Mechanism of selective packaging of primer tRNALys3 by HIV-1

项目总结： 艾滋病毒-1和艾滋病大流行仍然是对公共卫生的重大威胁。缺乏预防疾病的疫苗 HIV-1病毒和迫在眉睫的耐药突变幽灵促使人们寻找新的药物靶点。关键的一步 HIV-1生命周期的一部分是在病毒粒子组装过程中选择性包装宿主tRNALys 1、2、3，其中tRNALys3 同种异构体后来被用作启动逆转录的引物。这种选择性的tRNALys现象 HIV-1的包装在近30年前被发现，但其结构和分子细节 事件仍然难以捉摸。LysRS(Lysyl-tRNA合成酶)是tRNALys3氨酰化的细胞酶，具有 已被证明在HIV-1对tRNALys的包装中发挥了关键作用。LysRS主要被隔离为部分 多tRNA合成酶复合体(MSC)，但催化失活的S207磷酸化(PS207)池 当感染HIV-1时，细胞LysRS从MSC中释放出来。HIV-1 Gag在LysRS和LysRS中起主要作用 TRNALys3包装。目前的模型假设tRNALys3是由HIV-1 Gag通过一种 其衣壳蛋白(CA)与LysRS催化区的相互作用。但是，此模型基于 主要是在没有tRNALys 3和我们的初步数据的情况下使用未经修饰的LysRS进行的研究 与这一模型相矛盾。HIV-1已被证明选择性地包装不带电的tRNALys 3和过度带电的tRNALys 3 而pS207-LysRS被认为在这种包装偏向中发挥了作用。没有高分辨率的结构 关于pS207-LysRS的信息为了提供其缺乏氨基酰化活性的机制基础，其 与HIV-1 Gag的相互作用，或其执行的非规范功能。缺乏任何结构性的信息 HIV-1Gag招募LysRS和tRNALys3的方式排除了针对药物开发的这一步骤和 拟议的工作将解决这一知识差距。本研究的总体目标是建立一种 以及HIV-1选择性包装不带电的tRNALys 3的结构基础。在目标1中，我们将开展 报道的结合LysRS和tRNALys3的HIV-1因子的综合比较特征 定性和定量的生化和生物物理技术。比较分子解剖将 允许我们建立tRNALys3包装复合体的最低核心组件。我们将使用 拟磷突变LysRS(LysRSS207D)研究pS207-LysRS在选择包装中的作用 不带电荷的tRNALys 3，并研究三元tRNALys 3包装复合体的动力学，以探索任何协作性 在复杂的队形中。彻底的生化特性将有助于建立稳定的三元tRNALys 3 用于结构研究的包装复合体。在目标2中，我们将使用单粒子并行双管齐下的方法。 用低温电子显微镜和X射线结晶学确定三元tRNALys3包装的高分辨率结构 使用基于细胞的下拉、tRNALys包装和HIV-1传染性来复杂和验证我们的结构洞察力 化验。从这项研究中获得的见解将阐明艾滋病毒生物学的一个关键但尚未被开发的方面，用于设计 以及新型抗逆转录病毒药物的开发。

Project Summary: HIV-1 and the AIDS pandemic remains a significant threat to public health. The lack of a vaccine against HIV-1 and the looming specter of drug-resistant mutations urge the search for novel drug targets. A critical step of the HIV-1 life cycle is the selective packaging of host tRNALys1,2,3 during virion assembly, wherein the tRNALys3 isoacceptor is later used as a primer for initiating reverse transcription. This phenomenon of selective tRNALys packaging by HIV-1 was discovered nearly three decades ago, but the structural and molecular details of this event remain elusive. LysRS (lysyl-tRNA synthetase), the cellular enzyme for aminoacylation of tRNALys3, has been shown to play a crucial role in tRNALys packaging by HIV-1. LysRS is predominantly sequestered as part of the multi-tRNA synthetase complex (MSC) but a catalytically inactive S207 phosphorylated (pS207) pool of cellular LysRS is released from the MSC upon HIV-1 infection. HIV-1 Gag plays the primary role in LysRS and tRNALys3 packaging. The current model postulates that tRNALys3 is indirectly packaged by HIV-1 Gag via an interaction between its capsid protein (CA) and the catalytic domain of LysRS. However, this model is based primarily on studies carried out with unmodified LysRS in the absence of tRNALys3 and our preliminary data contradicts this model. HIV-1 has been shown to selectively package uncharged tRNALys3 over charged tRNALys3 and pS207-LysRS has been proposed to play a role in this packaging bias. There is no high-resolution structural information available on pS207-LysRS to provide a mechanistic basis for its lack of aminoacylation activity, its interactions with HIV-1 Gag, or the non-canonical functions it performs. The lack of any structural information on how HIV-1 Gag recruits LysRS and tRNALys3 has precluded targeting this step for drug development and the proposed work will address this knowledge gap. The overall goal of this research is to establish a mechanistic and structural basis for selective packaging of uncharged tRNALys3 by HIV-1. In Aim 1, we will carry out a comprehensive comparative characterization of the HIV-1 factors reported to bind LysRS and tRNALys3 using qualitative and quantitative biochemical and biophysical techniques. The comparative molecular dissection will allow us to establish the minimal core components of the tRNALys3 packaging complex. We will use a phosphomimetic mutant of LysRS (LysRSS207D) to study the role of pS207-LysRS in selective packaging of uncharged tRNALys3 and study the kinetics of the ternary tRNALys3 packaging complex to probe any cooperativity in complex formation. The thorough biochemical characterization will help establish a stable ternary tRNALys3 packaging complex for structural studies. In Aim 2, we will use a parallel two-pronged approach of single particle cryo-EM and X-ray crystallography to determine a high-resolution structure of the ternary tRNALys3 packaging complex and validate our structural insights using cell-based pull-down, tRNALys packaging, and HIV-1 infectivity assays. Insights gained from this study will elucidate a critical yet untapped aspect of HIV biology for the design and development of novel antiretroviral drugs.