Effect of HIV-1 Vpr on Basic Cellular Functions (II)

描述(申请人提供)：HIV-1 VPR在病毒发病机制中发挥关键作用，因为其功能与病毒前整合复合体的核运输、病毒复制和抑制人类免疫功能有关。然而，人们对这些效应背后的分子机制知之甚少。在本方案中，我们将重点研究VPR对细胞周期G2/M调控和蛋白降解的两个相关影响，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。在NIH R29第一奖的支持下，我们成功地实现了三个提出的具体目标，即：1)确定VPR负责核定位、G2期停滞和细胞杀伤的功能域；2)确定VPR阻断细胞周期时影响细胞的通路；3)研究PP2A在VPR诱导的G2期停滞中的具体作用。我们发现裂解酵母细胞中的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并不通过经典的DNA损伤或复制检查点诱导G2期停滞，而是使用另一种PP2A介导的调控途径。此外，我们还鉴定了一些基因，它们在过表达时抑制了VPR的G2期停滞和核定位，这些抑制基因使我们揭示了VPR在蛋白降解调节中的新角色。在所提出的研究中，我们假设vpr通过一种新的PP2A介导的调控途径(S)诱导G2期停滞，vpr通过与核外周的蛋白酶体相互作用来影响蛋白降解。为了检验这些假设，本文提出了三个新的具体目标。1)研究VPR诱导G2期细胞停滞的新的PP2A调控途径的细胞成分。2)检测VPR对蛋白酶体相关活性的影响，包括蛋白分解和MHC-I类抗原的处理和提呈。3)探讨VPR-HHR23A相互作用的生物学意义及其在蛋白降解中的特殊作用。拟议的研究结合了在哺乳动物和分裂酵母模型系统中使用的生化和遗传学方法，这将对VPR对这两种基本细胞功能的影响的基本方面产生重要的新见解。

DESCRIPTION (provided by applicant): HIV-1 Vpr plays a pivotal role in viral pathogenesis, as its functions are being linked to nuclear transport of viral 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 Vpr 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. With support of the NIH R29 First Award, we have successfully accomplished the three proposed Specific Aims, i.e., 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 role 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 classic DNA damage or replication checkpoints but instead uses an alternative PP2A-mediated regulatory pathway. In addition, we have identified a number of genes which when overexpressed suppress the G2 arrest and nuclear localization of Vpr, and these suppressors led us to uncover 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-mediated 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-mediated regulatory pathway by which Vpr induces G2 arrest. 2) To test the potential effect of Vpr on proteasome-related activities including proteolysis and MHC class I antigen processing and presentation. 3) To investigate biological significance of the Vpr-HHR23A interaction and its specific role in proteolysis. The proposed studies combine the use of biochemical and genetic approaches in both mammalian and fission yeast model system, which should yield important new insights into fundamental aspects of the effect of Vpr on these two basic cellular functions.