Characterization of Vpu-mediated degradation of BST-2

Vpu 介导的 BST-2 降解的表征

基本信息

批准号：
8646860
负责人：
ASHLEE V. MOSES
金额：
$ 40.59万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8646860
关键词：
AIDS/HIV problem Achievement Acquired Immunodeficiency Syndrome Adaptor Signaling Protein Address Affect Anti-HIV Therapy Antigens Antiviral Agents Binding Binding Proteins Binding Sites Biochemical Bone Marrow C-terminal CAMLG gene Cell surface Cells Chimera organism Co-Immunoprecipitations Complex Conflict (Psychology)Cysteine Data Degradation Pathway Down-Regulation Elements Endocytosis Generations Genetic Goals HIV HIV-1 Human Hybrids Immune Immune response Infection prevention Interferons Investigation Knowledge Lead Location Lysine Lysosomes Maps Mediating Membrane Modeling Mutagenesis Nature Pathogenesis Pathogenicity Pathway interactions Phage Display Play Process Proteins Proteomics Publications Recycling Research Role Sequence Analysis Serine Small Interfering RNA Surface Threonine Time Transmembrane Domain Ubiquitin Ubiquitination Variant Viral Viral Load result Virion Virus Yeasts base cell type comparative design inhibitor/antagonist insight interest knowledge base mutant novel pandemic disease particle prevent public health relevance research study screening vaccine development virus host interaction vpu Protein

项目摘要

DESCRIPTION (provided by applicant): Over the last 25 years, remarkable progress has been made in our basic knowledge and treatment of HIV-1, Despite such achievements however, AIDS remains a global pandemic that results in millions of fatalities each year. We must therefore continue to expand our knowledge of the virus and its interaction with the host in order to generate better options for both effective anti-HIV therapies and successful vaccine development. The human protein BST-2 was recently determined to possess the unique ability to restrict the egress of HIV-1 and other enveloped viruses. Coincident with this discovery was the finding that the HIV-1 protein Vpu counteracts BST-2, thereby allowing viral progeny to readily escape from infected cells. The goal of this application is to expand the current knowledge base by achieving a comprehensive understanding of how HIV Vpu subjugates the novel innate immune molecule BST-2, and by identifying other proteins involved in the host restriction and/or viral antagonism. The Specific Aims and Research Plan of this application are: S.A.1: To map the critical residues of Vpu outside of the TrCP-binding domain that are necessary for BST-2 interaction and degradation. This aim will be accomplished by exploiting the serendipitous discovery of a naturally occurring HIV-1 subtype C Vpu variant that has lost the ability to downregulate or interact with BST-2. A sequence comparison between the active subtype B Vpu and the inactive subtype C Vpu proteins has revealed potential regions and residues of interest. These findings will be used to direct the design and generation of Vpu chimeras and point mutants that will then be assessed for their impact upon BST-2 binding and degradation. S.A.2: To characterize the mechanism of Vpu-mediated degradation of BST-2. This aim will be achieved by determining the role that ubiquitin plays in BST-2 degradation, the timing and cellular location of Vpu action upon BST-2, the forms of BST-2 that are degraded in the presence of Vpu, and the requirement for ESCRT components in these processes. S.A.3: To identify BST-2 binding partners that are involved in HIV virion tethering or that act as adapters participating in Vpu-mediated BST-2 degradation. Three separate protein- interaction screens will be employed to accomplish this aim, including a modified yeast 2-hybrid approach, a phage display screen, and a proteomics-based co-immunoprecipitation strategy. All proteins that are found to interact with BST-2 will then be evaluated for their impact upon both viral egress and Vpu-dependent BST-2 degradation. Numerous publications have demonstrated that the ability of Vpu to enhance viral release is an important component of HIV pathogenicity, so the comprehensive analysis of this virus/host interaction proposed in this application will provide important insights that could lead to novel antiviral targets. The genetic and functional analyses proposed in this application will also provide valuable perspectives regarding the antiviral function of the newly identified immune modulator known as BST-2.

描述（由申请人提供）：在过去的 25 年里，我们在 HIV-1 的基础知识和治疗方面取得了显着的进步。然而，尽管取得了这些成就，艾滋病仍然是一种全球性流行病，每年导致数百万人死亡。因此，我们必须继续扩大对病毒及其与宿主相互作用的了解，以便为有效的抗艾滋病毒疗法和成功的疫苗开发提供更好的选择。最近确定人类蛋白 BST-2 具有限制 HIV-1 和其他包膜病毒流出的独特能力。与这一发现相一致的是，HIV-1 蛋白 Vpu 可以抵消 BST-2，从而使病毒后代能够轻松逃离受感染的细胞。该应用的目标是通过全面了解 HIV Vpu 如何抑制新型先天免疫分子 BST-2，并识别参与宿主限制和/或病毒拮抗作用的其他蛋白质，来扩展当前的知识库。本申请的具体目标和研究计划是： S.A.1：绘制 TrCP 结合域之外的 Vpu 关键残基图谱，这些残基对于 BST-2 相互作用和降解是必需的。这一目标将通过利用天然存在的 HIV-1 C 亚型 Vpu 变体的偶然发现来实现，该变体已丧失下调 BST-2 或与 BST-2 相互作用的能力。活性 B 亚型 Vpu 和失活 C 亚型 Vpu 蛋白之间的序列比较揭示了潜在的感兴趣区域和残基。这些发现将用于指导 Vpu 嵌合体和点突变体的设计和生成，然后评估它们对 BST-2 结合和降解的影响。 S.A.2：表征 Vpu 介导的 BST-2 降解机制。该目标将通过确定泛素在 BST-2 降解中所起的作用、Vpu 对 BST-2 作用的时间和细胞位置、在 Vpu 存在下降解的 BST-2 形式以及对 BST-2 的要求来实现。这些过程中的 ESCRT 组件。 S.A.3：鉴定参与 HIV 病毒颗粒束缚或充当参与 Vpu 介导的 BST-2 降解的接头的 BST-2 结合伴侣。将采用三种独立的蛋白质相互作用筛选来实现这一目标，包括改良的酵母2-杂交方法、噬菌体展示筛选和基于蛋白质组学的免疫共沉淀策略。然后将评估所有被发现与 BST-2 相互作用的蛋白质对病毒流出和 Vpu 依赖性 BST-2 降解的影响。许多出版物已经证明，Vpu 增强病毒释放的能力是 HIV 致病性的重要组成部分，因此本申请中提出的这种病毒/宿主相互作用的综合分析将提供重要的见解，从而可能导致新的抗病毒靶点。本申请中提出的遗传和功能分析还将为新鉴定的免疫调节剂 BST-2 的抗病毒功能提供有价值的观点。