VPR-INDUCED ENDOTHELIAL CYTOSKELETAL/FUNCTIONAL CHANGES

描述（根据申请人的描述改编） 我们的长期目标是了解HIV-1 Vpr在病毒感染过程中的功能。 复制和发病机制。 虽然Vpr的许多活动已经 尽管过去已经发现，Vpr功能的生化机制仍然存在， 难以捉摸。 在HIV-1感染过程中，内皮细胞层处于恒定状态， 接触HIV-1病毒粒子、HIV-1基因产物。 之间的相互作用 HIV-1和内皮细胞层显著影响宿主免疫 反应以及HIV-1在多个组织/器官中的发病机制。 期间 我们的初步研究，我们提出了四个新的发现， a）细胞外Vpr显著改变 内皮肌动蛋白结构。 体外试验也表明， 剂量响应性Vpr阻断肌动蛋白解聚; B）放射性标记的Vpr 与内皮细胞表面特异性结合。 突变分析 表明Vpr的C-末端基本结构域对于 c）与Vpr孵育的内皮细胞显著改变 通过RT-PCR的差异mRNA显示测量细胞基因表达， 和d）体外激酶测定显示Vpr特异性地与 磷酸化肌球蛋白轻链的蛋白激酶。 本研究 项目，我们建议扩展这些研究，以确定分子和 这些Vpr诱导的内皮细胞骨架和 功能变化。 我们将集中于四个具体目标：1） 表征Vpr诱导的内皮细胞中的细胞骨架修饰; 2） 对Vpr相关激酶进行生物化学和生理学表征 3）分子克隆内皮细胞的cDNA， vpr相关激酶，并检查它们对内皮细胞骨架的影响 结构和功能，以及4）识别和表征细胞基因 其表达受Vpr高度调控，并分析其作用 在Vpr诱导的细胞周期停滞和Vpr调节细胞凋亡期间。 我们 我认为，上述集中的具体目标应有助于理解 Vpr对HIV感染期间内皮功能障碍的贡献。 从这些研究中获得的知识也应该有助于理解 一般的HIV-1发病机制。 我们预计，该项目将 产生关于Vpr诱导机制的新知识 内皮细胞中的细胞骨架/功能变化，可以使 治疗策略的设计。

DESCRIPTION (Adapted from the applicant's description) Our long-term goal is to understand the function of HIV-1 Vpr during viral replication and pathogenesis. Although many activities of Vpr have been discovered in the past, the biochemical mechanism of Vpr function remains elusive. During HIV-1 infection, the endothelial cell layer is in constant contact with HIV-1 virion, HIV-1 gene products. The interaction between HIV-1 and the endothelial cell layer significantly impacts the host immune responses as well as HIV-1 pathogenesis in multiple tissues/organs. During our preliminary studies, we made four novel findings concerning the effect of Vpr on endothelial cells: a) extracellular Vpr dramatically altered endothelial actin structures. In vitro assays also suggested a dose-responsive Vpr block of actin depolymerization; b) radiolabeled Vpr specifically bound to endothelial cell surfact. Mutational analysis suggested that the C-terminal basic domain of Vpr is critical for the binding; c) incubation of endothelial cells with Vpr significantly changed cellular gene expression as measured by differential mRNA display by RT-PCR, and d) in vitro kinase assays showed that Vpr specifically interacted with a protein kinase that phosphorylated myosin light chain. In this research project, we propose to extend these studies to determine the molecular and biochemical mechanisms of these Vpr-induced endothelial cytoskeletal and functional changes. We will focus on four specific aims: 1) to characterize Vpr induced cytoskeletal modifications in endothelial cells; 2) to biochemically and physiologically characterize the Vpr-associated kinase present in endothelial cells; 3) to molecularly clone the cDNAs for the Vpr-associated kinase and examine their effects on endothelial cytoskeletal structure and function, and 4) to identify and characterize cellular genes whose expression is highly regulated by Vpr, and to analyze their roles during Vpr-induced cell cycle arrest and Vpr regulation of apoptosis. We believe that the above focused specific aims should help understand the contribution of Vpr to endothelial dysfunction during HIV infection. Knowledge gained from these studies should also be helpful for understanding HIV-1 pathogenesis in general. We anticipate that this project will generate novel knowledge about the mechanism of Vpr induced cytoskeletal/functional changes in endothelial cells that can benefit the designing of therapeutic strategies.