Nuclear export of the HIV genome: A Molecular Dynamics Study of the Interactions

描述（由申请人提供）：HIV基因组的核输出：输出过程中HIV Rev和宿主CRM 1相互作用和结构的分子动力学研究总结HIV病毒是一种流行性慢病毒，目前尚无治愈方法或疫苗。目前的治疗方法包括强大的药物鸡尾酒，其中含有针对病毒生命周期多个方面的多种药物。除了许多这些药物引起的毒副作用外，在大多数情况下，病毒对所用药物产生耐药性，迫使改用毒性更大、效果更差的药物，最终导致病毒逃脱药物管制。尽管有各种各样的药物可用，但目前没有药物靶向病毒生命周期的关键部分，即Rev和Exportin-1（Crm 1）介导的病毒活跃复制期间全病毒基因组的输出。在此，病毒蛋白Rev在病毒基因组的Rev反应元件（RRE）上形成六部分寡聚体。然后，这种寡聚体招募宿主蛋白crm 1的拷贝，绕过mRNA剪接和输出的标准途径，将基因组输出细胞核。针对该出口途径的尝试受到对所涉及蛋白质的结构和机制理解不足的限制。虽然实验方法已经成功地阐明了该过程的广泛细节，所涉及的蛋白质和RNA的结合区域，甚至结构和机制的一些细节，但迄今为止所获得的理解是不完整和不确定的，不足以用于真正深入的研究或支持修改途径的尝试。利用精细的时空分辨率的计算和分子动力学方法，这些问题可以克服。在本项目中，这些方法将用于研究输出复合物形成的整个过程，从第一个Rev单体与RRE的连接一直到多个crm 1拷贝的募集，并将使用它们来解决该领域的突出问题，并为进一步研究开辟道路。具体而言，本研究将验证先前提出的机制和结构，深入研究Rev寡聚体-RRE组装的机制，包括协同性的重要性，并提供这种组装的完整结构。此外，将研究组装与crm 1的结合，并将结果用于解决诸如结合crm 1蛋白的最大合理数量和所有六个Rev拷贝在结合中的重要性等主题。最后，也是最重要的是，整个出口复杂的结构将被生成，这可以用于未来的机械和计算研究，或作为未来实验研究的路标。从长远来看，甚至有可能将这些结果付诸实践。 公共卫生相关性：HIV基因组的核输出：HIV Rev和宿主CRM 1在输出过程中的相互作用和结构的分子动力学研究尽管进行了广泛的研究，HIV Rev在病毒基因组的Rev反应元件（RRE）序列上寡聚化并招募crm 1输出该基因组的过程还没有得到很好的表征。当考虑计算方法时，许多阻碍实验研究的障碍就会消失，这些障碍会阻碍人们充分理解这一过程。当使用这种方法时，将获得对Rev输出途径的更深入了解，这将证明对研究领域是非常宝贵的，并可能为探索HIV复制的少数步骤之一提供有趣的途径，而这还不是治疗的目标。

DESCRIPTION (provided by applicant): Nuclear export of the HIV genome: A Molecular Dynamics Study of the Interactions and Structure of HIV Rev and Host CRM1 during Export Summary The HIV virus is a pandemic lentivirus which has no existing cure or vaccine. Current treatments consist of powerful drug cocktails containing multiple drugs targeting multiple aspects of the viral lifecycle. Besides the toxic side effects caused by many of these drugs, in the majority of cases the virus develops resistance to the drugs used, forcing a switch to more toxic and less effective drugs, culminating in the escape of the virus from drug control. Despite the variety of drugs available, no current drugs target a critical part of the viral lifecycle, the Rev and Exportin-1 (Crm1) mediated export of the full viral genome during active viral replication. Here, the viral protein Rev forms a six-part oligomer on the Rev Response Element (RRE) of the viral genome. This oligomer then recruits copies of the host protein crm1 to export the genome out of the cell nucleus, bypassing the standard pathways of mRNA splicing and export. Attempts to target this export pathway are limited by a combination of poor structural and mechanistic understandings of the proteins involved. While experimental methods have been successful in elucidating broad details of the process, the binding regions of the proteins and RNA involved, and even some details of structure and mechanism, the understanding derived so far is incomplete and uncertain, inadequate for use in truly in-depth studies or to support attempts to modify the pathway. Utilizing the fine spatiotemporal resolution of computational and molecular dynamics approaches, these problems can be overcome. In this project, these methods will be used to investigate the entire process of export complex formation, from the attachment of the first Rev monomer to the RRE all the way up the recruitment of multiple crm1 copies, and will use them to address outstanding questions in the field and to open the way for further investigation. Specifically, this study will validate previous proposed mechanisms and structures, deeply investigate the mechanism of Rev oligomer-RRE assembly, including the importance of cooperativity, and provide a complete structure of this assembly. In addition, binding of the assembly with crm1 will be investigated, and the results used to address such topics as the maximal plausible number of bound crm1 proteins and the importance of all six Rev copies in binding. Finally, and most importantly, a structure of the entire export complex will be generated, which can be used in future mechanistic and computational studies, or as a guidepost for future experimental studies. In the long-run it may even be possible to put these results to practical use. PUBLIC HEALTH RELEVANCE: Nuclear export of the HIV genome: A Molecular Dynamics Study of the Interactions and Structure of HIV Rev and Host CRM1 during Export Narrative Despite extensive research, the process by which HIV Rev oligomerizes on the Rev Response Element (RRE) sequence of the viral genome and recruits crm1 to export this genome is not well-characterized. Many of the obstacles to experimental studies that frustrate attempts to fully understand the process fall away when computational methods are considered instead. The greater understanding of the Rev export pathway that will be obtained when such methods are used will prove invaluable to the field of study, and may provide intriguing avenues of exploration into one of the few steps of HIV replication which is not yet the target of therapy.