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: 6373846
• 负责人: Maria L. Zapp
• 金额: $ 25.3万
• 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 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6373846
• 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

The HIV-1 Rev protein regulates the nucleocytoplasmic distribution of viral precursor mRNAs that encode HIV-1 structural proteins. Biochemical features of Rev and its essential cis-acting binding element, the RRE, have been defined using functional in vivo assays. Yet, the molecular details of how Rev mediates nuclear viral mRNA export remains poorly understood. The major focus of this proposal is to understand the molecular basis of Rev-mediated nuclear RNA export. Here, we propose in vitro and in vivo studies for defining the cellular factors and molecular interactions essential for Rev function. These studies will provide additional insights into the mechanism of Rev action, as well as the cellular nuclear RNA export machinery. Specifically, we will analyze functional interactions between Rev and hCRM1, a beta-importin like protein, and the small nuclear GTPase Ran. Biochemical and immunological experiments will address whether hCRM1 mediates a direct interaction between Rev and hRIP/Rab. We will also explore whether hRIP/Rab interacts directly or indirectly with Ran-GTP, alpha importin, NTF2 or other cellular factors. In collaboration with established investigators, we will determine whether hRIP/Rab plays a role in modulating GTPase activity, NPC targeting and translocation or signal transmission between the nuclear envelope and nucleocytoplasmic transport components. Two genetic strategies are proposed to define interactions between Rev, hCRM1, and Ran during Rev-mediated nuclear RNA export. These genetic analyses will also be used to characterize the normal cellular function of hRIP/Rab. Lastly, we have established Rev assays based on microinjection in Xenopus oocytes and isolated germinal vesicles (GVs). We will use these systems to identify and characterize components of the Rev-mediated nuclear RNA pathway and test whether Rev function requires sequential interactions similar to nuclear protein import. Additionally, we will use factor competition and immuno-inhibition strategies to investigate the relationship between the Rev-mediated nuclear RNA export pathway and the nuclear RNA export pathway(s) used by different classes of cellular RNA.

HIV-1 REV蛋白调节核细胞质分布 编码HIV-1结构蛋白的病毒前体mRNA。 Rev的生化特征及其必需的顺式作用结合 元素，RRE已使用函数在体内测定中定义。 然而，REV如何介导核病毒mRNA输出的分子细节 仍然很了解。 该提议的主要重点是 了解Rev介导的核RNA输出的分子基础。 在这里，我们提出了体外和体内研究，以定义细胞 因素和分子相互作用对于转速功能必不可少。这些 研究将提供有关Rev机制的更多见解 作用以及细胞核RNA导出机械。 具体而言，我们将分析Rev和Rev之间的功能相互作用 HCRM1，类似β物质的蛋白质，小核GTPase运行。 生化和免疫学实验将解决HCRM1是否 介导Rev和HRIP/RAB之间的直接相互作用。 我们也会 探索HRIP/RAB是直接还是与RAN GTP直接或间接相互作用， alpha Importin，NTF2或其他细胞因子。 与 既定的研究人员，我们将确定HRIP/RAB是否扮演 在调节GTPase活性，NPC靶向和易位或 核包络与核细胞质之间的信号传递 运输组件。 提出了两种遗传策略来定义 Rev介导的核RNA期间Rev，HCRM1和RAN之间的相互作用 出口。 这些遗传分析也将用于表征 HRIP/RAB的正常细胞功能。 最后，我们基于对 爪蟾卵母细胞和分离的生发囊泡（GVS）。 我们将使用这些 识别和表征转速介导的组件的系统 核RNA通路和测试Rev功能是否需要顺序 类似于核蛋白进口的相互作用。 此外，我们会的 使用因子竞争和免疫抑制策略来调查 Rev介导的核RNA出口途径与 不同类别的细胞使用的核RNA导出途径 RNA。