Mechanism of selective packaging of primer tRNALys3 by HIV-1

HIV-1选择性包装引物tRNALys3的机制

基本信息

批准号：
10376353
负责人：
Karin M Musier-Forsyth
金额：
$ 24.27万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-22 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10376353
关键词：
Acquired Immunodeficiency Syndrome Address Affinity Amino Acyl-tRNA Synthetases Aminoacylation Anisotropy Anti-Retroviral Agents Binding Biochemical Biological Assay Biology C-terminal Calorimetry Capsid Proteins Catalytic Domain Cells Charge Chemicals Complex Coupled Cryoelectron Microscopy Crystallization Data Dependence Development Dissection Drug resistance EMSA Electron Microscopy Electrophoretic Mobility Shift Assay Enzymes Event Fluorescence Fluorescence Anisotropy Goals HIV HIV-1 Individual Infection Kinetics Knowledge Life Cycle Stages Lysine-Specific tRNA Lysine-tRNA Ligase Measures Modeling Molecular Molecular Sieve Chromatography Negative Staining Non-Small-Cell Lung Carcinoma Pharmaceutical Preparations Phosphorylation Play Process Public Health Recording of previous events Reporting Research Resolution Reverse Transcription Role SP1 gene Structure Testing Titrations Transcriptional Regulation Transfer RNA Vaccines Validation Viral Genome Viral Reverse Transcription Virion Work X-Ray Crystallography base biophysical techniques comparative crosslink design drug development experimental study flexibility insight light scattering mutant new therapeutic target novel pandemic disease particle pol Gene Products recruit resistance mutation screening success therapeutic development

项目摘要

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.

项目概要： HIV-1 和艾滋病流行仍然对公共健康构成重大威胁。缺乏针对的疫苗 HIV-1 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生命周期的关键是病毒粒子组装过程中宿主tRNALys1,2,3的选择性包装，其中tRNALys3 isoacceptor 随后用作启动逆转录的引物。这种选择性tRNALys的现象近三十年前就发现了 HIV-1 的包装，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）是一种用于 tRNALys3 氨酰化的细胞酶，具有已被证明在 HIV-1 的 tRNALys 包装中发挥着至关重要的作用。 LysRS 主要作为一部分被隔离多 tRNA 合成酶复合物 (MSC)，但催化失活的 S207 磷酸化 (pS207) 库 HIV-1 感染后，MSC 会释放细胞 LysRS。 HIV-1 Gag 在 LysRS 中起主要作用 tRNALys3 包装。目前的模型假设 tRNALys3 通过 HIV-1 Gag 间接包装其衣壳蛋白 (CA) 和 LysRS 催化结构域之间的相互作用。然而，这个模型是基于主要是在没有 tRNALys3 的情况下使用未经修饰的 LysRS 进行的研究以及我们的初步数据与这个模型相矛盾。 HIV-1 已被证明可以选择性地包装不带电的 tRNALys3，而不是带电的 tRNALys3 pS207-LysRS 被认为在这种包装偏差中发挥了作用。没有高分辨率的结构 pS207-LysRS 上的可用信息为其缺乏氨酰化活性提供了机制基础，其与 HIV-1 Gag 的相互作用，或其执行的非规范功能。缺乏任何结构性信息 HIV-1 Gag 如何招募 LysRS 和 tRNALys3 已排除了针对这一步骤的药物开发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个机制 HIV-1 选择性包装不带电荷的 tRNALys3 的结构基础。在目标 1 中，我们将开展使用报告结合 LysRS 和 tRNALys3 的 HIV-1 因子的综合比较表征定性和定量的生化和生物物理技术。比较分子解剖将让我们能够建立 tRNALys3 包装复合物的最小核心组件。我们将使用一个 LysRS (LysRSS207D) 的磷酸模拟突变体，用于研究 pS207-LysRS 在选择性包装中的作用不带电的 tRNALys3 并研究三元 tRNALys3 包装复合物的动力学以探测任何协同性在复杂的形成中。彻底的生化表征将有助于建立稳定的三元 tRNALys3 用于结构研究的包装综合体。在目标 2 中，我们将使用单粒子并行双管齐下的方法冷冻电镜和 X 射线晶体学确定三元 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 感染性来复杂化并验证我们的结构见解化验。从这项研究中获得的见解将阐明设计中艾滋病毒生物学的一个关键但尚未开发的方面和新型抗逆转录病毒药物的开发。