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: 8052890
• 负责人: Jacek Skowronski
• 金额: $ 38.47万
• 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/8052890
• 关键词: Acquired Immunodeficiency Syndrome; Adaptor Signaling Protein; Address; Affinity; Apoptotic; Binding; Binding Proteins; Biochemical; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cells; Chromatography; Complex; Coupled; Cullin Proteins; 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; programs; protein complex; public health relevance; 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（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，可以改变细胞环境，更有利于这些病毒在宿主中的复制。 Vpr 是一种小型多功能衔接蛋白，由所有灵长类慢病毒编码，干扰受感染细胞中的 DNA 代谢和细胞周期进程。这导致它们在 DNA 损伤检查点处停滞在 S 晚期/G2 早期，并激活细胞凋亡程序。 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。尽管 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。为了获得对 Vpr 和 Vpx 利用的机制的新见解，我们从细胞中免疫亲和纯化了它们，并通过多维色谱耦合串联质谱 (MudPIT) 鉴定了其相关蛋白。这些研究表明，Vpr 和 Vpx 与包含组装在 Cullin-4 支架 (Cul4-DDB1[VprBP]) 上的 E3 泛素连接酶亚基的蛋白质复合物特异性且丰富地关联，我们将其与细胞周期和 DNA 复制的控制联系起来，并确定了 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。该特性与 Vpr 将细胞阻滞在 G2 期的能力相关。综合证据表明，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组的泛素化来发挥其功能。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，对于在分子水平上了解它们的功能是必要的。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 Cul4-DDB1[VprBP] E3 复合物的调节。第二个具体目标是评估 Vpx 相关的 VprBP 及其相关的 Cul4 E3 促进巨噬细胞感染的能力。还将讨论使用质谱鉴定的其他精选 Vpx 相关蛋白的作用。第三个具体目标是结合生化纯化技术和 MudPIT 来鉴定 Vpr 和 VprBP 招募的细胞蛋白，以供 Cul4 泛素化。这些研究将为理解 Vpr 和 Vpx 功能背后的分子相互作用、它们如何侵占 Cul4-DDB1[VprBP] E3 泛素连接酶以促进灵长类慢病毒的复制周期提供一个框架，并将对有效策略的合理设计产生影响扰乱它们的功能。 公共卫生相关性：灵长类慢病毒的辅助蛋白（例如 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。