GAG Protein/RNA Interactions in the HIV Lifecycle

HIV 生命周期中的 GAG 蛋白/RNA 相互作用

• 批准号: 6408220
• 负责人: TRISTRAM G. PARSLOW
• 金额: $ 31.53万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6408220
• 关键词: RNA binding protein; RNA biosynthesis; Retroviridae; binding sites; biochemical evolution; dimer; functional /structural genomics; gag protein; genetic mapping; genetic recombination; genetic regulatory element; genetic transcription; human genetic material tag; human immunodeficiency virus 1; intermolecular interaction; ligands; murine leukemia virus; mutant; nucleic acid sequence; oligonucleotides; protein structure function; telomerase; tissue /cell culture; virus RNA; virus assembly; virus genetics; virus replication

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.

GAG蛋白是HIV-1病毒粒子核心的主要成分，它以多种方式与病毒RNA相互作用，这对病毒的生命周期至关重要。特异性Gag结合到HIV-1基因组的5'包装（或psi）区域对于将该RNA靶向新生病毒颗粒至关重要，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引物、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解剖Gag与HIV-1 RNA的各种分子相互作用，并评估它们在活病毒中的遗传功能。我们之前已经在psi位点上描述了gag结合和RNA二聚体接触的位点。我们现在建议详细探索这些位点之间的功能相互作用，绘制被认为位于上游的其他位点，并在基因组中寻找其他可能有助于病毒粒子组装和成熟的位点。我们将尝试在体内描述HIV-1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二聚化中具有选择性缺陷，与显著降低的传染性有关。通过分析这些突变体，我们将评估基因组二聚化对HIV-1核心内的包装、逆转录、序列重组、RNA稳定性和基因组构象的精确贡献。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。我们还发现了一种高亲和力结合HIV-1 Gag的异源RNA配体，并在体内具有包装活性；我们现在建议研究这种配体是否可以作为Gag/psi结合和HIV-1复制的有效竞争性抑制剂。总之，这些研究将阐明特定的Gag/RNA相互作用是脆弱的药物靶点，并将为利用这些靶点开发抗病毒药物提供新的体外检测方法。