/整联蛋白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结构，并确定通过与单价和多价配体结合在α-v β-5整联蛋白中诱导的构象变化。拟议的研究将明确测试的范例，广告已经发展了有效的途径感染特定的细胞类型和诱导整合素细胞信号事件。这些结果将弥补我们对Ad分子生物学的理解与迅速扩大的基于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，这些发现提供了更好的Ad结构及其与宿主细胞受体相互作用的表征。一个新兴的概念是，病毒及其相关受体的精确三维定位是病毒嗜性的一个促成因素。所提出的更高分辨率的研究将使我们能够表征的第三蛋白折叠的广告五邻体基础蛋白，它与α-V整合素在病毒细胞进入，以及α-V整合素的构象时，结合和聚集的多价广告五邻体基础蛋白相互作用。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整合素发生什么构象变化以启动信号通路？冷冻电子显微镜（cryo-EM）的进展，确定高分辨率的结构的二十面体病毒和冷冻电子断层扫描的Ad/受体囊泡复合物的可行性。这些进展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，和液氦冷却的标本台显微镜的发展。Cryo-EM方法最近也被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 β-5整联蛋白和Ad/α-v β-5整联蛋白复合物的结构。对Ad细胞进入过程的了解的增加可以提供开发阻断病毒细胞进入的抗病毒药物的机会，并且将促进靶向Ad载体的合理设计。

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.