Alpha-V Integrins and Adenovirus Cell Entry

描述(申请人提供)：尽管最初的期望，腺病毒(Ad)介导的基因治疗仍然受阻于缺乏宿主细胞相互作用的基本知识。尽管如此，广告仍然是揭示关键细胞和分子过程的重要工具。在以前的研究中，我们发现了一种整合素信号通路，可以促进宿主细胞对Ad的摄取。然而，在我们对进一步的细胞内贩运事件的理解方面仍然存在重大差距。特别是，Ad穿透早期内噬体体的机制仍不明确。 因此，一个经验丰富的研究团队将描述促进Ad介导的内吞体破碎化的细胞和病毒过程。一个关键的信号分子蛋白激酶C的作用将被研究，它通过整合素avB5促进Ad进入的后期步骤。生化和生物物理研究将确定膜结合和/或破坏所需的Ad颗粒的特定构象变化。改进的低温电子显微镜和X射线衍射技术将被用来揭示Ad颗粒的结构特征以及与膜缔合相关的构象变化。最后，我们将利用结构定义的大分子平台(即二十面体病毒)来研究整合素结合域(RGD序列)的精确空间定向如何调节病毒进入或细胞迁移。这些研究将提供更全面的Ad-宿主细胞进入的图景，以及揭示病毒内化和细胞迁移中的潜在信号事件。如果成功，这一新知识还可能促进病毒和非病毒基因传递系统的改进。

DESCRIPTION (provided by applicant): Despite initial expectations, adenovirus (Ad)- mediated gene therapy remains stymied by a lack of fundamental knowledge of host cell interactions. Nonetheless, Ad continues to serve as an important tool for uncovering key cellular and molecular processes. In previous studies, we discovered an integrin-signaling pathway that promotes Ad uptake into host cells. However, significant gaps remain in our understanding of further intracellular trafficking events. In particular, the mechanism by which Ad penetrates the early endosome remains poorly defined. Therefore, an experienced team of investigators will characterize the cellular and viral processes that facilitate Ad-mediated endosome disruption. The role of a key signaling molecule, protein kinase C, that promotes a late step in Ad entry via integrin avB5, will be investigated. Biochemical and biophysical studies will identify specific conformational changes in Ad particles required for membrane association and/or disruption. Improved cryo-electron microscopy and x-ray diffraction techniques will be used to reveal structural features of Ad particles as well as the conformational changes associated with membrane association. Finally, we will exploit structurally defined macromolecular platforms (i.e. icosahedral viruses) to investigate how the precise spatial orientation of integrin binding domains (RGD sequences) regulates virus entry or cell migration. These studies should provide a more comprehensive picture of Ad-host cell entry as well as reveal the underlying signaling events in virus internalization and cell migration. If successful, this new knowledge may also facilitate improvements in both viral and nonviral gene delivery systems.