Nuclear Trafficking of the Retroviral Gag Protein

描述（由申请方提供）：逆转录病毒利用细胞途径促进病毒颗粒的组装，从而传播感染。确定宿主因子篡夺逆转录病毒的病毒装配将提高我们的逆转录病毒细胞相互作用的理解，并可能发现新的抗病毒治疗的目标。虽然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，但组装的早期阶段仍然知之甚少，因为启动组装的分子事件一直难以研究。然而，我们的发现，劳斯肉瘤病毒（RSV）Gag蛋白，它协调的装配过程，经历核贩运，使我们能够研究在细胞核中发生的装配途径的早期事件。使用该系统，我们表明RSV Gag的核运输对于感染性病毒颗粒的形成至关重要，并且绕过核的突变体掺入减少量的病毒基因组RNA。此外，我们的结构研究表明，Gag结合到核输出机制是由Gag-RNA相互作用促进的，为病毒核糖核蛋白复合物的调节转运提供了机制。来自我们实验室的新数据表明，RSV Gag可以运输到活性转录位点，以识别和选择细胞核中的病毒基因组RNA。因此，我们的研究可能会为逆转录病毒如何选择和包装它们的基因组定义一个新的范式。在本申请中，我们提出了两个特定目的来测试Gag核运输促进基因组RNA的识别、选择和包装的新假设。这些机理实验利用了我们的Gag突变体的良好表征工具箱，其具有改变的核运输和RNA包装特性。我们将利用我们独特的试剂组来研究Gag是否被拴系在RNA合成位点附近以介导病毒基因组RNA的共转录包装。此外，我们将使用质谱法来鉴定与RSV Gag相互作用的宿主因子，并使用深度测序来分析Gag的细胞RNA靶标。从不同的逆转录病毒进行核定位的GAG相互作用因子的蛋白质组学分析将扩大这种应用的影响，并提供深入了解逆转录病毒Gag蛋白在细胞核中的潜在共同功能。通过这个精心设计的，重点突出的实验计划，我们将揭示逆转录病毒组装，基因组RNA包装和核事件的基本方面，揭示有关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.