Mechanism and Regulation of HIV-1 Uncoating

描述(申请人提供)：HIV-1感染依赖于一系列分子事件，涉及病毒组件和宿主因子之间的生化相互作用，导致病毒和宿主膜融合、逆转录、脱壳、核进入和整合。在感染的早期阶段，一个关键的知识缺口是病毒衣壳I促进逆转录的作用。CA蛋白的突变会破坏病毒衣壳的稳定性，导致逆转录受损，这表明病毒衣壳的完整性是有效合成病毒DNA的关键。HIV-1逆转录酶是一种低加工能力的酶，在逆转录链转移过程中与模板解离。利用纯化的HIV-1核心的生化方法，我们将检验病毒衣壳在逆转录过程中作为维持局部RT浓度的容器的假设，并将研究限制性宿主因素在HIV-1衣壳识别和脱壳中的作用。我们还将确定与HIV-1衣壳结合的新宿主因子。这项工作将由 目的1.确定病毒衣壳促进HIV-1逆转录的机制。目的2.确定对HIV-1脱壳至关重要的亚基间界面。目的3.检测CPSF6-358对HIV-1脱壳的影响。目的4.确定TRIM5限制因子识别功能衣壳的化学计量学要求。目的5.鉴定新的衣壳相互作用的宿主因子。这些研究将回答逆转录病毒生物学中的基本问题，并将阐明内源性宿主细胞因子的限制机制。

DESCRIPTION (provided by applicant): HIV-1 infection depends on a series of molecular events involving biochemical interactions between viral components and host factors, resulting in fusion of viral and host membranes, reverse transcription, uncoating, nuclear entry, and integration. A key knowledge gap in the early stages of infection is the role of the viral capsid i facilitating reverse transcription. Mutations in the CA protein that destabilize the viral capsid result in impaired reverse transcription, indicating that the integrity of the viral capsid is critcal for efficient viral DNA synthesis. HIV-1 reverse transcriptase is a low processivity enzyme and dissociates from the template during strand transfer steps of reverse transcription. Employing a biochemical approach involving purified HIV-1 cores, we will test the hypothesis that the viral capsid serves as a vessel to maintain the local concentration of RT during reverse transcription and will study the role of restrictive host factors in HIV-1 capsid recognition and uncoating. We will also identify novel host factors that bind the HIV-1 capsid. The work will be organized by the following Specific Aims: Aim 1. To determine the mechanism by which the viral capsid facilitates HIV-1 reverse transcription. Aim 2. Identification of intersubunit interfaces critical for HIV-1 uncoating. Aim 3. To determine the effect of CPSF6-358 on HIV-1 uncoating. Aim 4. To determine the stoichiometric requirement for functional capsid recognition by TRIM5 restriction factors. Aim 5. Identification of novel capsid-interacting host factors. These studies will answer fundamental question in retrovirus biology and will clarify the mechanisms of restriction by endogenous host cell factors.