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.

项目概要/摘要 拟议研究的目标是了解 HIV-1 序列多样性如何影响病毒基因组 二聚化是病毒复制的必要条件，与几乎所有逆转录病毒一样，HIV-1 选择性地包装两个病毒。 在形成二聚体后复制其全长基因组——这个过程不仅对于包装至关重要，而且对于 申办者的实验室也在逆转录和重组方面发挥了先锋作用。 高度保守的 HIV-1 二聚体 5'-前导序列的表征，该区域被认为启动了基因组 关于二聚化的机制和结构仍然存在一些悬而未决的问题。 当前的提案支持两步机制，首先是接吻环接口，然后是接吻环接口。 虽然体外研究支持这种机制，但大多数都是这样。 分离到 RNA 片段或忽略重要的细胞/病毒因素也研究了 HIV 基因组二聚化。 由突变和重组驱动的 HIV-1 巨大的基因组可塑性使情况变得复杂，导致 对二聚体界面内的多样性进行测序。这种多样性将菌株分为两个二聚体类别，即 是热力学稳定的（不稳定菌株）和那些容易解离的（不稳定菌株）。 通过研究上下文中的两种模型 HIV-1 毒株来表征这两种二聚体类别之间的差异 溶液、细胞和病毒中完整二聚体界面的分析：NL4-3（不稳定）和 MAL（不稳定）。 研究不稳定二聚体的方法是有限的，因为它们在天然凝胶电泳测定中很容易解离； 因此，建议需要依靠评估每种菌株的平衡、溶液状态的方法。 我们将从两种菌株的生物物理特征开始，特别是热力学特征， 使用荧光相关光谱 (FCS) 和核分析研究二聚体界面的动力学和结构 磁共振 (NMR)（目标 1）表明我们有能力监测二聚体形成。 使用我们的 2H 编辑 NMR 方法无法获得浓度和时间尺度。 建议我们可以直接探测全长二聚体 HIV-1 MAL 5′-前导序列 (>230 kDa），使我们能够直接与之前表征的 NL4-3 扩展二聚体进行比较，我也将进行比较。 这两种菌株在细胞和病毒中的二聚化过程（目标2）我们现在已经收集了初步的体外数据。 数据验证了我们区分分子间和分子内 RNA 的新型荧光标记策略 我们捕获了不稳定的二聚体，这些相互作用现在可以应用于细胞中的病毒复制。 在整个组装过程中主要表现出接吻二聚体结构，突出了接吻二聚体的更高稳定性 比之前想象的要好，并且暗示存在菌株特异性二聚化机制。 该项目的成功完成还将提供艾滋病毒的定量空间和时间特征 1 基因组二聚化，因为 RNA 被运输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。