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: 2655530
• 负责人: EDWARD M. JOHNSON
• 金额: $ 30.88万
• 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/2655530
• 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， 经常发生神经退行性疾病，进行性多灶性 白质脑病（PML）。PML由多瘤病毒JCV引起， 这种病毒通常潜伏在免疫力正常的人身上。激活JCV 在艾滋病患者的神经胶质细胞中， 1例感染。我们发现，刺激JCV晚期基因， HIV-1达特蛋白的转录由顺式作用达特- 反应元件，upTAR，含有细胞的识别位点， 单链DNA结合蛋白Puralpha。Puralpha也与RNA结合 HIV-1 TAR元件中的识别位点。此外，Puralpha形式 与达特的复合物，其可通过免疫共沉淀从 胶质细胞提取物。Tat-Puralpha相互作用差异 影响HIV-1和JCV晚期基因启动子启动的转录。 这项建议旨在利用两个独立的专业知识， 实验室协调对HIV-I、JCV和Puralpha的研究。我们将 确定JCV晚期基因转录的达特反式激活是否 依赖于达特与Puralpha的相互作用。我们将评估 达特和Puralpha影响HIV-1基因转录的能力 启动子和JCV晚期基因启动子在体外和神经胶质细胞中的表达 感染HIV-1或JCV。使用一系列的Puralpha删除 突变体，我们将确定哪些地区的Puralpha参与 与达特结合。我们将确定达特、Puralpha和TAR是否构成一个 稳定的三元复合物，以及这种复合物是否在 反式激活我们将确定哪些JCV序列与 在存在或不存在达特的情况下，体内人神经胶质细胞中的Puralpha。 最后，使用一系列puralpha衍生的合成肽，我们将 寻找抑制达特与Puralpha结合而不影响 Tat反应性JCV基因转录或HIV-1复制。结果将 提供关于HIV-1和JCV在人类神经胶质细胞中相互作用的信息。 这将有助于阐明HIV-1治疗的潜在靶点。