Nuclear Trafficking of the Retroviral Gag Protein

描述(申请人提供)：逆转录病毒在动物和人类中引起无法治愈的恶性肿瘤和免疫缺陷疾病。禽类病原体Rous肉瘤病毒(RSV)是第一种与癌症有关的病毒，是典型的肿瘤逆转录病毒，它作为模型系统的使用使人们在理解人类逆转录病毒HIV和HTLV-I方面取得了重大进展。所有逆转录病毒都必须进入细胞核，才能确定感染。在细胞质中的RNA基因组反转录后，前整合复合体(PIC)进入细胞核。在RSV和包括HIV在内的慢病毒中，核进入似乎是一个活跃的过程，因为有丝分裂并不是绝对必需的。这一核进口过程是病毒生命周期中最鲜为人知的方面之一。在上一个资助阶段，我们在RSV Gag蛋白的基质(MA)结构域中发现了一个核导入信号，这增加了MA可能在PIC的核导入中发挥作用的可能性。此外，我们还发现，在复制周期的组装阶段，MA中的核输入信号介导了Gag多聚蛋白的核定位，而p10序列中依赖CRM1的核输出信号将Gag返回到胞浆。有趣的是，在病毒RNA包装中有缺陷的突变Gag蛋白绕过了核隔间，这表明GAG的核运输和病毒RNA的封装之间存在功能联系。本提案的主要目的是利用这些关键发现作为一种手段来阐明(1)控制GAG核质运输的病毒因素，(2)介导GAG核进出的宿主辅助因子，以及(3)GAG短暂核定位的功能意义。在第一个具体目标中，我们建议定义MA中的NLS，核进入的机制，以及用于输入的细胞通路。在目标2中，将描述GAG NES的特征，并确定调解出口的宿主因素。具体目标3侧重于可测试的假说，这些假说解决了MA和GAG蛋白在PIC的核输入和选择基因组病毒RNA包装成病毒粒子中的作用。通过这些研究，我们希望深入了解病毒核进入和RNA包裹的基本机制，但了解得很少。通过阐明病毒复制的关键基本途径，我们希望为一类新的抗逆转录病毒疾病的药物找到靶点。

DESCRIPTION (provided by applicant): Retroviruses cause incurable malignancies and immunodeficiency diseases in animals and humans. The avian pathogen Rous sarcoma virus (RSV), the first virus linked to cancer, is the prototypic oncoretrovirus, and its use as a model system has led to significant advances in understanding the human retroviruses HIV and HTLV-I. All retroviruses must gain access to the nucleus in order to establish infection. Following reverse transcription of the RNA genome in the cytoplasm, the preintegration complex (PIC) enters the nucleus. In RSV and lentiviruses including HIV, nuclear entry appears to be an active process, as mitosis is not absolutely required. This process of nuclear import is one of the least understood aspects of the viral life cycle. In the previous funding period, we identified a nuclear import signal in the matrix (MA) domain of the RSV Gag protein, raising the possibility that MA might play a role in nuclear import of the PIC. In addition, we found that the nuclear import signal in MA mediates nuclear localization of the Gag polyprotein during the assembly phase of the replication cycle, and a CRMl-dependent nuclear export signal in the p10 sequence returns Gag to the cytosol. Interestingly, a mutant Gag protein that is defective in viral RNA packaging bypasses the nuclear compartment, suggesting a functional link between the nuclear trafficking of Gag and encapsidation of viral RNA. The major goal of the current proposal is to capitalize on these pivotal findings as a means to elucidate (1) the viral factors that control nucleocytoplasmic trafficking of Gag, (2) host cofactors that mediate nuclear entry and egress of Gag, and (3) the functional significance of transient nuclear localization of Gag. In the first Specific Aim, we propose to define the NLS in MA, the mechanism of nuclear entry, and the cellular pathways used for import. In Aim 2, the Gag NES will be characterized and host factors that mediate export will be identified. Specific Aim 3 focuses on testable hypotheses that address the role of MA and Gag proteins in nuclear import of the PIC and selection of genomic viral RNA for packaging into virions. Through these studies, we expect to gain insight into the essential but poorly understood mechanisms underlying viral nuclear entry and RNA encapsidation. By elucidating a fundamental pathway critical for virus replication, we hope to find targets for a new class of agents to combat retroviral diseases.