PROTEINS MEDIATING INTERACTION OF HIV 1 AND JCV IN CNS

介导中枢神经系统中 HIV 1 和 JCV 相互作用的蛋白质

• 批准号: 2333042
• 负责人: EDWARD M. JOHNSON
• 金额: $ 29.75万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2333042
• 关键词: DNA binding protein; DNA footprinting; Polyomavirus; central nervous system; gel mobility shift assay; gene deletion mutation; gene expression; genetic library; genetic promoter element; genetic transcription; glia; human immunodeficiency virus 1; immunoprecipitation; neurotropic virus; progressive multifocal leukoencephalopathy; protein structure function; reporter genes; synthetic peptide; tissue /cell culture; transfection; virus infection mechanism; virus protein; virus replication

Individuals infected with the human immunodeficiency virus, HIV-1, frequently develop the neurodegenerative disease, progressive multifocal leukoencephalopathy (PML). PML is caused by the polyomavirus, JCV, a virus normally latent in non-immunocompromised people. Activation of JCV in glial cells of AIDS individuals is thought to be a consequence of HIV- 1 infection. We have found that stimulation of JCV late-gene transcription by the HIV-1 Tat protein is mediated by a cis-acting Tat- responsive element, upTAR, containing recognition sites for the cellular single-stranded DNA-binding protein Puralpha. Puralpha also binds to RNA recognition sites in the HIV-1 TAR element. Furthermore, Puralpha forms a complex with Tat which can be detected by co-immunoprecipitation from extracts of glial cells. The Tat-Puralpha interaction differentially affects transcription initiated at HIV-1 and JCV late gene promoters. This proposal aims to capitalize on the expertise of two independent laboratories to coordinate studies on HIV-I, JCV and Puralpha. We shall determine whether Tat transactivation of JCV late-gene transcription is dependent upon interaction of Tat with Puralpha. We shall assess the ability of Tat and Puralpha to affect transcription at both the HIV-1 promoter and the JCV late-gene promoter in vitro and in glial cells infected with either HIV-1 or JCV. Using a series of Puralpha deletion mutants we shall determine which regions of Puralpha are involved in binding to Tat. We shall determine whether Tat, Puralpha and TAR form a stable ternary complex and whether such a complex plays a role in transactivation. We shall determine which JCV sequences are bound by Puralpha in vivo in human glial cells in the presence or absence of Tat. Finally, using a series of puralpha-derived synthetic peptides, we shall seek peptides which inhibit Tat binding to Puralpha without affecting Tat-responsive JCV gene transcription or HIV-1 replication. Results will provide information about the interaction of HIV-1 and JCV in human glial cells and will help illuminate a potential target for HIV-1 therapy.

感染人类免疫缺陷病毒HIV-1的个体 经常发展神经退行性疾病，进行性多灶 白血病（PML）。 PML是由多瘤病毒JCV，A引起的 病毒通常在非免疫力低下的人群中。 JCV的激活 在艾滋病的神经胶质细胞中，个体被认为是HIV的结果 1感染。我们发现JCV晚期的刺激 HIV-1 TAT蛋白的转录是由顺式作用的Tat-介导的 响应元素，uptar，包含细胞的识别位点 单链DNA结合蛋白puralpha。 puralpha也与RNA结合 HIV-1 TAR元素中的识别站点。此外，puralpha形成 带有TAT的复合物，可以通过共免疫沉淀从 神经胶质细胞的提取物。 tat-puralpha相互作用差异 影响在HIV-1和JCV晚期基因启动子上启动的转录。 该建议旨在利用两个独立的专业知识 实验室协调有关HIV-I，JCV和Puralpha的研究。我们将 确定JCV晚期转录的TAT反式激活是否为 取决于TAT与puralpha的相互作用。我们将评估 Tat和Puralpha影响HIV-1的转录的能力 启动子和JCV晚期基因启动子体外和神经胶质细胞 感染HIV-1或JCV。使用一系列puralpha删除 突变体我们将确定紫色的哪些区域参与 与tat结合。我们将确定tat，puralpha和tar是否形成 稳定的三元络合物以及这种复杂的综合体是否在 反式激活。我们将确定哪些JCV序列由 在存在或不存在TAT的情况下，人类神经胶质细胞中的puralpha体内。 最后，使用一系列紫色的合成肽，我们将 寻求抑制tat结合puralpha而不会影响的肽 TAT响应JCV基因转录或HIV-1复制。结果将 提供有关人神经胶质中HIV-1和JCV相互作用的信息 细胞并将有助于阐明HIV-1治疗的潜在靶标。