Nuclear Trafficking of the Retroviral Gag Protein

描述（由申请方提供）：逆转录病毒在动物和人类中引起不可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）是第一种与癌症相关的病毒，是原型肿瘤逆转录病毒，其作为模型系统的使用导致了对人类逆转录病毒HIV和HTLV-I的理解的重大进展。所有逆转录病毒必须进入细胞核才能建立感染。RNA基因组在细胞质中逆转录后，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。在上一个资助期，我们在RSV Gag蛋白的基质（MA）结构域中确定了核输入信号，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，并且plO序列中的CRMl依赖性核输出信号将Gag返回到胞质溶胶。有趣的是，在病毒RNA包装中有缺陷的突变Gag蛋白绕过了核区室，表明Gag的核运输和病毒RNA的胞苷化之间存在功能联系。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核质运输的病毒因子，（2）介导Gag核进入和流出的宿主辅因子，以及（3）Gag瞬时核定位的功能意义的手段。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导入的细胞途径。在目标2中，将对Gag内斯ES进行表征，并确定介导输出的宿主因子。具体目标3侧重于可验证的假设，解决了MA和Gag蛋白在PIC的核输入和选择基因组病毒RNA包装成病毒体中的作用。通过这些研究，我们希望能够深入了解病毒核进入和RNA沉积的基本但知之甚少的机制。通过阐明病毒复制的一个关键的基本途径，我们希望找到一类新的药物的目标，以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.