GAG Protein/RNA Interactions in the HIV Lifecycle

Gag蛋白是HIV-1病毒核心的主要组成部分，它以多种对病毒生命周期至关重要的方式与病毒RNA相互作用。与HIV-1基因组中5‘包装(或psi)区域的特异性Gag结合对于将该RNA靶向新生病毒颗粒是必不可少的，并有助于启动基因组二聚体的形成。Gag/RNA相互作用还促进tRNA启动、逆转录、病毒粒子组装和其他关键过程，因此为新的抗病毒治疗提供了一个特别有希望的靶点。我们的目标是在体外剖析Gag与HIV-1RNA的各种分子相互作用，并从遗传学角度评估它们在活病毒中的功能。我们以前已经在psi基因座上鉴定了Gag结合和RNA二聚体接触的位置。我们现在建议详细地探索这些位置之间的功能相互作用，绘制更多据信位于上游的位置，并在基因组的其他地方寻找可能有助于病毒粒子组装和成熟的其他位置。我们将尝试描述HIV-1在体内包装所需的所有顺式作用信号。我们早期的研究揭示了一类新的HIV-1突变体，它们在RNA二聚化方面存在选择性缺陷，与显著降低的传染性有关。通过对这些突变体的分析，我们将评估基因组二聚化对包装、反转录、序列重组、RNA稳定性和HIV-1核心内基因组构象的准确贡献。对小鼠白血病病毒和人类端粒酶复合体的比较研究正在计划中。我们还鉴定了一种异源RNA配体，它与HIV-1 Gag高亲和力结合，并在体内具有包装活性；我们现在建议研究该配体是否可以作为Gag/psi结合和HIV-1复制的有效竞争性抑制物。总之，这些研究将阐明作为脆弱药物靶点的特定GAG/RNA相互作用，并将为在抗病毒药物发现中利用这些靶点提供新的体外分析方法。

GAG protein is the major constituent of the HIV-1 virion core, and it interacts with viral RNA in a number of ways that are critical for the viral lifecycle. Specific Gag binding to the 5' packaging (or psi) region in the HIV-1 genome is essential for targeting this RNA into nascent viral particles, and helps initiate formation of the genomic dimer. Gag/RNA interactions also promote tRNA priming, reverse transcription, virion assembly, and other crucial processes, and so offer an especially promising target for new antiviral therapies. Our goal is to dissect the various molecular interactions of Gag with HIV-1 RNA in vitro, and to evaluate their functions genetically in the live virus. We have previously characterized sites of Gag-binding and RNA dimer contact in the psi locus. We now propose to explore the functional interactions among those sites in detail, to map additional sites believed to lie upstream, and to search for others elsewhere in the genome that may contribute to virion assembly and maturation. We will attempt to delineate all of the cis-acting signals required for HIV-1 packaging in vivo. Our earlier studies revealed a new class of HIV-1 mutants that have a selective defect in RNA dimerization, associated with markedly reduced infectivity. By analyzing those mutants, we will evaluate the precise contributions of genomic dimerization to packaging, reverse transcription, sequence recombination, RNA stability, and the conformation of the genome within the HIV-1 core. Comparative studies in murine leukemia virus and the human telomerase complex are planned. We have also identified a heterologous RNA ligand that binds HIV-1 Gag with high affinity and has packaging activity in vivo; we now propose to investigate whether this ligand can act as an effective competitive inhibitor of Gag/psi binding and of HIV-1 replication. Together, these studies will illuminate particular Gag/RNA interactions that are vulnerable drug targets, and will provide novel in vitro assays for exploiting those targets in antiviral drug discovery.