Regulation of HIV-1 RNA Processing

HIV-1 RNA 加工的调控

• 批准号: 6591090
• 负责人: MASSIMO CAPUTI
• 金额: $ 28.26万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-15 至 2003-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6591090
• 关键词: RNA splicing; gene expression; genetic regulation; genetic regulatory element; genetic transcription; human immunodeficiency virus 1; intermolecular interaction; intracellular transport; posttranscriptional RNA processing; protein structure function; transfection; virus RNA; virus genetics; virus infection mechanism; virus protein; virus replication

DESCRIPTION (provided by applicant): The integrated HIV-1 proviral genome is transcribed by the host transcription machinery into a single 9.2 kb primary transcript. To express the nine different gene products required for viral replication, HIV has developed a number of strategies to regulate splicing and to circumvent cellular mechanisms restricting unspliced RNA in the cytoplasm. The regulatory protein Rev promotes transport of the incompletely spliced viral RNAs to the cytoplasm, binding to the Rev Responsive Element (RRE) on the viral RNA. Mechanisms regulating the appearance of unspliced and spliced viral RNAs, and their transport to the cytoplasm, involve interactions between viral RNA sequences and host cell factors. The characterization of these viral/host interactions could allow the design of novel therapeutic compounds. hnRNP H proteins have been shown to be necessary for HIV-1 RNA splicing in a series of in vitro experiments. They may also regulate RNA export and trafficking in infected cells. The role of single members of the hnRNP H protein family in HIV-1 RNA processing in vivo will be investigated by modulating hnRNP H protein expression in cells transfected with HIV-1-derived plasmids, bearing mutations in regulatory sequences. Several viral sequences that regulate viral RNA splicing in vitro have been identified. These will be tested to determine whether they are necessary for the regulation of viral RNA splicing in vivo. Preliminary data indicates that viral sequences interact with the RRE-Rev complex and, possibly, have a role in the Rev-dependent viral RNA transport pathway. These interactions will be characterized by a combination of in vivo and in vitro assays. Transcription and splicing have been shown to be closely coupled in other systems. Preliminary data indicates that the viral promoter influences splicing of viral substrates in vitro. Possible coupling of HIV-1 transcription and splicing will be investigated, using both a coupled transcription-splicing assay and transient transfection experiments.

描述(由申请人提供)：整合的HIV-1前病毒基因组由宿主转录机制转录成单个9.2kb的初级转录本。为了表达病毒复制所需的九种不同的基因产物，HIV开发了许多策略来调节剪接，并绕过限制细胞质中未剪接RNA的细胞机制。调节蛋白REV促进未完全剪接的病毒RNA向细胞质的运输，与病毒RNA上的REV反应元件(RRE)结合。调节未剪接和剪接的病毒RNA的外观以及它们向细胞质的运输的机制涉及病毒RNA序列和宿主细胞因子之间的相互作用。对这些病毒/宿主相互作用的表征可以使设计新的治疗化合物成为可能。在一系列体外实验中，hnRNP H蛋白被证明是HIV-1RNA剪接所必需的。它们还可能对受感染细胞的RNA出口和贩运进行监管。HnRNP H蛋白家族的单个成员在体内HIV-1 RNA加工中的作用将通过调节HIV-1衍生的质粒转染的细胞中hnRNP H蛋白的表达来研究，该细胞带有调控序列的突变。已经确定了几种在体外调节病毒RNA剪接的病毒序列。将对它们进行测试，以确定它们是否对体内病毒RNA剪接的调节是必要的。初步数据表明，病毒序列与RRE-REV复合体相互作用，并可能在依赖REV的病毒RNA运输途径中发挥作用。这些相互作用将通过体内和体外试验相结合来表征。转录和剪接已被证明在其他系统中紧密耦合。初步数据表明，病毒启动子在体外影响病毒底物的剪接。HIV-1转录和剪接的可能偶联将通过转录-剪接偶联实验和瞬时转染实验进行研究。