Interaction of Oncoretroviral Gag Protein with Host Nuclear Factors

肿瘤逆转录病毒 Gag 蛋白与宿主核因子的相互作用

• 批准号: 8909970
• 负责人: Breanna Lynn Rice
• 金额: $ 2.93万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8909970
• 关键词: Active Sites; Activities of Daily Living; Adenoviruses; Affinity; Affinity Chromatography; Animals; Binding; Binding Proteins; Biochemical; Biological Models; Birds; Cancer Etiology; Cell Fractionation; Cell Nucleus; Cell membrane; Cells; Chromatin; Chromosomes; Complex; Cytoplasm; DNA; Data; Disease; Drosophila genus; Euchromatin; Feline Immunodeficiency Virus; Fluorescence Resonance Energy Transfer; Fractionation; Future; Gagging; Genes; Genetic Transcription; Genome; Genomics; Genus Alpharetrovirus; Goals; HIV-1; Health; Hereditary Disease; Herpesvirus 1; Herpesvirus Type 3; Heterochromatin; Human; Human Spumavirus; Human T-lymphotropic virus 1; Immunodeficiency and Cancer; Immunofluorescence Immunologic; Infectious Agent; Integration Host Factors; Knowledge; Laboratories; Light; Link; Mediator of activation protein; Medicine; Mouse Mammary Tumor Virus; Murine leukemia virus; Nuclear; Nuclear Proteins; Nucleoplasm; Oncogenes; Oncogenic; Organism; Pharmacotherapy; Play; Positioning Attribute; Protein Splicing; Proteins; Proteomics; RNA; RNA Binding; RNA Polymerase II; RNA chemical synthesis; RNA-Directed DNA Polymerase; Recombinants; Retrotransposon; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Role; Rous sarcoma virus; Science; Site; Spumavirus; Structural Protein; Techniques; Transcription Coactivator; Viral; Viral Structural Proteins; Virion; Virus Replication; Work; chromatin immunoprecipitation; chromatin protein; ds-DNA; gag Gene Products; genomic RNA; mutant; overexpression; particle; pathogen; protein function; prototype; research study; trafficking; transcription factor; unpublished works; viral DNA; viral RNA; viral vector development

DESCRIPTION (provided by applicant): Retroviruses induce cancer and immunodeficiency diseases in both humans and animals. Some commonly known human retroviruses are HIV-1 and human T-lymphotropic virus type I. Our laboratory uses Rous sarcoma virus (RSV), an oncogenic avian retrovirus, as a model system to study viral trafficking and pathogen- host interactions. The major structural protein of retroviruses, Gag, directs the encapsidation of genomic viral RNA into virus particles during assembly. Retroviral assembly occurs at the plasma membrane; however, our laboratory has shown that the RSV Gag protein transiently traffics through the nucleus. It was found that nuclear trafficking of RSV Gag is linked to efficiet packaging of genomic RNA, suggesting that Gag may bind the RNA genome in the nucleus. Through studies performed with prototype foamy virus and murine leukemia virus, it was discovered the Gag was able to interact with host chromatin during proviral integration. The hypothesis of this proposal is that RSV Gag tethers to chromatin or chromatin-bound proteins as a strategy for capturing the newly transcribed viral genomic RNA. The goal of this proposal is to delineate the role of RSV Gag interaction with host chromatin-associated factors. Using the techniques of biochemical fractionation, chromatin immunoprecipitation, and immunofluorescence, it will be determined whether RSV Gag associates with chromatin or chromatin-bound proteins. In unpublished work, we found that RSV Gag is present in chromatin-associated protein fractions when subcellular fractionations were performed. To determine whether this phenomenon is conserved among other retroviruses, subcellular fractionations will be performed on a variety of Gag proteins from different retroviral genera. To identify Gag-associated host proteins, proteomic studies using affinity purification with affinity tagged RSV Gag and nuclear lysates were performed. Included in this proteomic analysis were proteins associated with the mediator complex, which is necessary for RNA polymerase II transcription. To determine whether mediator proteins have a crucial role in RSV genomic RNA packaging, mediator knockdown and overexpression experiments will be performed. Effects on viral genomic RNA packaging will be determined through RT-PCR of the RNA within virus particles collected after mediator knockdown or overexpression. To further identify other chromatin proteins that could interact with RSV Gag, tandem affinity purification experiments will be performed. These studies will shed some light into the early steps of retroviral genome recognition, possibly leading to the identification of future targets for drug therapies.

描述（由申请人提供）：逆转录病毒会在人类和动物中诱发癌症和免疫缺陷疾病。一些常见的人类逆转录病毒是 HIV-1 和人类 T 淋巴细胞病毒 I 型。我们的实验室使用劳斯肉瘤病毒 (RSV)（一种致癌禽逆转录病毒）作为模型系统来研究病毒贩运和病原体-宿主相互作用。逆转录病毒的主要结构蛋白 Gag 在组装过程中指导基因组病毒 RNA 衣壳化成病毒颗粒。逆转录病毒组装发生在质膜上；然而，我们的实验室已经证明 RSV Gag 蛋白可以短暂地穿过细胞核。结果发现，RSV Gag 的核运输与基因组 RNA 的有效包装有关，这表明 Gag 可能与细胞核中的 RNA 基因组结合。通过对原型泡沫病毒和鼠白血病病毒进行的研究，发现 Gag 能够在原病毒整合过程中与宿主染色质相互作用。该提议的假设是，RSV Gag 与染色质或染色质结合蛋白结合，作为捕获新转录的病毒基因组 RNA 的策略。该提案的目标是描述 RSV Gag 与宿主染色质相关因子相互作用的作用。 使用生化分级分离、染色质免疫沉淀和免疫荧光技术，将确定 RSV Gag 是否与染色质或染色质结合蛋白结合。在未发表的工作中，我们发现在进行亚细胞分级分离时，RSV Gag 存在于染色质相关蛋白组分中。为了确定这种现象在其他逆转录病毒中是否保守，将对来自不同逆转录病毒属的多种 Gag 蛋白进行亚细胞分级。为了鉴定 Gag 相关宿主蛋白，使用亲和标记的 RSV Gag 和核裂解物进行亲和纯化进行蛋白质组学研究。该蛋白质组分析包括与介体复合物相关的蛋白质，这是 RNA 聚合酶 II 转录所必需的。为了确定介体蛋白是否在 RSV 基因组 RNA 包装中发挥关键作用，将进行介体敲除和过表达实验。对病毒基因组 RNA 包装的影响将通过对介质敲低或过度表达后收集的病毒颗粒内的 RNA 进行 RT-PCR 来确定。为了进一步鉴定可以与 RSV Gag 相互作用的其他染色质蛋白，将进行串联亲和纯化实验。这些研究将为逆转录病毒基因组识别的早期步骤提供一些线索，可能有助于确定未来药物治疗的靶点。