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

项目总结/摘要 这项研究的目的是了解HIV-1序列多样性如何影响病毒基因组 二聚化是病毒复制的必要条件。像几乎所有的逆转录病毒一样，HIV-1选择性地包装两个 它的全长基因组的拷贝后，形成一个二聚体-一个过程中必不可少的不仅包装， 还有逆转录和重组。赞助商的实验室在结构上发挥了先锋作用， 高度保守的HIV-1二聚体5′-前导序列的特征，该区域被认为是启动基因组 二聚化。在参与《公约》的机制和结构方面，仍有几个悬而未决的问题。 过程目前的建议支持一个两步机制，首先是接吻环接口，然后 成熟为更广泛的分子间界面。虽然体外研究支持这种机制，但大多数研究都是 分离到RNA片段或忽略重要的细胞/病毒因子。艾滋病毒基因组二聚化的研究也是 由突变和重组驱动的HIV-1巨大的基因组可塑性使其复杂化， 二聚体界面内的序列多样性。这种多样性将菌株分为两种二聚体类型， 是代谢稳定的（非不稳定菌株）和容易解离的（不稳定菌株）。我会 通过研究两种HIV-1模型毒株，描述这两种二聚体类别之间的差异 在溶液、细胞和病毒中完整二聚体界面：NL 4 -3（非不稳定）和MAL（不稳定）。电流 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分析中容易解离; 因此，这表明需要依赖于评估每种菌株的平衡溶液状态的方法。 我们将开始与两种菌株的生物物理特性，特别是表征热力学， 动力学和结构的二聚体界面使用荧光相关光谱（FCS）和核 磁共振（NMR）（目的1）。初步的FCS数据表明，我们有能力监测二聚体的形成， 浓度和时间尺度以前无法达到。使用我们的2 H编辑NMR方法的初步数据 这表明我们可以直接探测全长二聚体HIV-1 MAL 5′-前导序列中的分子间相互作用（>230 kDa），从而允许我们与先前表征的NL 4 -3延伸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（目的2）。我们现在已经收集了最初的体外 数据验证了我们新的荧光标记策略，以区分分子间和分子内RNA 这些相互作用现在可以应用于细胞中病毒复制的背景下。我们假设不稳定的二聚体 在整个组装过程中主要表现出对接二聚体结构，突出了对接二聚体的较高稳定性 比以前认为的，以及暗示存在一个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基因组二聚化作为RNA被贩运到质膜上的组装位点，以及允许我们 以监测在病毒组装和成熟过程中可能发生的二聚体界面的变化。

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.