Effect of HIV-1 Vpr on Basic Cellular Functions

HIV-1 Vpr在病毒的发病机制中起着关键作用，其功能与视觉预整合复合体的核转运、病毒复制和人体免疫功能的抑制有关。然而，人们对这些作用背后的分子机制知之甚少。在本课题中，我们将重点研究Bpr对细胞周期G2/M控制和蛋白水解的两个相关作用，这将有助于我们进一步了解这些病毒对宿主细胞功能影响的分子基础。我们已经成功地完成了三个提出的具体目标：1)确定Vpr负责核定位、G2阻滞和细胞杀伤的功能域；2)确定Vpr中断细胞周期时受其影响的细胞途径；3)研究PP2A在Vpr诱导的G2阻滞中的具体作用。我们发现分裂酵母细胞中的Vpr活性与哺乳动物细胞中的Vpr活性非常相似。我们还发现Vpr不会通过两个经典的DNA损伤或复制检查点诱导G2阻滞，而是通过依赖pp2a的新型调控途径。此外，我们已经确定了一些基因，当过表达时抑制Vpr的G2阻滞和核定位，这些抑制表明Vpr在蛋白质水解调控中的新作用。在我们提出的研究中，我们假设Vpr通过一种新的依赖pp2a的调控途径诱导G2阻滞，Vpr通过与核周围的蛋白酶体相互作用影响蛋白质水解。提出了三个新的具体目标来检验这些假设。1)定义和表征Vpr诱导G2阻滞的新PP2A依赖调控途径的细胞成分。2)研究Vpr与核外周蛋白酶体的潜在相互作用。3)评估前蛋白酶体相互作用在蛋白水解调控中的可能作用，包括vpr诱导的G2阻滞与蛋白酶体活性之间的关系。这些拟议的研究将在人类细胞中得到证实，并为Vpr对这两种基本细胞功能的影响的基本方面提供重要的新见解。

HIV-1 Vpr plays a pivotal role in viral pathogenesis, as its functions are being linked to nuclear transport of visual pre-integration complex, viral replication and suppression of human immune function. However, little is known about the molecular mechanisms underlying these effects. In this proposal, we will focus on studying two related effects of Bpr on cell cycle G2/M control and proteolysis, which will help us to further understand the molecular basis of these viral effects on the host cellular functions. We have successfully accomplished three proposed Specific Aims. 1) to define the functional domains of Vpr responsible for nuclear localization, G2 arrest and cell killing, 2) to identify the cellular pathways affected by Vpr when it interrupts the cell cycle, and 3) to investigate the specific roles of PP2A in Vpr-induced G2 arrest. We showed that Vpr activities in fission yeast cells are very similar to those in mammalian cells. We also found that Vpr does not induce G2 arrest through the two classic DNA damage or replication checkpoints but instead through a PP2A-dependent novel regulatory pathway. In addition, we have identified a number of genes which when over- expressed suppress the G2 arrest and nuclear localization of Vpr, and these suppressions suggest a new role for Vpr in the regulation of proteolysis. For the proposed studies, we hypothesize that Vpr induces G2 arrest through a novel PP2A-dependent regulatory pathway(s), and Vpr affects proteolysis by interaction with the proteasome on the nuclear periphery. Three new specific aims are proposed to test these hypotheses. 1) To define and characterize the cellular components of the new PP2A- dependent regulatory pathway by which Vpr induces G2 arrest. 2) To investigate the potential interaction of Vpr with the proteasome at the nuclear periphery. 3) To evaluate the possible role of pr-proteasome interaction in the regulation of proteolysis including a relationship between Vpr-induced G2 arrest and proteasome activity. These proposed studies will be confirmed in human cells and provide important new insights into fundamental aspects of the Vpr's effects on these two basic cellular functions.