RNA Aptamers that Differentiate Among HIV-1 Capsid Assembly States

项目摘要 成熟的HIV-1衣壳核心因其在HIV感染性中的关键作用而成为关键的抗病毒靶点 以及发现衣壳特异的寄主限制因子，如TRIM5α和MX2。最近的工作有 证明衣壳不只是存放病毒遗传物质，还需要 复制酶。它还参与并可以调解几个关键复制事件， 包括脱壳、启动反转录、核导入、整合和逃避宿主免疫 回应。对于这些过程中的每一个，衣壳的稳定性和解离之间的微妙平衡 必须保持，证明衣壳和病毒传染性之间存在复杂的联系。 成熟的衣壳格子是在蛋白酶介导的Gag多聚蛋白被切割后形成的。最基本的 成熟晶格的结构元件是衣壳蛋白(CA)六聚体，由CA二聚体三聚体组成。 富勒烯锥体结构包含约250个六角体，以及12个五聚体，以便于关闭。突变 改变衣壳蛋白(CA)组装状态的相对稳定性(二聚体、五聚体、六聚体或 组装的晶格)由于一个或多个复制事件的中断而导致严重的传染性缺陷。 复制也可以通过宿主因子结合的改变而受到影响。值得注意的是，CA在 这些事件并未得到很好的理解，也不清楚各种CA程序集状态如何促成 衣壳的功能或与复制中涉及的宿主因子的相互作用。目前还没有工具可用于 区分体内的CA组装状态，以评估它们在复制事件中的作用。这个项目将 鉴定和鉴定与组装的六聚体晶格特异结合位点的RNA适配子，并 通过绑定到独特的暴露于溶剂的缝隙来区分CA组装状态，这些缝隙定义 每个独立的CA程序集状态。拟议中的实验充分利用了研究小组的 多靶标适体选择方面的专业知识，选择后生物信息学分析的进展，细胞内 适配子表达、艾滋病毒生物学和先天免疫。重要的是，拟议的方法可以广泛应用 适用于其他对公众健康有重要影响的病毒。

Project Summary The mature HIV-1 capsid core has emerged as a key antiviral target because of its critical role in HIV infectivity and the discovery of capsid-specific host restriction factors, such as TRIM5α and MX2. Recent work has demonstrated that capsid does much more than simply house the viral genetic material and required replication enzymes. It also participates in and may mediate several critical replication events, including uncoating, initiation of reverse transcription, nuclear import, integration, and evasion of host immune responses. For each of these processes to occur, a delicate balance between capsid stability and dissociation must be maintained, demonstrating an intricate link between capsid and viral infectivity. The mature capsid lattice is formed following protease-mediated cleavage of the Gag polyprotein. The basic structural element of the mature lattice is a capsid protein (CA) hexamer, comprising a trimer of CA dimers. The fullerene cone structure contains ~250 hexamers, along with 12 pentamers to facilitate closing. Mutations that alter the relative stability of capsid protein (CA) assembly states (dimers, pentamers, hexamers, or the assembled lattice) result in severe infectivity defects due to disruption of one or more replication events. Replication can also be impacted through alteration of host factor binding. Notably, the specific roles of CA in these events is not well understood, and it is unknown how the various CA assembly states contribute to capsid function or interactions with host factors involved in replication. There are currently no tools available to differentiate CA assembly states in vivo to assess their role during replication events. This project will identify and characterize RNA aptamers that bind sites specific to the assembled hexamer lattice and differentiate among CA assembly states by binding to unique solvent-exposed crevices that define each independent CA assembly state. The proposed experiments capitalize on the research team's expertise in poly-target aptamer selection, advances in post-selection bioinformatics analysis, intracellular aptamer expression, HIV biology, and innate immunity. Importantly, the proposed approach could be widely applicable to other viruses of importance to public health.