Nuclear Trafficking of the Retroviral Gag Protein

描述(申请人提供)：逆转录病毒与宿主密切相互作用，篡夺细胞途径以完成其生命周期。识别有助于病毒复制的新宿主因子将增强我们对逆转录病毒与细胞相互作用的理解，并可能为抗病毒治疗提供新的靶点。宿主因素已知在病毒颗粒形成和萌发的后期阶段发挥主要作用，但组装的早期步骤在很大程度上仍未得到充分研究，因为很难识别不同的分子事件。最近，我们发现了劳斯肉瘤病毒(RSV)组装的早期步骤：指导组装过程的病毒因子Gag蛋白的核质穿梭。这种核转运事件对感染性病毒颗粒的形成至关重要，而绕过核隔室的突变体合并了数量减少的病毒基因组RNA。我们假设，RSV基因组识别和选择，粒子组装的最早步骤之一，发生在细胞核内。这一想法挑战了目前逆转录病毒将基因组包装在细胞质中的教条；因此，这项研究项目有可能改变范式。我们的具体目标是围绕这一新的假设，解决RSV Gag核穿梭的机制方面。我们有一个定义明确的模型系统和一个信息量丰富的Gag突变体集合，这些突变体的核运输性质发生了变化，可以用作工具。在Aim1中，我们将剖析核输入途径，定义特定重要蛋白的作用，并使用进入抑制物来研究Gag核运输在病毒复制中的生物学作用。目的2重点研究GAG蛋白、GAG-RNA复合体、核仁因子和亚核小体之间的核内相互作用。在目标3中，我们将利用创新的成像方法来可视化Gag-RNA复合体在细胞核内以及从核膜到质膜的运动，以确定参与Gag-RNA运输的其他宿主因素。我们还将寻找GAG核穿梭影响基本细胞过程的证据，因为许多病毒最近被证明影响核输出、进口因子的活性以及必要的核/核仁蛋白的定位。通过这一重点实验计划，我们希望阐明逆转录病毒组装、基因组包装和参与逆转录病毒病原体及其宿主细胞之间相互作用的核事件的基本方面。 公共卫生相关性：逆转录病毒会导致人和动物的癌症和免疫缺陷综合征。这项应用侧重于确定逆转录病毒Gag蛋白的细胞内运输，GAG蛋白是逆转录病毒的主要结构蛋白。这项研究的结果可能会加深我们对逆转录病毒如何与其感染的细胞相互作用的理解，可能会导致新的抗病毒靶点和增强的逆转录病毒载体用于基因治疗。

DESCRIPTION (provided by applicant): Retroviruses interact intimately with their hosts, usurping cellular pathways to complete their life cycles. Identification of novel host factors that contribute to virus replication will enhance our understanding of retrovirus-cell interactions and may provide new targets for antiviral therapy. Host factors are known to play a major role in the late stages of virus particle formation and budding, but the early steps of assembly have remained understudied largely because distinct molecular events have been difficult to identify. Recently, we discovered an early step in Rous sarcoma virus (RSV) assembly: nucleocytoplasmic shuttling of the Gag protein, the viral factor that directs the assembly process. This nuclear trafficking event is crucial for the formation of infectious virus particles, and mutants that bypass the nuclear compartment incorporate reduced amounts of viral genomic RNA. We hypothesize that RSV genome recognition and selection, one of the earliest steps in particle assembly, occurs within the nucleus. This idea challenges the current dogma that retroviruses package their genomes in the cytoplasm; thus this research project has the potential to be paradigm-shifting. Our specific aims are centered on this novel hypothesis and address mechanistic aspects of nuclear shuttling of RSV Gag. We have a well-defined model system and an informative collection of Gag mutants with altered nuclear transport properties to use as tools. In Aim1, we will dissect the nuclear import pathway, define the roles of specific importins, and use inhibitors of entry to investigate the biological role of Gag nuclear trafficking in virus replication. Aim 2 focuses on intranuclear interactions between Gag proteins, Gag-RNA complexes, nucleolar factors, and subnuclear bodies. In Aim 3, we will utilize innovative imaging methods to visualize the movement of Gag-RNA complexes within the nucleus and from the nuclear envelope to the plasma membrane, identifying additional host factors involved in Gag- RNA trafficking. We will also be looking for evidence that Gag nuclear shuttling influences basic cellular processes, as many viruses have recently been shown to compromise nuclear export, activities of import factors, and localization of essential nuclear/nucleolar proteins. Through this focused experimental plan, we hope to shed light on fundamental aspects of retrovirus assembly, genome packaging, and nuclear events engaged in the interplay between retroviral pathogens and their host cells. PUBLIC HEALTH RELEVANCE: Retroviruses cause cancer and immunodeficiency syndromes in people and animals. This application focuses on defining the intracellular trafficking of the retroviral Gag protein, the major structural protein of retroviruses. Results of this research may enhance our understanding of how retroviruses interact with the cells they infect, potentially leading to new antiviral targets and enhanced retroviral vectors for gene therapy.