Molecular Interactions Between Rev and Cellular Factors

Rev 和细胞因子之间的分子相互作用

• 批准号: 7120450
• 负责人: Maria L. Zapp
• 金额: $ 31.84万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-15 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7120450
• 关键词: RNA; Xenopus; Xenopus oocyte; guanosinetriphosphatases; human immunodeficiency virus 1; intermolecular interaction; intracellular transport; microinjections; nucleoproteins; protein structure function; tissue /cell culture; transfection; transport proteins; virus protein

DESCRIPTION (provided by applicant): Replication of human immunodeficiency virus type 1 (HIV-1) entails a tightly regulated, complex life cycle, which is dependent upon specific interactions between viral RNAs and viral and cellular proteins. An essential and characteristic step in the viral replication cycle is the export of the intron-containing gag-pol and env mRNAs from the nucleus to the cytoplasm. The viral regulatory protein Rev mediates this event, in conjunction with the cellular nuclear export machinery and several protein cofactors. Cellular factors that are essential for Rev-directed RNA export offer alternate targets for inactivation and are potential candidates for antiviral drug development. During the past funding period, we have implicated the human Rev Interacting Protein (hRIP) and other cellular factors in a protein interaction network by which Rev mediates export of HIV RNAs from the nucleus to the cytoplasm. Our studies have revealed that hRIP is an essential cellular HIV cofactor that acts at a previously unanticipated step in Rev-directed RNA export: release of RNAs from the nuclear periphery to the cytoplasm. This perinuclear step may link nuclear export of Rev-directed RNAs with their cytoplasmic localization and function, in particular, virus assembly. Experiments are proposed to delineate the physical and functional protein interaction network mediating Rev-directed RNA export in living cells. We will use biochemical, genetic, and cell-based approaches to continue to study the function of hRIP and analyze how hRIP promotes movement of HIV RNAs from the nuclear periphery. Additionally, our studies of hRIP led us to investigate interactions between Rev and proteins that mediate cellular trafficking pathways. Experiments are proposed to explore the possibility of a functional link between Rev-directed RNA export and cellular trafficking pathways involved in HIV budding and assembly. hRIP is not required for cell viability and thus, may be an attractive target for the development of new antiviral strategies. During the past funding period, we have shown that ablation of hRIP activity using RNA interference (RNAi) results in the loss of HIV replication in mammalian cells. We will use several approaches to optimize the efficiency and specificity of RNAi-mediated silencing of hRIP expression and validate its inhibitory effect on HIV replication in mammalian cell lines and primary cells. The ultimate goal of these experiments is to validate cellular hRIP activity as a target for new antiviral strategies.

描述（由申请人提供）：人类免疫缺陷病毒类型1（HIV-1）的复制需要严格调节，复杂的生命周期，这取决于病毒RNA与病毒和病毒和细胞蛋白之间的特定相互作用。病毒复制周期中的重要和特征步骤是从核从细胞核到细胞质的含有内含子的GAG-POL和ENV mRNA的导出。病毒调节蛋白REV与细胞核输出机械和几种蛋白质辅因子一起介导了这一事件。对于转向导向的RNA导出至关重要的细胞因子提供了替代靶标的灭活靶标，并且是抗病毒药物开发的潜在候选者。在过去的资金期间，我们牵涉到蛋白质相互作用网络中的人类Rev相互作用蛋白（HRIP）和其他细胞因子，通过该网络，REV将HIV RNA从核中介导了从细胞核到细胞质的出口。我们的研究表明，HRIP是一种必不可少的细胞HIV辅助因子，它在先前意外的REV指导RNA导出的步骤中起作用：RNA从核周围释放到细胞质。这个核周步骤可能将REV定向RNA的核出口与其细胞质定位和功能，尤其是病毒组装联系起来。提出了实验来描述介导活细胞中的REV定向RNA导出的物理和功能蛋白相互作用网络。我们将使用生化，遗传和基于细胞的方法继续研究HRIP的功能，并分析HRIP如何促进HIV RNA从核周围的运动。此外，我们对HRIP的研究使我们研究了介导细胞运输途径的Rev和蛋白质之间的相互作用。提出了实验，以探讨REV指导的RNA输出与涉及HIV萌芽和组装的细胞运输途径之间的功能联系的可能性。细胞活力不需要HRIP，因此，可能是开发新抗病毒药策略的有吸引力的目标。在过去的资金期间，我们表明使用RNA干扰（RNAI）消融HRIP活性导致哺乳动物细胞中HIV复制的丧失。我们将使用几种方法来优化RNAi介导的HRIP表达沉默的效率和特异性，并验证其对哺乳动物细胞系和原代细胞中HIV复制的抑制作用。这些实验的最终目标是验证细胞HRIP活性，作为新的抗病毒药策略的目标。