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附属蛋白的功能。辅助蛋白是重要的毒力因子，可以改变细胞环境，使其更有利于这些病毒在宿主体内的复制。Vpr是一种小的多功能接头蛋白，由所有灵长类慢病毒编码，并干扰受感染细胞的DNA代谢和细胞周期进程。这导致它们在DNA损伤检查点的S晚期/早期G2阶段被阻止并激活凋亡程序。Vpx是一个与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尚不清楚。为了进一步了解Vpr和Vpx的作用机制，我们从细胞中对它们进行了免疫亲和纯化，并通过多维色谱耦合串联质谱（MudPIT）对它们的相关蛋白进行了鉴定。这些研究表明，Vpr和Vpx特异且丰富地与一个由组装在Cullin-4支架上的E3泛素连接酶亚基组成的蛋白复合物（Cul4-DDB1[VprBP]）相关联，我们将其与细胞周期和DNA复制的控制联系起来，并鉴定了Vpr和Vpx靶向的其他细胞蛋白。重要的是，Vpr和Vpx似乎可以调节它们所结合的Cul4 E3复合物的泛素连接酶活性。这种特性与Vpr阻滞G2细胞的能力有关。有证据表明，Vpr和Vpx通过篡位Cul4-DDB1[VprBP] E3泛素连接酶来调节特定但不同的细胞蛋白的泛素化，从而发挥其功能。因此，了解这些毒力因子与Cul4-DDB1[VprBP] E3连接酶复合物的相互作用，并鉴定它们改变泛素化的细胞蛋白，是了解它们在分子水平上的功能所必需的。为了实现这些目标，第一个具体目标将描述Vpr和Vpx辅助因子对Cul4- DDB1[VprBP] E3复合物的调节。第二个特定目标将评估vpx相关的VprBP及其相关的Cul4 E3在促进巨噬细胞感染方面的作用。使用质谱鉴定的其他选择vpx相关蛋白的作用也将得到解决。第三个具体目标是通过结合生化纯化技术和MudPIT技术，鉴定由Vpr和VprBP募集的被Cul4泛素化的细胞蛋白。这些研究将为理解Vpr和Vpx功能背后的分子相互作用提供一个框架，它们如何篡夺Cul4-DDB1[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的策略来破坏其功能产生影响。

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.