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

基本信息

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
项目类别：
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RNA mutant overexpression particle pathogen protein function prototype research study trafficking transcription factor unpublished works viral DNA viral RNA viral vector development

项目摘要

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）：逆转录病毒诱导人和动物的癌症和免疫缺陷疾病。一些众所周知的人逆转录病毒是HIV-1和人类T-淋巴细胞病毒I型I型。我们的实验室使用肌肉瘤病毒（RSV），一种致癌的逆转录病毒，作为研究病毒贩运和病原体宿主相互作用的模型系统。逆转录病毒的主要结构蛋白（GAG）将基因组病毒RNA的封装在组装过程中。逆转录病毒组件发生在质膜；但是，我们的实验室表明，RSV GAG蛋白通过细胞核瞬时运输。发现RSV GAG的核运输与基因组RNA的效率包装有关，这表明GAG可以结合细胞核中的RNA基因组。通过使用原型泡沫病毒和鼠白血病病毒进行的研究，发现在病毒整合过程中，GAG能够与宿主染色质相互作用。该提议的假设是，RSV堵嘴将染色质或染色质结合的蛋白作为捕获新转录的病毒基因组RNA的策略。该提案的目的是描述RSV GAG相互作用与宿主染色质相关因子的作用。使用生化分馏，染色质免疫沉淀和免疫荧光的技术，将确定RSV GAG是否与染色质或染色质结合的蛋白相关。在未发表的工作中，我们发现在亚细胞分馏时，在染色质相关蛋白分数中存在RSV GAG。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蛋白进行亚细胞分馏。为了鉴定与GAG相关的宿主蛋白，进行了使用亲和力纯化具有亲和力标记的RSV GAG和核裂解物的蛋白质组学研究。该蛋白质组学分析中包括与介质复合物相关的蛋白质，这对于RNA聚合酶II转录是必需的。为了确定介体蛋白在RSV基因组RNA包装中是否具有至关重要的作用，将进行介质敲低和过表达实验。对病毒基因组RNA包装的影响将通过在调解器敲低或过表达后收集的病毒颗粒中的RNA的RT-PCR确定。为了进一步识别可能与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。这些研究将介绍逆转录病毒基因组识别的早期步骤，可能导致鉴定未来的药物疗法靶标。