Mechanistic studies of genomic RNA dimerization in an oncoretrovirus

致癌逆转录病毒基因组RNA二聚化的机制研究

• 批准号: 9259362
• 负责人: Eunice C Chen
• 金额: $ 3.05万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-09 至 2021-01-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9259362
• 关键词: 5' Untranslated Regions; Address; Animals; Anti-Retroviral Agents; Binding; Biochemical; Cancer Etiology; Cell Compartmentation; Cell Nucleus; Cell membrane; Cells; Cis-Acting Sequence; Collection; Cytoplasm; Dimerization; Disease; Future; Gammaretrovirus; Genetic; Genetic Transcription; Genetic Variation; Genome; Genus Alpharetrovirus; HIV-1; Homo; Human; Image; Immunodeficiency and Cancer; Immunologic Deficiency Syndromes; Label; Laboratories; Laboratory Study; Link; Location; Mechanics; Mentors; Methods; Modeling; Molecular; Murine leukemia virus; Mutation; Nuclear; Nuclear Export; Parents; Physicians; Play; Population; RNA; RNA Viruses; Research; Retroviridae; Role; Rous sarcoma virus; Scientist; Series; Signal Transduction; Solid Neoplasm; Structural Protein; Subfamily lentivirinae; System; Testing; Therapeutic; Time; Training; Travel; Viral; Viral Genome; Virion; Virus; Virus Replication; Widespread Disease; Work; base; career development; design; dimer; drug development; experience; experimental study; fluorophore; gag Gene Products; genomic RNA; imaging modality; imaging study; inhibitor/antagonist; monomer; mutant; preference; programs; stable cell line; trafficking; viral RNA

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.

项目摘要 逆转录病毒是单个环链的RNA病毒，引起癌症和免疫方面的探测 人类和动物。 驯化的家禽中的肿瘤，是剖析复古组件的分子基础的模型。 发现RSV结构蛋白GAG透射会在CRM1依赖性中传播细胞核 时尚挑战了用CRM1抑制剂治疗的传统视图 核出口信号（NES）有效地将RSV插入核中的RSV插入到核中，使我们能够进一步研究Rolee 核中的GAG最近的研究显示了额外的逆转录病毒GAG蛋白 本地化，建议在核中可能存在插孔的常见功能。 逆转录病毒是独一无二的 基因组二聚体。 病毒RNA。 尽管在复制中至关重要，但受感染的基因组二聚化的机制 细胞仍然很少了解。 我们广泛的遗传，生化和成像方法的工具箱，因为我们可以准备好操纵您 在最近的工作中，插科打术的亚细胞定位 开发了可视化荧光团标签的RSV GAG蛋白与病毒基因组RNA共定位的方法 我们的实验表明，逆转录病毒需要核运输的核运输 基因组RNA。 我们将检验以下假设：核运输核的核能在促进基因组二聚体中起作用。 在这些研究中，我们将利用MS2和BGL RNA标记系统可视化两个不同的病毒RNA 检查基因组二聚体的种群将使我们确定亚细胞局部 二聚化；这是异二聚体的优先形成 病毒基因组或同二聚体，由两个相同的病毒基因组组成； 共转录方式。 RSV GAG最初结合单体还是二聚体。 研究GAG是否促进了细胞不同区域内的基因组二聚化。 提案将在复制周期和 可以主要支持未来的抗逆转录病毒疗法。 全面的经验，包括基于实验室的研究，科学职业的机会 发展，教育研讨会和指导，以推进医师科学家培训。