Structural Basis of Retroviral Restriction by TRIM5alpha

描述(申请人提供)：TRIM5pha蛋白在进入细胞后与逆转录病毒衣壳结合，并通过阻止病毒遗传物质的逆转录和/或整合来限制逆转录病毒感染[Nisole，2005#305；Towers，2007#310}。这种针对逆转录病毒的新的细胞免疫机制似乎决定了当今活跃的灵长类免疫缺陷病毒的物种取向。实验证据表明，TRIM5α活性的物种差异源于TRIM5α对衣壳的亲和力不同。衣壳识别是由TRIM5pha的B30.2结构域介导的，但TRIM5pha与CA相互作用的结构基础尚不清楚。这一建议的中心假设是，B30.2-衣壳界面的结构差异解释了TRIM5pha活性的物种差异。为了阐明TRIM5α与衣壳结合的机制，我将致力于以下目标：1.TRIM5αB30.2结构域的结构：将建立表达和纯化方案，以生产和标记足够数量的TRIM5α蛋白，用于结构和生物物理研究。恒河猴和人类TRIM5Alpha B30.2结构域的结构将使用核磁共振确定。将研究TRIM5pha特异性改变突变对B30.2结构的影响。将确定导致huTRIM5pha无法限制HIV的rhB30.2和huB30.2之间的结构差异。将测量涉及衣壳结合的B30.2可变环的动态参数。2.TRIM5pha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核磁共振进行鉴定。将确定B30.2和CA在络合物中的相对取向。将产生B30.2-CA复合体的模型，并确定关键的B30.2-CA相互作用。结构模型将使用诱变、生物物理和体内测试进行测试。核磁共振将用于检测CypA-CA和TRIM5α-CA结合之间的协同性，并检查CypA催化的HIV-1衣壳G89-P90肽键的顺反异构化是否影响B30.2-CA相互作用。与公共卫生的相关性：艾滋病毒逆转录病毒引起的艾滋病流行是传染病病原体对全球健康构成的主要威胁之一。高等生物对逆转录病毒病原体有多层免疫，但艾滋病毒已经进化出特定的机制来克服宿主防御。本研究的目的是研究灵长类TRIM5pha蛋白限制逆转录病毒的机制，阐明病毒逃避策略，并探索能否通过药理学手段恢复人类TRIM5pha不能限制HIV的状态。

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.