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介导的含有内含子的病毒mRNA的运输对于HIV-1逆转录病毒的复制是必不可少的。目前的低聚REV与其同源RRE结合的模型不足以完全解释调节蛋白REV的功能。虽然REV寡聚的重要性已经被证实，但没有明确的分子基础来理解核运输的要求以及与核出口因子CRM1的相互作用。我们提出了几个机制和结构问题，旨在弥合REV齐聚、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节HIV-1蛋白Rev获得其生物活性结构的分子机制上。REV如何发挥作用的全面模型可能会促进针对这种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)确定的结构和2)齐聚能力作为细胞输出机制的底物。单体REV本质上是无序的。在途径上，部分折叠的REV中间体和有序的齐聚核相互转化；良好的REV结构需要通过与RRE结合、与其他辅因子结合或纤颤来稳定。提出的全面结构-活性关系的发展也将检验这一假设，即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下赋予正选择优势，进而与艾滋病的缓慢发展和逃离免疫系统有关。关于HIV-1调节蛋白REV出口活性的根本问题是：REV结构、REV寡聚、寡聚REV-RRE组装以及与细胞CRM1和RAN-GTP的相互作用有什么分子和机制特征？我们打算通过以下具体目标来回答这个问题：1)了解REV齐聚的功能和分子机制。2)阐明了单体和寡聚体REV及其茎II RRE底物的结构和动力学。3)建立了CRM1/RAN-GTP介导的含低聚REV-RRE底物复合体的核输出的机理和结构模型。与公共卫生相关：艾滋病毒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。我们将研究必要的HIV调节蛋白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依赖的RNA运输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.