Characterization of HIV-1 Genome Dimerization: A Strain Specific Dimerization Mechanism

HIV-1 基因组二聚化的表征：毒株特异性二聚化机制

• 批准号: 10618005
• 负责人: Saif Yasin
• 金额: $ 3.9万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10618005
• 关键词: Address; Anti-Retroviral Agents; Area; Biological; Biological Assay; Biology; Cell membrane; Cells; Color; Cytoplasm; Data; Diffusion; Dimerization; Discrimination; Dissociation; Equilibrium; Exhibits; Fluorescence; Fluorescence Resonance Energy Transfer; Gel; Genetic Recombination; Genetic Transcription; Genome; Goals; HIV; HIV Genome; HIV-1; Image; Impairment; In Vitro; Kinetics; Label; Length; Location; Mediating; Membrane; Methods; Modeling; Molecular; Monitor; Mutation; NMR Spectroscopy; Nature; Nuclear Magnetic Resonance; Patients; Population; Process; Property; Publishing; RNA; Resistance; Retroviridae; Reverse Transcription; Role; Site; Spectrum Analysis; Structure; System; Therapeutic; Thermodynamics; Time; Universities; Validation; Variant; Viral; Viral Genome; Viral Proteins; Virus; Virus Assembly; Virus Replication; Wisconsin; Work; antiretroviral therapy; biophysical properties; data quality; dimer; gag Gene Products; gel electrophoresis; intermolecular interaction; novel; novel strategies; therapeutic target; validation studies

PROJECT SUMMARY/ABSTRACT The goal of the proposed studies is to understand how HIV-1 sequence diversity impacts viral genome dimerization, a requirement for viral replication. Like nearly all retroviruses, HIV-1 selectively packages two copies of its full-length genome after the formation of a dimer – a process essential not only to packaging, but also reverse transcription and recombination. The sponsor’s lab took a pioneering role in the structural characterization of the highly conserved HIV-1 dimeric 5′-leader, the region believed to initiate genome dimerization. Several outstanding questions remain regarding the mechanism and structures involved in the process. Current proposals support a two-step mechanism beginning with a kissing-loop interface that then matures into a more extensive intermolecular interface. While in vitro studies support this mechanism, most are isolated to RNA fragments or ignore important cellular/viral factors. Studies of HIV genome dimerization are also complicated by the enormous genome plasticity of HIV-1 that is driven by mutation and recombination, leading to sequence diversity within the dimer interface. This diversity stratifies strains into two dimer classes, those that are thermodynamically stable (nonlabile strains) and those that readily dissociate (labile strains). I will characterize the differences between these two dimer classes by studying two model HIV-1 strains in the context of the intact dimeric interface in solution, in cells, and in viruses: NL4-3 (nonlabile) and MAL (labile). Current methods to study labile dimers are limited, as they readily dissociate in native gel electrophoresis assays; therefore, suggesting a need to rely upon methods that will assess the equilibrium, solution state for each strain. We will begin with a biophysical characterization of both strains, specifically characterizing the thermodynamics, kinetics, and structures of the dimeric interface using Fluorescence Correlation Spectroscopy (FCS) and Nuclear Magnetic Resonance (NMR) (Aim 1). Preliminary FCS data has shown our ability to monitor dimer formation at concentrations and timescales previously inaccessible. Preliminary data using our 2H-edited NMR approach suggest we can directly probe for intermolecular interactions in the full-length, dimeric HIV-1 MAL 5′-leader (>230 kDa), allowing us direct comparison with the previously characterized NL4-3 extended dimer. I will also compare the dimerization process of these two strains in cells and viruses (Aim 2). We have now collected initial in vitro data validating our novel fluorescent labeling strategy to discriminate intermolecular and intramolecular RNA interactions that can now be applied in the context of viral replication in cells. We hypothesize that labile dimers exhibit primarily a kissing dimer structure throughout assembly, highlighting a higher stability to the kissing dimer than was previously thought, as well as implying the existence of a strain specific dimerization mechanism. Successful completion of this project will also provide quantitative spatial and temporal characterization of HIV- 1 genome dimerization as RNAs are trafficked to assembly sites on the plasma membrane as well as allow us to monitor changes in the dimer interface that potentially occur during virus assembly and maturation.

项目摘要/摘要 拟议的研究的目的是了解HIV-1序列多样性如何影响病毒基因组 二聚，病毒复制的要求。像几乎所有逆转录病毒一样，HIV-1有选择地包装两个 形成二聚体后其全长基因组的副本 - 不仅是包装的过程，而且还必须 还逆转录和重组。赞助商的实验室在结构中发挥了开创性的作用 高度保守的HIV-1二聚体5'-Leader的表征，该区域被认为启动基因组 二聚化。关于涉及的机制和结构仍然存在几个重大问题 过程。当前的建议支持一个以接吻环接口开始的两步机制，然后 成熟到更广泛的分子间界面。在体外研究支持这种机制的同时，大多数是 分离为RNA片段或忽略重要的细胞/病毒因子。 HIV基因组二聚化的研究也是 由突变和重组驱动的HIV-1的巨大基因组可塑性复杂 在二聚体界面内序列多样性。这种多样性将条纹分为两个二聚体类别， 是热力学稳定的（非法比菌株）和容易分离的那些（不稳定菌株）。我会 通过在上下文中研究两个模型HIV-1菌株来表征这两个二聚体类别之间的差异 溶液，细胞和病毒中完整的二聚体界面的完整界面：NL4-3（非法比勒）和MAL（不稳定）。当前的 研究不稳定二聚体的方法是有限的，因为它们很容易在天然凝胶电泳测定中分离。 因此，提出需要依靠可以评估每个菌株的等效溶液状态的方法。 我们将从两种菌株的生物物理表征开始，专门表征热力学， 动力学和使用荧光相关光谱（FC）和核界面的结构 磁共振（NMR）（AIM 1）。初步FCS数据显示了我们在监测二聚体形成的能力 浓度和时间尺度以前无法访问。使用我们的2H编辑的NMR方法的初步数据 建议我们可以直接探测全长二聚体HIV-1 MAL 5'-LEADER中分子间相互作用（> 230 KDA），使我们与先前表征的NL4-3扩展二聚体进行直接比较。我也会比较 这两种菌株在细胞和病毒中的二聚化过程（AIM 2）。我们现在已经在体外收集了初始 数据验证了我们新型的荧光标记策略，以区分分子间和分子内RNA 现在可以在细胞中的病毒复制背景下应用的相互作用。我们假设那个不稳定的二聚体 在整个组装中展示主要的接吻二聚体结构，突出了接吻二聚体的稳定性 比以前认为的，也意味着存在特异性二聚化机制。 该项目的成功完成还将提供艾滋病毒的定量空间和临时表征 1基因组二聚化，因为RNA被贩运到质膜上的组装位点，并允许我们 监测病毒组装和成熟过程中可能发生的二聚体界面的变化。