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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研制提供更好的选择。人类蛋白BST-2最近被确定具有限制HIV-1和其他包膜病毒流出的独特能力。与这一发现相一致的是，HIV-1蛋白Vpu抵消BST-2，从而使病毒后代容易从感染的细胞中逃逸。本申请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，并通过鉴定参与宿主限制和/或病毒拮抗作用的其他蛋白质，来扩展现有的知识基础。本申请的具体目的和研究计划为：S.A.1：绘制BST-2相互作用和降解所必需的TrCP结合结构域之外的Vpu关键残基。这一目标将通过利用偶然发现的天然存在的HIV-1 C亚型Vpu变体来实现，该变体已失去下调BST-2或与BST-2相互作用的能力。活性亚型B Vpu和非活性亚型C 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嵌合体和点突变体的设计和产生，然后评估其对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。这一目标将通过确定泛素在BST-2降解中发挥的作用、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组分的要求来实现。S.A.3：鉴定参与HIV病毒体束缚或作为衔接子参与Vpu介导的BST-2降解的BST-2结合伴侣。将采用三种单独的蛋白质相互作用筛选来实现这一目标，包括改良的酵母双杂交方法、噬菌体展示筛选和基于蛋白质组学的共免疫沉淀策略。然后将评价发现与BST-2相互作用的所有蛋白质对病毒排出和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提供关于新鉴定的称为BST-2的免疫调节剂的抗病毒功能的有价值的观点。