MOLECULAR BIOLOGY OF LENTIVIRAL VPR AND VPX PROTEINS

慢病毒 VPR 和 VPX 蛋白的分子生物学

• 批准号: 7994402
• 负责人: Jacek Skowronski
• 金额: $ 22.44万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7994402
• 关键词: Acquired Immunodeficiency Syndrome; Adaptor Signaling Protein; Address; Affinity; Apoptotic; Binding; Binding Proteins; Biochemical; Cell Culture Techniques; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cells; Cellular biology; Chromatography; Complex; DNA; DNA biosynthesis; DNA damage checkpoint; Development; Environment; G2 Phase; Goals; HIV-2; Infection; Link; Mass Spectrum Analysis; Mediating; Metabolism; Methods; Molecular; Molecular Biology; Primate Lentiviruses; Property; Proteins; Recruitment Activity; Regulation; Role; SIV; Techniques; Technology; Therapeutic Agents; Ubiquitination; Virulence Factors; Virus; Virus Replication; base; design; insight; macrophage; positional cloning; programs; protein complex; scaffold; ubiquitin ligase; ubiquitin-protein ligase

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.

描述（由申请人提供）：我们的长期目标是了解灵长类动病毒的辅助蛋白在分子水平上的功能。辅助蛋白是重要的毒力因子，可修饰细胞环境更有利于在宿主中复制这些病毒。 VPR是一种小的多功能衔接蛋白，由所有灵长类动病毒编码，并干扰感染细胞中的DNA代谢和细胞周期进程。这导致他们在DNA损伤检查点上的S/早期G2阶段被捕并激活凋亡程序。 VPX是与VPR密切相关的因素，是HIV-2和SIVSM/MAC病毒在巨噬细胞中复制的能力所必需的。尽管VPR和VPX是重要的毒力因素，但介导其功能的分子机制尚不清楚。为了获得对VPR和VPX利用的机制的新见解，它们是从细胞中纯化的免疫亲和力及其通过多维色谱耦合串联串联质谱（MUDPIT）鉴定的相关蛋白质。这些研究表明，VPR和VPX与包含E3泛素连接酶的亚基的蛋白质复合酶特异性和大量相关联，该蛋白质组合在Cullin-4支架上（CUL4-DDB1 [VPRBP]），我们将其与细胞周期和DNA复制的控制链接到其他细胞复制，并鉴定出其他细胞蛋白的对照。重要的是，VPR和VPX似乎调节其结合的CUL4 E3复合物的泛素连接酶活性。该特性与VPR在G2中逮捕细胞的能力相关。证据共同表明，VPR和VPX通过篡夺CUL4-DDB1 [VPRBP] E3泛素连接酶来调节特定但不同的细胞蛋白集的泛素化来执行其功能。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以及对它们在分子水平上了解其功能的理解所必需的细胞蛋白的鉴定。朝向这些目标，第一个具体目标将表征VPR和VPX附件因子对CUL4-DDB1 [VPRBP] E3复合物的调节。第二个特定目的将评估与VPX相关的VPRBP及其相关的CUL4 E3的作用，以促进巨噬细胞感染。还将解决使用质谱鉴定的其他选择VPX相关蛋白的作用。第三个具体目的将通过使用生化纯化技术和泥浆的组合来识别VPR和VPRBP募集的CUL4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，如何篡夺CUL4-DDB1 [VPRBP] E3泛素连接酶，以促进灵长类动物慢病毒的复制周期，并将对有效策略的有效策略破坏其功能。 公共卫生相关性：灵长类动病毒（例如VPR和VPX）的附属蛋白是重要的毒力因素。拟议的研究旨在理解这些蛋白质用来促进灵长类动物复制的分子机制，并可能为开发会延迟艾滋病发展的治疗剂提供基础。