ALPHA-V INTEGRINS AND ADENOVIRUS CELL ENTRY

腺病毒（Ad）是引起人类呼吸道疾病的重要原因， 胃肠道和眼部感染;然而，复制缺陷 Ad形式目前用于基因治疗的临床试验。 尽管这种方法取得了一些成功，但缺乏对广告如何传播的了解， 识别宿主细胞共受体（α v整合素）， 整联蛋白促进病毒摄取阻碍了Ad载体的最佳使用 在诊所里该提案旨在定义精确的整合素 结合事件和细胞内信号通路参与 腺病毒进入宿主细胞。本提案的目标是：（1） 确定Ad的三维结构和Ad的动力学， α v整合素结合，2）表征Ad介导的整合素结合的机制。 内体破坏和α v整联蛋白在该过程中的作用，3） 确定调节Ad内化的信号分子， 4)利用细胞信号通路增强Ad介导的基因表达， 体外和体内递送。 冷冻电子显微镜和动力学分析将用于确定 负责α v整联蛋白结合的结构元件 不同的Ad血清型和细胞外基质蛋白。一组 表达α v整联蛋白修饰形式的上皮细胞系 将被用来确定精确的序列和机制， 在Ad介导内体破坏中，病毒 渗透进细胞。生物化学和分子遗传学方法 将用于研究信号分子在病毒中的作用， 内吞作用（Endocytosis）。最后，一种改良的腺病毒载体 具有触发多种细胞信号通路的能力， 用于增强向新生血管组织和实体瘤的基因递送， vivo.如果成功，这些研究有可能增加我们的 了解几种基本的细胞和分子生物学 流程以及优化广告载体的使用。

Adenovirus (Ad) represent a significant causes of human respiratory, gastrointestinal and ocular infections; however, replication-defective forms of Ad are currently in use in clinical trials for gene therapy. Despite some success with this approach, a lack of knowledge of how Ad recognizes host cell co-receptors (alphav integrins) and how alphav integrins promote virus uptake has prevented optimal use of Ad vectors in the clinic. This proposal seeks to define the precise integrin binding events and intracellular signaling pathways involved in adenovirus entry into host cells. The goals of this proposal are to 1) determine the three-dimensional architecture of Ad and the kinetics of alphav integrin binding, 2) to characterize the mechanism of Ad-mediated endosome disruption and the role of alphav integrins in this process, 3) to define the signaling molecules that regulate Ad internalization and 4) to exploit cell signaling pathways to enhance Ad-mediated gene delivery in vitro and in vivo. Cryoelectron microscopy and kinetic analyses will be used to define the structural elements responsible for alphav integrin association with different Ad serotypes and extracellular matrix proteins. A panel of epithelial cells lines expressing modified forms of alphav integrins will be used to identify the precise sequences and mechanisms involved in Ad-mediated endosome disruption, a key step required for virus penetration into the cell. Biochemical and molecular genetic approaches will be used to investigate the role of signaling molecules in virus internalization (endocytosis). Finally, a modified adenovirus vector with the capacity to trigger multiple cell signaling pathways will be used to enhance gene delivery to neovascular tissue and solid tumors in vivo. If successful, these studies have the potential to increase our understanding of several fundamental cell and molecular biological processes as well as optimize the use of Ad vectors.