Identification of functional inhibitors of HIV-1 Rev in astrocytes

星形胶质细胞中 HIV-1 Rev 功能抑制剂的鉴定

• 批准号: 5419952
• 负责人: Professorin Dr. Ruth Brack-Werner
• 金额: --
• 依托单位: Institut für Virologie (VIRO)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2004
• 资助国家: 德国
• 起止时间: 2003-12-31 至 2008-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5419952?language=en
• 关键词: Identification; functional; inhibitors; HIV; Rev

The HIV Rev protein stimulates expression of HIV RNAs by enhancing stability, export, and translation of HIV target RNAs. Rev is crucial for replication of HIV. Rev accumulates in the nucleus and shuttles between nuclear and cytoplasmatic campartments by using active transport pathways. We have demonstrated that inefficient production of HIV in human astrocytes is associated with cytoplasmatic accumulation and retarded nuclear import kinetics of the Rev protein. Cell fusion experiments indicate that the inhibiton of nuclear import in astrocytes is caused by dominant negative factors. The goal of this collaborative project is to isolate and characterize Rev-interacting factors from astrocytes and to analyze their effects on nuclear import of Rev in an attempt to identify cellular inhibitors of Rev. We will adapt previously established nuclear import assay systems to monitor the inhibitory activity of cytoplasmatic extracts obtained from astrocytes on nuclear import of Rev. Rev-interacting factors will be isolated from cytoplasmatic extracts of astrocytes by affinity chromatography with immobilized Rev. In parallel we plan to isolate Rev-associated protein complexes from the cytoplasm of intact astrocytes, using tandem affinity purification (TAP) methodoloy. Isolated factors will be characterized with regard to their capacity to bind Rev, their influence on nuclear import and localization of Rev and their specific occurence in astrocytes or HeLa cells. Proteins will be identified by mass spectrometry. This work will contribute to understanding mechanisms of cellular control of HIV replication and may lead to novel anti-viral therapies.

HIV Rev 蛋白通过增强 HIV 靶 RNA 的稳定性、输出和翻译来刺激 HIV RNA 的表达。 Rev 对于 HIV 的复制至关重要。 Rev 在细胞核中积累，并通过主动转运途径在核区和细胞质区之间穿梭。我们已经证明，人星形胶质细胞中 HIV 的低效产生与 Rev 蛋白的细胞质积累和核输入动力学延迟有关。细胞融合实验表明，星形胶质细胞核输入的抑制是由显性负因子引起的。该合作项目的目标是从星形胶质细胞中分离和表征 Rev 相互作用因子，并分析它们对 Rev 核输入的影响，以试图鉴定 Rev 的细胞抑制剂。我们将采用先前建立的核输入测定系统来监测从星形胶质细胞获得的细胞质提取物对 Rev 核输入的抑制活性。Rev 相互作用因子将从星形胶质细胞的细胞质提取物中分离出来。 同时，我们计划使用串联亲和纯化（TAP）方法从完整星形胶质细胞的细胞质中分离Rev相关蛋白复合物。分离的因子将被表征为它们结合 Rev 的能力、它们对 Rev 的核输入和定位的影响以及它们在星形胶质细胞或 HeLa 细胞中的特定出现。蛋白质将通过质谱法进行鉴定。这项工作将有助于理解艾滋病毒复制的细胞控制机制，并可能带来新的抗病毒疗法。