Structural Basis of Retroviral Restriction by TRIM5alpha

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，并通过阻断逆转录和/或病毒遗传物质的整合来限制逆转录病毒感染{Nisole， 2005 #305；塔，2007 #310}。这种抗逆转录病毒的细胞免疫新机制似乎决定了今天活跃的灵长类免疫缺陷病毒的物种倾向。实验证据表明，TRIM5alpha活性的物种特异性差异源于TRIM5alpha对衣壳亲和力的差异。衣壳识别是由TRIM5alpha的B30.2结构域介导的，但TRIM5alpha- ca相互作用的结构基础尚不清楚。该建议的中心假设是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trim5 α -衣壳结合的机制，我将追求以下目标：1。TRIM5alpha B30.2结构域结构：将开发表达和纯化方案，以便生产和标记足够数量的TRIM5alpha蛋白，用于结构和生物物理研究。恒河猴和人类TRIM5alpha B30.2结构域的结构将通过核磁共振测定。TRIM5alpha特异性改变突变对B30.2结构的影响将被研究。将确定rhB30.2和huB30.2之间的结构差异，这些差异导致huTRIM5alpha无法限制HIV。B30.2可变环参与衣壳结合的动态参数将被测量。2. TRIM5alpha识别衣壳的分子基础：参与b30.2衣壳结合的相互作用表面将使用NMR进行鉴定。B30.2和CA在配合物中的相对取向将被确定。将生成B30.2-CA复合物的模型并确定关键的B30.2-CA相互作用。结构模型将使用诱变、生物物理和体内分析进行测试。NMR将用于检测CypA-CA和trim5 α -ca结合之间的协同性，并检查cypa催化的G89-P90肽键的顺反异构化是否影响B30.2-CA相互作用。公共卫生相关性：由艾滋病毒逆转录病毒引起的艾滋病流行是传染性病原体对全球健康构成的主要威胁之一。高等生物体对逆转录病毒病原体有多层免疫力，但HIV已经进化出克服宿主防御的特定机制。本研究的目的是研究灵长类TRIM5alpha蛋白限制逆转录病毒的机制，阐明病毒逃避策略，并探讨是否可以通过药理学手段恢复人类TRIM5alpha蛋白限制HIV的能力。

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.