Rev-dependent Nuclear Export of RNA

描述（由申请人提供）：Rev-RRE相互作用和随后由细胞输出受体CRM1介导的含内含子的病毒mRNA的转运对HIV-1逆转录病毒的复制至关重要。目前的低聚物Rev与其同源RRE结合的模型不足以充分解释调节蛋白Rev的功能。虽然Rev寡聚化的重要性之前已经建立，但对于理解核运输的要求以及与核输出因子CRM1的相互作用还没有明确的分子基础。我们建议解决几个机制和结构问题，旨在弥合rev -寡聚化，rre结合和crm1介导的RNA输出之间的差距。我们的综合生化、生物物理和核磁共振结构研究集中在调控HIV-1蛋白Rev获得其生物活性结构的分子机制上。Rev如何工作的综合模型可能有助于针对这一重要病毒基因的合理药物设计。我们的中心假设是Rev功能需要：1)一个确定的结构和2)寡聚化能力作为细胞输出机制的底物。单体Rev本质上是无序的。通路上部分折叠的Rev中间体与有序寡聚化核相互转化；一个有序的Rev结构需要通过与RRE的寡聚结合、与其他辅因子的结合或纤颤来稳定。全面的结构-活性关系的发展也将验证Rev活性的调节是否可以在宿主免疫反应或抗逆转录病毒治疗中赋予积极的选择优势，而这反过来又与艾滋病的缓慢进展和逃离免疫系统有关。关于HIV-1调控蛋白Rev输出活性的基本问题变成：Rev结构、Rev寡聚、寡聚物Rev- rre组装以及与细胞CRM1和Ran-GTP相互作用的分子和机制特征是什么？我们建议从以下几个方面来回答这个问题：1)了解Rev寡聚化的功能和分子机制。2)阐明单体和低聚Rev及其茎II RRE底物的结构和动力学。3)建立CRM1/ ran - gtp介导的涉及低聚Rev-RRE底物复合物的核出口机制和结构模型。公共卫生相关性：HIV逆转录病毒编码少量蛋白质，因此必须利用特定的细胞辅助因子才能成功复制。我们将研究HIV基本调控蛋白Rev与参与病毒RNA核输出的细胞蛋白之间的相互作用。了解rev依赖性RNA转运的分子细节将为旨在根除病毒的治疗提供新的途径。

DESCRIPTION (provided by applicant): The Rev-RRE interaction and the subsequent transport of intron-containing viral mRNA mediated by the cellular export receptor CRM1 are essential to the replication of the HIV-1 retrovirus. The current model for oligomeric Rev binding to its cognate RRE is inadequate to fully explain the function of the regulatory protein Rev. While the importance of Rev oligomerization has been previously established, there is no clear molecular basis for understanding the requirements for nuclear transport and the interactions with the nuclear export factor CRM1. We propose to address several mechanistic and structural questions aiming to bridge the gap between Rev-oligomerization, RRE-binding and CRM1-mediated RNA export. Our integrated biochemical, biophysical, and NMR structural studies are centered on the molecular mechanisms by which the regulatory HIV-1 protein Rev attains its biologically active structures. A comprehensive model of how Rev works could potentially facilitate rational drug design targeting this essential viral gene. Our central hypothesis is that Rev function requires: 1) a defined structure and 2) oligomerization ability to serve as a substrate for the cellular export machinery. Monomeric Rev is intrinsically disordered. An on-pathway, partially folded Rev intermediate and the ordered oligomerization nucleus interconvert; a well-ordered Rev structure requires stabilization by either oligomeric binding to RRE, binding to other cofactors, or fibrillation. The proposed development of a comprehensive structure-activity relationship will also test the hypothesis whether modulation of Rev activity could confer positive selection advantages under either host immune responses or antiretroviral therapy that in turn can be linked to the slow progression of AIDS and escape from the immune system. The fundamental question concerning the export activity of the HIV-1 regulatory protein Rev becomes: What are the molecular and mechanistic features connecting Rev structure, Rev oligomerization, oligomeric Rev-RRE assembly and the interaction with cellular CRM1 and Ran-GTP? We propose to answer this question by pursuing the following Specific Aims: 1) Understand the function and molecular mechanism of Rev oligomerization. 2) Elucidate the structures and dynamics of monomeric and oligomeric Rev and of its stem II RRE substrate. 3) Develop mechanistic and structural models for CRM1/Ran-GTP-mediated nuclear export involving oligomeric Rev-RRE substrate complexes. PUBLIC HEALTH RELEVANCE: The HIV retrovirus encodes a small number of proteins and thus must exploit specific cellular cofactors for successful replication. We will investigate the interactions between the essential HIV regulatory protein Rev and cellular proteins involved in nuclear export of viral RNA. Understanding the molecular details of Rev-dependent RNA transport will provide new avenues to therapies that aim at viral eradication.