Structural Basis of Retroviral Restriction by TRIM5alpha

TRIM5alpha 限制逆转录病毒的结构基础

• 批准号: 7755507
• 负责人: DMITRI N IVANOV
• 金额: $ 9.48万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-23 至 2010-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7755507
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Affinity; Binding; Biological Assay; Capsid; Cells; Cellular Immunity; Complex; Epidemic; Genetic Materials; Goals; HIV; HIV-1; Health; Host Defense; Human; Immunity; In Vitro; Infection; Infectious Agent; Investigation; Label; Macaca mulatta; Measurement; Measures; Mediating; Modeling; Molecular; Monitor; Mutagenesis; Mutation; NMR Spectroscopy; Organism; Peptides; Phase; Primate Lentiviruses; Primates; Production; Property; Protein Binding; Proteins; Protocols documentation; Recombinant Proteins; Recombinants; Relative (related person); Resolution; Retroviridae; Reverse Transcription; Specificity; Structural Models; Structure; Surface; Testing; Tropism; Viral; Virus-like particle; base; chaperonin; cis trans isomerization; human TFRC protein; human TRIM5alpha; in vivo; mutant; novel; pathogen; public health relevance; virus genetics

DESCRIPTION (provided by applicant): TRIM5alpha proteins bind retroviral capsids after cell entry and restrict retroviral infection by blocking reverse transcription and/or integration of the viral genetic material {Nisole, 2005 #305; Towers, 2007 #310}. This novel mechanism of cellular immunity against retroviruses appears to determine the species tropism of the primate immunodeficiency viruses active today. Experimental evidence suggests that species-specific differences in TRIM5alpha activity arise from differences in TRIM5alpha affinity for the capsid. Capsid recognition is mediated by the B30.2 domain of TRIM5alpha, but the structural basis of TRIM5alpha-CA interactions is unknown. The central hypothesis of this proposal is that structural differences at the B30.2- capsid interface explain species-specific differences in TRIM5alpha activity. In order to elucidate the mechanism of the TRIM5alpha-capsid binding I will pursue the following objectives: 1. TRIM5alpha B30.2 domain structure: Expression and purification protocols will be developed in order to produce and label TRIM5alpha proteins in sufficient quantities for structural and biophysical studies. Structures of the rhesus and human TRIM5alpha B30.2 domains will be determined using NMR. Effects of the TRIM5alpha specificity-altering mutations on B30.2 structure will be investigated. Structural differences between rhB30.2 and huB30.2 responsible for the inability of the huTRIM5alpha to restrict HIV will be determined. Dynamic parameters of the B30.2 variable loops involved in capsid binding will be measured. 2. Molecular basis of capsid recognition by TRIM5alpha: Interaction surfaces involved in B30.2-capsid binding will be identified using NMR. Relative orientation of B30.2 and CA in the complex will be determined. A model of the B30.2-CA complex will be produced and key B30.2-CA interactions identified. Structural models will be tested using mutagenesis, biophysical and in-vivo assays. NMR will be used to detect cooperativity between CypA-CA and TRIM5alpha-CA binding and to check whether CypA-catalyzed cis-trans isomerisation of the G89-P90 peptide bond of the HIV-1 capsid affects B30.2-CA interactions. PUBLIC HEALTH RELEVANCE: The AIDS epidemic caused by the HIV retrovirus is one of the leading threats posed to global health by an infectious agent. Higher organisms have multiple layers of immunity against retroviral pathogens, but the HIV has evolved specific mechanisms to overcome host defenses. The goal of this proposal is to investigate the mechanism of retroviral restriction by the primate TRIM5alpha proteins, to elucidate viral evasion strategies and to explore whether the inability of the human TRIM5alpha to restrict HIV could be restored by pharmacological means.

描述（由申请人提供）：Trim5alpha蛋白在细胞进入后结合逆转录病毒衣壳，并通过阻断病毒遗传材料的逆转录和/或整合来限制逆转录病毒感染{Nisole，2005＃305；塔，2007＃310}。这种针对逆转录病毒的细胞免疫力的新型机制似乎决定了今天活跃的灵长类动物免疫缺陷病毒的物种对潮流。实验证据表明，Trim5alpha活性的物种特异性差异是由Capsid的Trim5alpha亲和力的差异引起的。衣壳识别是由trim5alpha的B30.2结构域介导的，但是Trim5alpha-CA相互作用的结构基础尚不清楚。该提议的中心假设是，b30.2-衣壳界面的结构差异解释了Trim5alpha活性的物种特异性差异。为了阐明Trim5alpha-Capsid结合的机制，我将追求以下目标：1。Trim5alphab30.2域结构：将开发表达和纯化方案，以生产和标记Trim5alpha蛋白，以足够的结构和生物物理研究的量。将使用NMR确定恒河类和人类Trim5alpha B30.2结构域的结构。将研究Trim5alpha特异性突变对B30.2结构的影响。 RHB30.2和HUB30.2之间的结构差异将确定Hutrim5alpha限制HIV的能力。将测量与衣壳结合的B30.2变量回路的动态参数。 2。trim5alpha的衣壳识别的分子基础：将使用NMR鉴定与B30.2-Capsid结合所涉及的相互作用表面。将确定复合物中B30.2和Ca的相对取向。将产生B30.2-CA复合物的模型，并确定键B30.2-Ca相互作用。结构模型将使用诱变，生物物理和体内测定法进行测试。 NMR将用于检测CYPA-CA和TRIM5ALPHA-CA结合之间的协同性，并检查HIV-1 Capsid的G89-P90肽键的CYPA催化的顺式Trans异构化是否会影响B30.2-CA的相互作用。公共卫生相关性：HIV逆转录病毒引起的艾滋病流行是传染病患者对全球健康构成的主要威胁之一。较高的生物体对逆转录病毒病原体具有多种免疫力，但是艾滋病毒已经发展了特定的机制以克服宿主防御能力。该提案的目的是研究灵长类动物Trim5alpha蛋白的逆转录病毒限制机制，以阐明病毒逃避策略，并探索是否可以通过药理手段来恢复人类Trim5alpha限制HIV的能力。