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.
