Vpr as a Mediator Proteasomal Degradation

描述（由申请人提供）：HIV1蛋白Vpr在主要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 以生成两个开放阅读框 vpr 和 vpx，这一观察结果强调了 Vpr 对灵长类全病毒感染的重要性。这种重复允许 Vpr 功能的分歧和进一步优化。该复制还支持 SLV1 Vpr 具有至少两个功能的假设。 Vpr/x 产生被禁用的实验感染表明致病性减弱。我们的工作重点是通过识别 HIV1 Vpr 所结合的细胞蛋白来发现 HIV1 Vpr 所发挥的作用。我们将这些蛋白质与其 HIV2 对应物靶向的蛋白质进行比较，假设虽然这些蛋白质具有不同的 hi 序列，但它们的功能仍然重叠，就像 then* 蛋白质伙伴的身份一样。我们采用免疫共沉淀和串联质谱进行的初步研究表明，HTV1 和 2 Vpr 与某些副粘病毒的 V 蛋白一样，都与包含 DDB1 的泛素连接酶复合物结合。我们假设 Vpr 与 V 蛋白一样，通过使用 DDB1 招募泛素连接酶来靶向抗病毒因子进行蛋白酶体降解。具体目标 1 将重点测试 Vpr 的能力，如 V 蛋白，充当泛素连接酶机制的适配器，从而促进 Stat 信号蛋白的降解，或在先前描述的 Vpr 介导的尿嘧啶-N-糖基化酶破坏中充当中间体。具体目标 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。具体目标 3 将重点确定对 Vpr 介导的细胞周期停滞很重要的 Vpr 介导的泛素化靶标的身份，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.