MOLECULAR BIOLOGY OF LENTIVIRAL VPR AND VPX PROTEINS

描述（由申请人提供）：我们的长期目标是在分子水平上了解灵长类慢病毒辅助蛋白的功能。辅助蛋白是重要的毒力因子，其改变细胞环境以更有利于这些病毒在宿主中的复制。Vpr是一种多功能的衔接蛋白，由所有灵长类慢病毒编码，并干扰感染细胞中的DNA代谢和细胞周期进程。这导致它们在DNA损伤检查点的S期晚期/G2期早期停滞，并激活凋亡程序。Vpx是与Vpr密切相关的因子，是HIV-2和SIVsm/mac病毒在巨噬细胞中复制的能力所必需的。虽然Vpr和Vpx是重要的毒力因子，但介导其功能的分子机制还不清楚。为了获得Vpr和Vpx所利用的机制的新见解，将它们从细胞中免疫亲和纯化，并通过多维色谱耦合串联质谱（MudPIT）鉴定其相关蛋白。这些研究表明，Vpr和Vpx特异性地和大量地与蛋白质复合物结合，该蛋白质复合物包含在Cullin-4支架上组装的E3泛素连接酶的亚基（Cul 4-DDB 1 [VprBP]），我们将其与细胞周期和DNA复制的控制相关联，并鉴定了Vpr和Vpx靶向的其他细胞蛋白。重要的是，Vpr和Vpx似乎调节它们结合的Cul 4 E3复合物的泛素连接酶活性。这种特性与Vpr将细胞阻滞在G2期的能力相关。共同的证据表明，Vpr和Vpx通过篡夺Cul 4-DDB 1 [VprBP] E3泛素连接酶来调节特定但不同的细胞蛋白组的泛素化来执行其功能。因此，了解这些毒力因子与Cul 4-DDB 1 [VprBP] E3连接酶复合物的相互作用，并鉴定它们改变其泛素化的细胞蛋白质，是在分子水平上了解其功能所必需的。为了实现这些目标，第一个具体目标将表征Vpr和Vpx辅助因子对Cul 4-DDB 1 [VprBP] E3复合物的调节。第二个具体目标将评估Vpx相关的VprBP及其相关Cul 4 E3促进巨噬细胞感染的能力的作用。其他选择Vpx相关的蛋白质鉴定使用质谱的作用也将得到解决。第三个具体目标将通过使用生化纯化技术和MudPIT的组合来鉴定由Vpr和VprBP募集的用于Cul 4泛素化的细胞蛋白。这些研究将为理解Vpr和Vpx功能的分子相互作用提供一个框架，它们如何篡夺Cul 4-DDB 1 [VprBP] E3泛素连接酶以促进灵长类慢病毒的复制周期，并将对合理设计有效策略以破坏其功能产生影响。 公共卫生相关性：灵长类慢病毒的辅助蛋白，如Vpr和Vpx，是重要的毒力因子。拟议的研究旨在了解这些蛋白质用于促进灵长类慢病毒复制的分子机制，并可能为开发延缓艾滋病发展的治疗药物提供基础。

DESCRIPTION (provided by applicant): Our long-term objective is to understand the functions of accessory proteins of primate lentiviruses at the molecular level. Accessory proteins are important virulence factors that modify cellular environments to be more conducive for replication of these viruses in the host. Vpr is a small multifunctional adaptor protein, that is encoded by all primate lentiviruses and interferes with DNA metabolism and cell cycle progression in the infected cells. This leads to their arrest in late S/early G2 phase at the DNA damage checkpoint and activates apoptotic program. Vpx, a factor closely related to Vpr, is required for the ability of HIV-2 and SIVsm/mac viruses to replicate in macrophages. Although Vpr and Vpx are important virulence factors, the molecular mechanisms that mediate their functions are not well understood. To gain new insights into mechanisms exploited by Vpr and Vpx, they were immuno-affinity purified from cells and their associated proteins identified by multidimensional chromatography-coupled tandem mass spectroscopy (MudPIT). These studies revealed that Vpr and Vpx specifically and abundantly associate with a protein complex comprising subunits of a E3 ubiquitin ligase assembled on Cullin-4 scaffold (Cul4-DDB1[VprBP]), which we linked to the control of cell cycle and DNA replication, and identified additional cellular proteins targeted by Vpr and Vpx. Importantly, Vpr and Vpx appear to regulate ubiquitin ligase activity of the Cul4 E3 complex they bind. This property correlates with the ability of Vpr to arrest cells in G2. Together evidence suggests that Vpr and Vpx carry out their functions by usurping the Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate ubiquitination of specific but distinct sets of cellular proteins. Therefore understanding the interactions of these virulence factors with the Cul4-DDB1[VprBP] E3 ligase complex and identification of cellular proteins whose ubiquitination they alter is required for the understanding of their functions at the molecular level. Towards these goals the first specific aim will characterize the regulation of Cul4- DDB1[VprBP] E3 complex by Vpr and Vpx accessory factors. The second specific aim will assess the role of Vpx-associated VprBP, and its associated Cul4 E3, for its ability to facilitate macrophage infection. The roles of other select Vpx-associated proteins identified using mass spectroscopy will also be addressed. The third specific aim will identify cellular proteins recruited by Vpr and VprBP for ubiquitination by Cul4, by using a combination of biochemical purification techniques and MudPIT. These studies will provide a framework for understanding the molecular interactions that underlie the functions of Vpr and Vpx, how they usurp Cul4-DDB1[VprBP] E3 ubiquitin ligase to facilitate the replication cycle of primate lentiviruses and will have implications for rational design of effective strategies to disrupt their functions. PUBLIC HEALTH RELEVANCE: Accessory proteins of primate lentiviruses, such as Vpr and Vpx, are important virulence factors. The proposed studies are aimed at understanding molecular mechanism that these proteins use to facilitate replication of primate lentiviruses and may provide the basis for developing therapeutic agents that will delay development of AIDS.