Mechanistic studies of genomic RNA dimerization in an oncoretrovirus

项目摘要 逆转录病毒是单链RNA病毒，可导致癌症和免疫缺陷疾病， 人类和动物。我们的实验室研究劳斯肉瘤病毒（RSV），一种鸟类逆转录病毒， 家禽的肿瘤，作为解剖逆转录病毒组装的分子基础的模型。我们 发现RSV结构蛋白Gag以Crm 1依赖性的方式瞬时通过细胞核， 时尚挑战了逆转录病毒组装的传统观点。使用Crm 1抑制剂或突变的治疗 核输出信号（内斯）有效地将RSV Gag捕获在细胞核中，使我们能够进一步研究RSV Gag在细胞核中的作用。 在细胞核中的加格。最近的研究揭示了额外的逆转录病毒Gag蛋白， 定位，这表明可能有共同的功能的Gag细胞核内。 逆转录病毒的独特之处在于它们将两个拷贝的基因组包装成非共价连接的 基因组RNA二聚体。基因组二聚化通过位于基因组的5 'UTR中的顺式作用序列来促进。 病毒RNA基因组的二聚化在正逆转录病毒中是保守的，并且是病毒复制所需的。 尽管它在复制中至关重要，但感染者体内基因组二聚化的潜在机制 细胞仍然知之甚少。RSV提供了一个独特的机会，研究基因组二聚化， 我们广泛的遗传、生化和成像方法工具箱，因为我们可以很容易地操纵 Gag的亚细胞定位与我们的良好表征的病毒突变体的集合。在最近的工作中，我们 开发了可视化荧光团标记的RSV Gag蛋白与病毒基因组RNA共定位的方法， 牢房我们的实验表明Gag的核运输是逆转录病毒有效包装所必需的 基因组RNA。因此，该提议旨在了解RSV中基因组二聚化的机制， 我们将检验Gag的核运输在促进基因组二聚化中起作用的假设。 在这些研究中，我们将利用MS 2和Bgl RNA标记系统来可视化两种不同的病毒RNA， 研究基因组二聚化。这种方法将使我们能够确定亚细胞位置 二聚化;是否有异二聚体的优先形成，其中包含两个遗传上不同的 病毒基因组，或同源二聚体，由两个相同的病毒基因组组成;以及二聚化是否发生在一个 共转录方式。我们还将研究核Gag在基因组二聚化中的作用，并确定 RSV Gag最初是结合单体还是二聚体。表达病毒RNA和Gag的研究将 研究Gag是否促进细胞不同区室内的基因组二聚化。这些发现 将大大有助于理解复制周期中的这一关键步骤， 可能为未来的抗逆转录病毒疗法提供支持。总的来说，该计划旨在提供一个 全面的经验，包括在实验室为基础的研究，科学生涯的机会 发展，教育研讨会和指导，以促进医生科学家的培训。

PROJECT ABSTRACT Retroviruses are single-stranded RNA viruses that cause cancers and immunodeficiency diseases in humans and animals. Our laboratory studies Rous sarcoma virus (RSV), an avian retrovirus that causes solid tumors in domesticated fowl, as a model for dissecting the molecular underpinnings of retroviral assembly. Our discovery that the RSV structural protein Gag transiently travels through the nucleus in a Crm1-dependent fashion challenged the traditional view of retroviral assembly. Treatment with a Crm1 inhibitor or mutation of the nuclear export signal (NES) effectively traps RSV Gag in the nucleus, allowing us to further study the role of Gag in the nucleus. Recent studies have revealed additional retroviral Gag proteins that undergo nuclear localization, suggesting there may be common functions of Gag within the nucleus. Retroviruses are unique in that they package two copies of their genome as non-covalently linked genomic RNA dimers. Genome dimerization is facilitated by cis-acting sequences located in the 5’UTR of the viral RNA. Dimerization of the genome is conserved in orthoretroviruses and is required for viral replication. Despite its critical importance in replication, the mechanisms underlying genome dimerization within infected cells remains poorly understood. RSV affords a unique opportunity to investigate genome dimerization using our extensive toolbox of genetic, biochemical and imaging methods because we can readily manipulate the subcellular localization of Gag with our well-characterized collection of viral mutants. In recent work, we have developed methods to visualize fluorophore-tagged RSV Gag proteins co-localized with viral genomic RNA in the cell. Our experiments suggest nuclear trafficking of Gag is required for the efficient packaging of retroviral genomic RNA. This proposal thus aims to understand the mechanism governing genome dimerization in RSV, and we will test the hypothesis that nuclear trafficking of Gag plays a role in facilitating genome dimerization. In these studies, we will utilize the MS2 and Bgl RNA labeling system to visualize two distinct viral RNA populations to examine genome dimerization. This approach will allow us to determine the subcellular location of dimerization; whether there is preferential formation of heterodimers, which contain two genetically distinct viral genomes, or homodimers, composed of two identical viral genomes; and whether dimerization occurs in a co-transcriptional manner. We will also examine the role of nuclear Gag in genome dimerization and determine whether RSV Gag initially binds monomers or dimers. Studies that express the viral RNA and Gag in trans will investigate whether Gag facilitates genome dimerization within different compartments of the cell. The findings from this proposal will greatly contribute to the understanding of this critical step in the replication cycle and may provide support for future anti-retroviral therapeutics. Overall, this program is designed to provide a comprehensive experience consisting of opportunities in laboratory-based research, scientific career development, educational seminars, and mentoring to advance physician-scientist training.