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

HIV-1 Vpr在病毒的发病机制中起着关键作用，因为它的功能与细胞核蛋白有关。 病毒整合前复合物的转运、病毒复制和人免疫功能的抑制。 然而，人们对这些效应背后的分子机制知之甚少。在本提案中，我们 将重点研究Vpr对细胞周期G2/M控制和蛋白水解的两个相关影响，这将有助于 我们可以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 R29一等奖的支持下，我们成功地完成了三项建议， 具体目标，即：1)为了确定Vpr负责核定位的功能域，G2 阻滞和细胞杀伤，2）鉴定当Vpr中断细胞周期时受其影响的细胞通路， （3）研究PP 2A在Vpr诱导的G2期阻滞中的作用。我们发现， 裂殖酵母细胞与哺乳动物细胞非常相似。我们还发现Vpr并不诱导 通过经典的DNA损伤或复制检查点进行G2阻滞，但使用替代方法 PP 2A介导的调节途径。此外，我们已经确定了一些基因， 过表达抑制Vpr的G2期阻滞和核定位，这些抑制因子使我们 揭示Vpr在调节蛋白水解中的新作用。 对于所提出的研究，我们假设Vpr通过一种新的PP 2A介导的 调节途径，Vpr通过与细胞核上的蛋白酶体相互作用影响蛋白水解 外围提出了三个新的具体目标来检验这些假设。1)定义和描述 新PP 2A介导的Vpr诱导G2期阻滞的调节途径的细胞组分。 2)检测Vpr对蛋白酶体相关活性（包括蛋白水解和MHC类）的潜在影响 I抗原加工和呈递。3)探讨Vpr-HHR 23 A的生物学意义 相互作用及其在蛋白质水解中的特殊作用。拟议的研究将生物化学和生物医学的使用联合收割机结合起来， 遗传方法在哺乳动物和裂殖酵母模型系统，这应该产生重要的 对Vpr对这两种基本细胞功能的影响的基本方面的新见解。

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.