Vpr as a Mediator Proteasomal Degradation

描述（由申请方提供）：HIV 1蛋白Vpr在原代患者分离株中高度保守，并且在体外对于在巨噬细胞中建立低滴度感染和增强病毒在分裂细胞中的复制很重要。VP如何在这些能力和体内发挥作用仍然知之甚少。Vpr对于灵长类动物病毒感染的重要性通过以下观察而得到强调，即这些病毒中的几种，包括HIV 2，已经复制了vpr以产生两个开放阅读框架，vpr和vpx。这种重复允许了Vpr功能的发散和进一步优化。这种重复也支持了SLV 1 Vpr至少有两种功能的假设。Vpr/x生产已被禁用的实验感染表明致病性降低。我们的工作重点是通过识别它所参与的细胞蛋白来发现HIV 1 Vpr所起的作用。我们将这些蛋白与HIV 2对应物靶向的蛋白进行比较，假设这些蛋白虽然在序列上有分歧，但它们的功能仍然重叠，因为它们的蛋白伴侣的身份也应该重叠。我们的初步研究，采用免疫共沉淀和串联质谱，揭示了HTV 1和2 Vpr，像一些副粘病毒的V蛋白，都从事泛素连接酶复合物，包括DDB 1。我们假设Vpr，像V蛋白，靶向抗病毒因子的蛋白酶体降解使用DDB 1招募泛素连接酶。特异性AIM 1将集中于测试Vpr的能力，像V蛋白一样，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-糖基化酶破坏的中间体。特异性AIM 2将确定Vpr与泛素连接酶复合物的相互作用如何影响HIV感染。特异性目的3将侧重于确定Vpr介导的泛素化的靶标的身份，这些靶标对Vpr介导的细胞周期停滞很重要，并检查Vpr参与促进泛素化的复合物的正常功能。

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.