Characterization of Vpu-mediated degradation of BST-2

描述（由申请人提供）：在过去25年中，我们在艾滋病毒-1的基本知识和治疗方面取得了显着进展，然而，尽管取得了这些成就，艾滋病仍然是一种全球流行病，每年导致数百万人死亡。因此，我们必须继续扩大我们对这种病毒及其与宿主相互作用的认识，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。人类蛋白BST-2最近被确定具有限制HIV-1和其他包膜病毒出口的独特能力。与这一发现相一致的是，HIV-1蛋白Vpu抵消了BST-2，从而使病毒的后代很容易从被感染的细胞中逃脱。本应用程序的目标是通过全面了解HIV Vpu如何抑制新的先天免疫分子BST-2，以及通过识别参与宿主限制和/或病毒拮抗的其他蛋白质，来扩展现有的知识库。本应用的具体目的和研究计划是：S.A.1：绘制BST-2相互作用和降解所必需的trcp结合域外Vpu的关键残基。这一目标将通过利用偶然发现的自然发生的HIV-1亚型C Vpu变体来实现，该变体失去了下调BST-2或与BST-2相互作用的能力。通过对活性的B亚型Vpu和无活性的C亚型Vpu蛋白的序列比较，发现了潜在的区域和残基。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降解的机制。这一目标将通过确定泛素在BST-2降解中的作用、Vpu作用于BST-2的时间和细胞位置、Vpu存在下BST-2降解的形式以及这些过程中对ESCRT成分的需求来实现。目的：确定BST-2结合伴侣参与HIV病毒粒子的捆绑或作为适配器参与vpu介导的BST-2降解。三种单独的蛋白质相互作用筛选将被用于实现这一目标，包括改良的酵母2-杂交方法，噬菌体显示屏幕和基于蛋白质组学的共免疫沉淀策略。所有被发现与BST-2相互作用的蛋白质将被评估其对病毒输出和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致病性的一个重要组成部分，因此在本应用中提出的对这种病毒/宿主相互作用的综合分析将提供可能导致新的抗病毒靶点的重要见解。本应用程序中提出的遗传和功能分析也将为新发现的免疫调节剂BST-2的抗病毒功能提供有价值的视角。

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. PUBLIC HEALTH RELEVANCE: Approximately 2 million people die from HIV/AIDS every year. Understanding how HIV is able to overcome the host's innate immune response to restrict viral release may lead to new strategies to treat or prevent infection or its consequences. To this end, this application seeks to define the mechanism through which the HIV-1 protein Vpu counteracts the activity of a newly identified cellular virion release inhibitor, BST-2. Strategies that neutralize Vpu activity or sustain BST-2 function may help to reduce viral burden and restrict HIV spread within or between susceptible hosts.