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

描述(申请人提供)：艾滋病毒基因组的核出口：出口过程中艾滋病毒Rev和宿主CRM1相互作用和结构的分子动力学研究艾滋病毒病毒是一种大流行慢病毒，目前还没有治愈方法或疫苗。目前的治疗方法包括含有多种药物的强大药物鸡尾酒，这些药物针对病毒生命周期的多个方面。除了这些药物引起的毒副作用外，在大多数情况下，病毒对所使用的药物产生抗药性，迫使人们转向毒性更强、效果更差的药物，最终导致病毒逃脱药物控制。尽管有各种各样的药物可用，但目前还没有药物针对病毒生命周期的关键部分，即REV和Exportin-1(CRM1)介导的病毒基因组在活跃的病毒复制过程中的输出。在这里，病毒蛋白REV在病毒基因组的REV反应元件(RRE)上形成一个由六个部分组成的寡聚体。然后，这种寡聚体招募宿主蛋白CRM1的副本，绕过标准的mRNA剪接和输出途径，将基因组输出到细胞核外。针对这一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佳的限制。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，甚至结构和机制的一些细节，但到目前为止所得到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该途径的尝试。利用计算和分子动力学方法的精细时空分辨率，这些问题是可以克服的。在本项目中，这些方法将用于调查出口复合体形成的整个过程，从第一个REV单体到RRE一直到招募多个CRM1拷贝，并将使用它们来解决现场悬而未决的问题，并为进一步研究铺平道路。具体地说，本研究将验证以前提出的机制和结构，深入研究REV齐聚物-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，还将研究该组件与CRM1的结合，结果将用于解决诸如结合的CRM1蛋白的最大可能数量以及所有六个REV拷贝在结合中的重要性等主题。最后，也是最重要的是，将生成整个出口综合体的结构，该结构可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，甚至有可能将这些成果付诸实践。 公共卫生相关性：HIV基因组的核输出：出口过程中HIV REV和宿主CRM1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，但HIV REV在病毒基因组的REV反应元件(RRE)序列上进行寡聚并招募CRM1输出该基因组的过程尚未得到很好的描述。当考虑计算方法时，阻碍完全理解这一过程的实验研究的许多障碍都消失了。当使用这些方法时，对REV输出途径的更多了解将被证明对研究领域非常宝贵，并可能为探索艾滋病毒复制的少数步骤之一提供耐人寻味的途径，而该步骤尚未成为治疗的目标。

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.