Nuclear Trafficking of the Retroviral Gag Protein

描述(由申请人提供)：逆转录病毒选择细胞途径，以促进传播感染的病毒颗粒的组装。识别被逆转录病毒侵占的宿主因子进行病毒组装将增强我们对逆转录病毒与细胞相互作用的理解，并可能发现抗病毒治疗的新靶点。尽管参与逆转录病毒组装和释放的后期步骤的宿主因素已经被确定，但由于启动组装的分子事件一直难以研究，因此组装的早期阶段仍然知之甚少。然而，我们发现协调组装过程的劳斯肉瘤病毒(RSV)Gag蛋白经历了核运输，这使我们能够研究发生在细胞核中的组装途径中的早期事件。使用这个系统，我们证明了RSV Gag的核运输对于感染性病毒颗粒的形成是至关重要的，而绕过核的突变体合并了数量减少的病毒基因组RNA。此外，我们的结构研究表明，Gag与核出口机制的结合是通过Gag-RNA相互作用促进的，为病毒核糖核蛋白复合体的调控运输提供了一种机制。我们实验室的新数据表明，RSV Gag可能通过运输到活跃的转录位点来识别和选择细胞核中的病毒基因组RNA。因此，我们的研究可能为逆转录病毒如何选择和包装它们的基因组定义一种新的范式。在这一应用中，我们提出了两个具体的目标来检验这一新的假设，即Gag核贩运促进了基因组RNA的识别、选择和包装。这些机械实验利用了我们特征良好的Gag突变体工具箱，这些突变体具有改变的核运输和RNA包装属性。我们将利用我们独特的试剂集来调查GAG是否被拴在RNA合成位点附近，以介导病毒基因组RNA的共转录包装。此外，我们将使用质谱仪来鉴定与RSV Gag相互作用的宿主因子，并进行深度测序来分析Gag的细胞RNA靶标。对来自不同逆转录病毒的Gag相互作用因子进行蛋白质组学分析将扩大这一应用的影响，并提供对逆转录病毒Gag蛋白在细胞核中潜在的共同功能的洞察。通过这个精心设计的、有重点的实验计划，我们将阐明逆转录病毒组装、基因组RNA包装和核事件的基本方面，以揭示关于逆转录病毒病原体及其宿主细胞之间相互作用的新信息。

DESCRIPTION (provided by applicant): Retroviruses co-opt cellular pathways to facilitate the assembly of virus particles that spread infection. Identifying the host factors usurped by retroviruses for virus assembly will enhance our understanding of retrovirus-cell interactions and may uncover new targets for antiviral therapy. Although host factors engaged in late steps of retrovirus assembly and release have been identified, the early stages of assembly remain poorly understood because the molecular events that initiate assembly have been difficult to study. However, our discovery that the Rous sarcoma virus (RSV) Gag protein, which orchestrates the assembly process, undergoes nuclear trafficking has allowed us to study early events in the assembly pathway that occur in the nucleus. Using this system, we showed that nuclear trafficking of RSV Gag is crucial for the formation of infectious virus particles, and mutants that bypass the nucleus incorporate reduced amounts of viral genomic RNA. Furthermore, our structural studies demonstrate that Gag binding to nuclear export machinery is facilitated by Gag-RNA interactions, providing a mechanism for the regulated transport of viral ribonucleoprotein complexes. New data from our laboratory suggest that RSV Gag may traffic to sites of active transcription to recognize and select viral genomic RNA in the nucleus. Therefore, our research may define a novel paradigm for how retroviruses select and package their genomes. In this application, we propose two Specific Aims to test the novel hypothesis that Gag nuclear trafficking facilitates recognition, selection, and packaging of the genomic RNA. These mechanistic experiments take advantage of our well-characterized toolbox of Gag mutants with altered nuclear trafficking and RNA packaging properties. We will utilize our unique set of reagents to investigate whether Gag is tethered near sites of RNA synthesis to mediate co-transcriptional packaging of viral genomic RNA. In addition, we will use mass spectrometry to identify host factors that interact with RSV Gag and deep sequencing to analyze cellular RNA targets of Gag. Proteomic analysis of Gag-interacting factors from diverse retroviruses that undergo nuclear localization will broaden the impact of this application and provide insight into potential common functions of retroviral Gag proteins in the nucleus. Through this well-crafted, focused experimental plan, we will shed light on fundamental aspects of retrovirus assembly, genomic RNA packaging, and nuclear events to reveal new information about the interplay between retroviral pathogens and their host cells.