Characterization of Vpu-mediated degradation of BST-2

描述(申请人提供)：在过去的25年里，我们对艾滋病毒-1的基本知识和治疗取得了显著的进展，尽管取得了这些成就，但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该病毒及其与宿主的互动的了解，以便为有效的抗艾滋病毒疗法和成功研制疫苗提供更好的选择。人类蛋白BST-2最近被确定具有独特的能力来限制HIV-1和其他包膜病毒的出口。与这一发现一致的是，HIV-1蛋白VPU与BST-2相互作用，从而使病毒后代容易从受感染的细胞中逃脱。这项应用的目标是通过全面了解HIV VPU如何征服新的天然免疫分子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当前的知识库。本申请的具体目标和研究计划是：S.A.1：定位BST-2相互作用和降解所必需的TrCP结合域之外的VPU的关键残基。这一目标将通过偶然发现一种自然产生的HIV-1 C型VPU变异株来实现，该变异株已经失去了下调BST-2或与BST-2相互作用的能力。活性B亚型VPU和非活性C亚型VPU蛋白之间的序列比较揭示了潜在的感兴趣的区域和残基。这些发现将用于指导VPU嵌合体和点突变的设计和生成，然后将评估它们对BST-2结合和降解的影响。S.A.2：研究VPU介导的BST-2降解机制。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、在VPU存在下降解的BST-2的形式以及这些过程中对ESCRT成分的需求来实现。S.A.3：确定参与HIV病毒粒子拴系或作为接头参与VPU介导的BST-2降解的BST-2结合伙伴。将使用三个独立的蛋白质相互作用屏幕来实现这一目标，包括改进的酵母双杂交方法、噬菌体显示屏和基于蛋白质组学的免疫共沉淀策略。所有被发现与BST-2相互作用的蛋白质都将被评估它们对病毒出口和依赖VPU的BST-2降解的影响。大量文献表明，VPU促进病毒释放的能力是HIV致病性的重要组成部分，因此本申请中提出的对这种病毒/宿主相互作用的全面分析将提供重要的见解，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前景。 公共卫生相关性：每年约有200万人死于艾滋病毒/艾滋病。了解艾滋病毒是如何克服宿主的先天免疫反应来限制病毒释放的，可能会导致治疗或预防感染或其后果的新策略。为此，本申请试图确定HIV-1蛋白VPU通过何种机制中和新发现的细胞病毒粒子释放抑制物BST-2的活性。中和VPU活性或维持BST-2功能的策略可能有助于减少病毒负担，限制艾滋病毒在易感宿主内部或之间传播。

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.