Vpr as a Mediator Proteasomal Degradation

描述(申请人提供)：HIV1蛋白VPR在原发患者分离株中高度保守，在体外对建立巨噬细胞的低滴度感染和促进分裂细胞中的病毒复制非常重要。VPR如何在这些能力和体内发挥作用仍然知之甚少。观察到，包括HIV2在内的几种病毒复制了VPR，产生了两个开放阅读框架VPR和VPX，这突显了VPR对灵长类全病毒感染的重要性。这种重复允许了VPR功能的分歧，并可能进一步优化VPR功能。该复制还支持SLV1 VPR至少具有两个功能的假设。在实验感染中，VPR/x的产生已被禁用，显示出致病性降低。我们的工作重点是发现HIV1 VPR通过识别它参与的细胞蛋白所起的作用。我们将这些蛋白质与其HIV2对应物靶向的蛋白质进行了比较，假设尽管这些蛋白质具有不同的hi序列，但它们的功能仍然重叠，当时*蛋白质伙伴的身份也应该如此。我们的初步研究，利用免疫共沉淀和串联质谱学，发现HTV1和2VPR，像一些副粘病毒的V蛋白一样，都与包括DDB1在内的泛素连接酶复合体结合。我们假设，VPR和V蛋白一样，利用DDB1到新的泛素连接酶来靶向蛋白酶体降解的抗病毒因子。特定的目标1将集中于测试VPR的能力，如V-蛋白，作为泛素连接酶机制的适配器，从而促进Stat信号蛋白的降解，或作为先前描述的VPR介导的尿嘧啶-N-糖基酶特异性破坏的中间产物。AIM 2将确定VPR与泛素连接酶复合体的相互作用如何影响HIV感染。具体目标3将集中于确定VPR介导的泛化靶标的身份，这对VPR介导的细胞周期停滞至关重要，并检查VPR参与促进泛化的复合体的正常功能。

DESCRIPTION (provided by applicant): The HIV1 protein Vpr is highly conserved hi primary patient isolates and is, in vitro, important for establishing low titer infections in macrophages and enhancing virus replication in dividing cells. How Vpr unctions in these capacities and in vivo remains poorly understood. The importance of Vpr for primate entivirus infections is underscored by the observation that several of these viruses, including HIV2, have duplicated vpr to generate two open reading frames, vpr and vpx. This duplication has permitted divergence and presumably, further optimization of Vpr functions. The duplication also supports the supposition that SLV1 Vpr has at least two functions. Experimental infections in which Vpr/x production has been disabled demonstrate diminished pathogenicity. Our work has focused on discovering the role that HIV1 Vpr plays by dentifying cellular proteins that it engages. We compare these with the proteins targeted by its HIV2 counterparts under the hypothesis that although these proteins have diverged hi sequence, their functions still overlap as should the identity of then* protein partners. Our preliminary studies, employing co- immunoprecipitation and tandem mass spectroscopy, have revealed that HTV1 and 2 Vpr, like the V proteins of some paramyxoviruses, both engage an ubiquitin ligase complex that includes DDB1. We hypothesize that Vpr, like the V proteins, targets anti-viral factors for proteasomal degradation using DDB1 to recrui ubiquitin ligases. SPECIFIC AIM 1 will focus on testing the ability of Vpr to, like the V-proteins, act as an adaptor to the ubiquitin ligase machinery and thereby to promote degradation of Stat signaling proteins, or to function as an intermediate in the previously described Vpr-mediated destruction of uracil-N-glycosylase SPECIFIC AIM 2 will determine how the Vpr interactions with ubiquitin ligase complexes impact HIV infections. SPECIFIC AIM 3 will focus on determining the identity of targets of Vpr-mediated ubiquitilation important for Vpr-mediated cell cycle arrest and examining the normal function of the complexes that Vpr engages to promote ubiquitilation.