Mechanism of selective packaging of primer tRNALys3 by HIV-1

项目摘要： HIV-1和艾滋病流行病仍然是对公共卫生的重大威胁。由于缺乏疫苗， HIV-1和抗药性突变的阴影迫使人们寻找新的药物靶点。一个关键步骤 HIV-1生命周期的一个重要特征是在病毒体组装期间宿主tRNALys1、2、3的选择性包装，其中tRNALys3 随后将同种受体用作启动逆转录的引物。这种选择性tRNALys的现象 HIV-1的包装是在近三十年前发现的，但这种包装的结构和分子细节 事件仍然难以捉摸。LysRS（赖氨酰-tRNA合成酶），tRNALys3氨酰化的细胞酶，具有 已经显示在HIV-1包装tRNALys中起关键作用。LysRS主要作为一种 的多tRNA合成酶复合物（MSC），但无催化活性的S207磷酸化（pS207）库， 细胞LysRS在HIV-1感染时从MSC释放。HIV-1 Gag在LysRS中起主要作用， tRNALys3包装。目前的模型假设tRNALys3是由HIV-1 Gag通过一个 其衣壳蛋白（CA）和LysRS的催化结构域之间的相互作用。然而，该模型基于 主要是在tRNALys3不存在的情况下用未修饰的LysRS进行的研究和我们的初步数据 与这个模型相矛盾。已经显示HIV-1选择性地包装不带电荷的tRNALys3而不是带电荷的tRNALys3 而pS207-LysRS被认为在这种包装偏倚中起作用。没有高分辨率的结构 关于pS207-LysRS的可用信息提供了其缺乏氨酰化活性的机理基础，其 与HIV-1 Gag的相互作用，或其执行的非规范功能。缺乏任何结构性信息， HIV-1 Gag如何招募LysRS和tRNALys3已经排除了药物开发的这一步骤， 拟议的工作将弥补这一知识差距。本研究的总体目标是建立一个机制， 以及HIV-1选择性包装不带电荷的tRNALys3的结构基础。在目标1中，我们将实施 使用以下方法对报告结合LysRS和tRNALys3的HIV-1因子进行全面比较表征： 定性和定量生物化学和生物物理技术。比较分子解剖将 允许我们建立tRNALys3包装复合物的最小核心组分。我们将使用一个 LysRS的磷酸化模拟突变体（LysRSS207D），以研究pS207-LysRS在选择性包装 不带电荷的tRNALys3并研究三元tRNALys3包装复合物的动力学以探测任何协同性 形成复杂的队形彻底的生化表征将有助于建立稳定的三元tRNALys3 包装复杂的结构研究。在目标2中，我们将使用单粒子的并行双管齐下的方法 cryo-EM和X射线晶体学，以确定三元tRNALys3封装的高分辨率结构 使用基于细胞的下拉，tRNALys包装和HIV-1感染性来复杂和验证我们的结构见解 测定。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开发的方面， 和开发新型抗逆转录病毒药物。

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.