Nuclear Trafficking of the Retroviral Gag Protein

描述（由申请人提供）：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。禽类病原体劳斯肉瘤病毒（RSV）是第一个与癌症相关的病毒，是一种典型的逆转录病毒，将其作为一种模型系统，在了解人类逆转录病毒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产生感染。随着细胞质中RNA基因组的逆转录，整合前复合物（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，因为有丝分裂不是绝对必需的。这种核输入过程是病毒生命周期中最不为人所知的方面之一。在之前的资助期内，我们在RSV Gag蛋白的基质（MA）结构域中发现了一个核输入信号，这提高了MA可能在PIC的核输入中发挥作用的可能性。此外，我们发现MA中的核输入信号在复制周期的组装阶段介导Gag多蛋白的核定位，p10序列中依赖于crml的核输出信号将Gag返回到细胞质中。有趣的是，在病毒RNA包装中有缺陷的Gag蛋白突变绕过核室，这表明Gag的核运输与病毒RNA的封装之间存在功能联系。当前提案的主要目标是利用这些关键发现作为阐明(1)控制Gag核胞质运输的病毒因子，(2)介导Gag核进入和出口的宿主辅助因子，以及(3)Gag瞬时核定位的功能意义。在第一个特定目标中，我们建议定义MA中的NLS，核进入机制以及用于输入的细胞途径。在目标2中，将对Gag NES进行表征，并确定介导出口的宿主因素。特异性目标3侧重于可测试的假设，这些假设解决了MA和Gag蛋白在PIC的核输入和基因组病毒RNA包装成病毒粒子的选择中的作用。通过这些研究，我们期望深入了解病毒核进入和RNA封装的基本但尚不清楚的机制。通过阐明对病毒复制至关重要的基本途径，我们希望找到一类新的药物来对抗逆转录病毒疾病。

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.