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的个人， 频发神经退行性疾病，进行性多灶性 白质脑病(PML)。PML是由多瘤病毒JCV，一种 病毒通常潜伏在非免疫功能受损的人群中。JCV的激活 在艾滋病人的神经胶质细胞中，被认为是艾滋病毒的结果- 感染1例。我们已经发现JCV晚期基因的刺激 HIV-1Tat蛋白的转录是由顺式作用的Tat-1介导的。 响应元件，upTAR，包含细胞的识别位点 单链DNA结合蛋白Purpha。Purpha也能与RNA结合 HIV-1TAR元件中的识别位点。此外，Purpha形式 与TAT的络合物可通过免疫共沉淀检测到 神经胶质细胞的提取物。差分的TAT-Purpha相互作用 影响HIV-1和JCV晚期基因启动子启动的转录。 这项提议旨在利用两家独立的 实验室协调关于艾滋病毒-I、JCV和Purpha的研究。我们会 确定JCV晚期基因转录的TAT反式激活是否 依赖于TAT与Purpha的相互作用。我们将评估 Tat和Purpha对HIV-1转录的影响能力 启动子和JCV晚期基因启动子在体外和胶质细胞中的表达 感染了HIV-1或JCV。使用一系列PurAlpha删除 我们将确定PurAlpha的哪些区域参与突变 与TAT绑定。我们将确定TAT、Purpha和Tar是否形成 稳定的三元络合物以及这种络合物是否在 激活。我们将确定哪些JCV序列由 体内人脑胶质细胞中存在或不存在TAT。 最后，使用一系列由Purpha衍生的合成肽，我们将 寻找既能抑制TAT与Purpha结合又不影响TAT的多肽 TAT反应的JCV基因转录或HIV-1复制。结果将 提供有关HIV-1和JCV在人神经胶质细胞中相互作用的信息 并将有助于阐明HIV-1治疗的潜在靶点。