Interaction of Oncoretroviral Gag Protein with Host Nuclear Factors

描述(申请人提供)：逆转录病毒导致人类和动物的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。我们实验室使用Rous肉瘤病毒(RSV)作为模型系统，研究病毒的传播和病原体-宿主的相互作用。逆转录病毒的主要结构蛋白GAG在组装过程中指导病毒基因组RNA被包裹到病毒颗粒中。逆转录病毒组装发生在质膜；然而，我们的实验室已经表明，RSV Gag蛋白瞬时通过细胞核运输。研究发现，RSV Gag的核转运与基因组RNA的有效包装有关，提示Gag可能与核内的RNA基因组结合。通过对原型泡沫病毒和小鼠白血病病毒进行的研究，发现GAG能够在前病毒整合过程中与宿主染色质相互作用。这一建议的假设是，RSV Gag与染色质或染色质结合蛋白捆绑在一起，作为捕获新转录的病毒基因组RNA的一种策略。这项提议的目的是描述RSV Gag与宿主染色质相关因子相互作用的作用。利用生化分离、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相结合。在未发表的工作中，我们发现当进行亚细胞分离时，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各种Gag蛋白进行亚细胞分级。为了鉴定与Gag相关的宿主蛋白，用亲和标记的RSV Gag和核裂解物进行了亲和纯化的蛋白质组学研究。蛋白质组学分析中包括与中介复合体相关的蛋白质，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包装中起关键作用，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RT-PCR对介体敲除或过度表达后收集的病毒颗粒内的RNA进行确定。为了进一步鉴定其他可以与RSV Gag相互作用的染色质蛋白，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，可能导致确定未来药物治疗的目标。

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 In unpublished work, we found that RSV Gag is present in chromatin-associated protein fractions when subcellular fractionations were performed.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.