Structural studies of HIV Capsid with host factors and Capsid-targeting antivirals

 描述(由申请人提供)：HIV-1衣壳蛋白(CA)在病毒生命周期的多个步骤中发挥关键作用。它是Gag多聚蛋白的一部分，在成熟过程中被裂解，形成基质、CA和核衣壳。到目前为止，已知CA结构及其对核心稳定性的影响在脱壳、反转录、核进入、整合位置和组装过程中是关键的，也是隐藏DNA产物免受免疫监视的重要因素。HIV-1 CA与宿主因子相互作用，包括TRIM5a、裂解和多聚腺苷化特异因子6(CPSF6)、核孔蛋白153和358(NUP153、Nup358)、MXB和亲环素A。所有这些不同的功能和相互作用都受一种结构的调节。 单一的，非常灵活的蛋白质，CA，因此可以被称为生物瑞士军刀。尽管在过去的20年里已经解决了HIV-1 CA片段或重型工程变体的几种结构，但这些结构都没有提供控制衣壳稳定性的关键六聚体间接触的全部原子细节。萨拉菲亚诺斯实验室最近解决了HIV-1 CA的晶体结构，包括完整的天然全长六聚体CA的难以捉摸的结构。这些结构揭示了以前未知的分子细节，特别是在影响核心稳定性的3倍和2倍内六聚体界面上。它们证明了六角体之间的界面是可塑性的，并可以通过新发现的适应性水化层改变，这被认为是岩心稳定的关键。它们揭示了一种显著的可塑性，这解释了在病毒粒子中观察到的多态现象。他们还验证了我们的实验系统是在没有人工制品的自然环境中研究CA相互作用的最相关的系统。最后，在其他初步研究中，该系统已被用于研究a) 与CA结合的抗病毒药物的结合，b)宿主因子多肽的结合，以及c)到目前为止增加或降低核心稳定性并影响病毒学功能的三个CA突变体。与以往的CA结构不同，我们的结构揭示了六聚体间界面结构变化的新信息，并通过以下目标建立了一个全面的框架，将有助于显著推进该领域的研究：目的1：表征核心稳定性增强或降低的结构决定因素目标2：CA与宿主因子的结构相互作用目标3：使用CA靶向抗病毒探针了解CA生物学功能的机制这些研究将揭开核心稳定性的结构基础，提供关于CA结构如何控制与宿主因子和小分子效应物相互作用的独特信息，并可能揭示各种生物功能的脆弱结构中间体。

 DESCRIPTION (provided by applicant): The HIV-1 capsid protein (CA) plays a critical role in multiple steps of the virus life cycle. It is part of the Gag polyprotein, which is cleaved during maturation to give matrix, CA, and nucleocapsid. So far it is known that CA structure and its effects on core stability are critical for the processes of uncoating, reverse transcription, nucler entry, sites of integration, and assembly, and it is also important for cloaking the DNA product from immune surveillance. HIV-1 CA interacts with host factors including TRIM5a, cleavage and polyadenylation specific factor 6 (CPSF6), nucleoporins 153 and 358 (NUP153, NUP358), MxB and Cyclophilin A. All these diverse functions and interactions are regulated by the structure of a single, remarkably flexible protein, CA, which can thus be termed a biological Swiss Army knife. Although several structures of HIV-1 CA fragments or heavily engineered variants have been solved over the past 20 years, none of these has provided the full atomic details of the critical inter-hexameric contacts that govern capsid stability. The Sarafianos lab recently solved crystal structures of HIV-1 CA, including the elusive structure of the intact native full length hexameric HIV-1 CA. The structures reveal previously unknown molecular details, especially at the 3-fold and 2-fold intra-hexamer interfaces that affect core stability. They demonstrate that the inter-hexamer interfaces are malleable and can change through a newly discovered adaptable hydration layer, which is hypothesized to be critical for core stabilization. They reveal a remarkable plasticity which explains the polymorphism observed in virions. They also validate our experimental system as the most relevant for the study of CA interactions in a native context free of artifacts. Finally, in additional preliminary studies this system has been used to study a) binding of a CA-binding antiviral, b) binding of a host factor peptide, and c) so far three CA mutants that increase or decrease core stability and affect virological functions. Unlike all previous CA structures our structures reveal novel information of the structural changes at the inter-hexamer interfaces and establish a comprehensive framework that will help significantly advance the field through the following Aims: AIM 1: Characterize the structural determinants of increased or decreased core stability AIM 2: Structural interactions of CA with host factors AIM 3: Understanding the mechanisms of CA biological functions using CA-targeting antiviral probes These studies will unravel the structural basis of core stability, provide unique information on how CA structure controls interactions with host factors and small molecule effectors, and may reveal vulnerable structural intermediates of various biological functions.