VPR-INDUCED ENDOTHELIAL CYTOSKELETAL/FUNCTIONAL CHANGES

描述(改编自申请人的描述) 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。尽管VPR的许多活动已经被 过去发现的VPR功能的生化机制仍然存在 难以捉摸。在HIV-1感染期间，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。两国之间的相互作用 HIV-1和内皮细胞层显著影响宿主免疫 HIV-1在多个组织/器官中的反应和致病机制。在.期间 在我们的初步研究中，我们对这种影响有四个新的发现 VPR对内皮细胞的影响：a)细胞外VPR显著改变 内皮肌动蛋白结构。体外试验也表明， 肌动蛋白解聚的剂量反应Vpr阻断；b)放射性标记的vpr 与内皮细胞表面特异性结合。突变分析 提示VPR的C-末端基本结构域对 结合；c)内皮细胞与vpr的孵育显著改变 RT-PCR差异显示法检测细胞基因表达， 和d)体外激酶分析表明，vpr与 使肌球蛋白轻链磷酸化的蛋白激酶。在这项研究中 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血管内皮细胞骨架的生化机制 功能变化。我们将重点关注四个具体目标：1) 表征VPR诱导内皮细胞的细胞骨架改变；2) VPR相关激酶的生化和生理学特征 存在于内皮细胞中；3)从分子上克隆 VPR相关激酶及其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 它们的表达受vpr高度调控，并分析它们的作用 在VPR诱导的细胞周期停滞和VPR对细胞凋亡的调控过程中。我们 我认为以上重点关注的具体目标应该有助于理解 VPR在HIV感染过程中内皮功能障碍中的作用。 从这些研究中获得的知识也应该有助于理解 HIV-1的一般发病机制。我们预计这个项目将 产生关于VPR诱导机制的新知识 内皮细胞的细胞骨架/功能变化有助于 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.