CYP4F12促进丙型肝炎病毒（HCV）复制及受病毒诱导表达机制研究

发布时间：2018-03-07 18:09

本文选题：丙型肝炎病毒　切入点：病毒复制　出处：《武汉大学》2016年博士论文　论文类型：学位论文

【摘要】：丙型肝炎病毒(HCV)隶属黄病毒科,其毒粒仅包含单一的9.6kb正链基因组RNA作为遗传信息的载体和病毒早期开始翻译的模板,外层由Core蛋白组成的核衣壳和结合了E1、E2糖蛋白的包膜组成。HCV从二十世纪开始传播至今,已在全世界范围内感染大约1.7亿人口,成为人类健康的严重威胁,对社会造成了巨大的卫生和经济损失。HCV在肝脏内造成感染的细胞仅占很小比例,但由它逃逸宿主免疫形成慢性感染和宿主的持续性炎症应答,将会造成长期的肝脏损伤,是导致病情发展为脂肪肝、肝硬化和肝细胞癌的主要诱因。HCV感染在肝脏内调节多种脂肪代谢基因,诱发的肝细胞质大量积累脂滴,也是推动病程发展,削弱干扰素联合治疗效果的重要原因。病毒在肝细胞内复制的过程中通过自身表达非结构蛋白诱导和组建其复制复合物的产生,这个过程中也需要招募众多宿主蛋白行使功能。本研究的初衷即寻找人肝组织中表达,但是参与到HCV复制过程中的宿主蛋白,研究其中原理和抑制病毒复制的新方向。首先我们通过酵母双杂交实验筛选出若干可能与HCV蛋白相互作用的宿主因子,从中选取了与病毒基因组复制必须的——以RNA为模板的RNA多聚酶,即HCV NS5B相互作用的细胞色素P450家族4亚族F多肽12(CYP4F12)作为继续研究的靶标。经过CoIP,荧光共聚焦和分离HCV CRC实验,我们确定了CYP4F12蛋白通过NS5B中段的手掌部结构实现相互作用。在活细胞内模拟NS5B结合病毒负链RNA上逆向5’-UTR合成基因组RNA的过程,发现CYP4F12能够提高NS5B的聚合酶活性。并且在HCV复制子系统和JFH-1活病毒感染体系中,我们观察到外源表达CYP4F12对病毒RNA水平的蛋白水平的增强作用,敲减内源CYP4F12则会降低HCV复制水平。由于没有CYP4F12启动子结构和调控的具体研究报道,我们经过筛选构建了它的启动子报告质粒,并通过一系列的实验分析确定了上面的p50和SREBP1结合位点的具体位置,以及两者之间不存在协同激活,反而在相互竞争结合空间的关系。并以这些结果为基础,研究发现了：HCV在感染细胞过程中上调p50和SREBP1水平；HCV NS3/4A特异的增强CYP4F12启动子上SRE和SREBP1的结合,而p7蛋白则对p50结合有一定促进。从而揭示HCV感染中对CYP4F12的诱导调控方式。同时,初步探索了HCV对CYP4F亚族其他成员的影响,发现CYP4F11受到和CYP4F12类似的调节,并且幅度更大。本实验首次将细胞色素酶P450和HCV联系在一起,为进一步理解HCV在细胞内复制的过程提供了新的信息,并指出了HCV影响细胞脂类代谢的另一个途径。
[Abstract]:Hepatitis C virus (HCV) belongs to the family Flavoviridae and contains only a single 9.6kb positive strand genomic RNA as a carrier of genetic information and a template for early translation of the virus. The outer core-capsid composed of Core protein and the envelope composed of E1OE2 glycoprotein. HCV has been infected about 170 million people worldwide since 20th century, and has become a serious threat to human health. Great health and economic losses to society. HCV causes only a small percentage of infected cells in the liver, but chronic infection and persistent inflammatory response from which it escapes will cause long-term liver damage. HCV infection regulates a variety of fat metabolism genes in the liver, and induces a large accumulation of lipid droplets in the liver cytoplasm, which also promotes the development of the course of disease. The important reason for weakening the effect of interferon combination therapy is that the virus induces and forms its replication complex by self-expression of non-structural proteins during the process of replication in hepatocytes. The original purpose of this study is to look for host proteins expressed in human liver tissues but involved in the process of HCV replication. First, we screened out some host factors which may interact with HCV protein by yeast two-hybrid experiment. RNA polymerase based on RNA was selected as the target for further study. It was the cytochrome P450 family member F polypeptide 12CYP 4F12 that interacted with HCV NS5B. After CoIP, fluorescence confocal focusing and isolation of HCV CRC, We have determined that the CYP4F12 protein interacts through the palm structure of the midpiece of NS5B. In living cells, we mimic the synthesis of genomic RNA by reverse 5H-UTR on the negative RNA of NS5B binding virus. It was found that CYP4F12 could increase the polymerase activity of NS5B, and in the HCV replication subsystem and JFH-1 virus infection system, we observed that exogenous expression of CYP4F12 could enhance the protein level of viral RNA. Knockout of endogenous CYP4F12 reduces the level of HCV replication. Since there are no specific studies on the promoter structure and regulation of CYP4F12, we have screened and constructed its promoter reporter plasmid. Through a series of experimental analyses, the specific position of the above p50 and SREBP1 binding sites is determined, and the relationship between the above p50 and SREBP1 binding sites is not synergistically activated, but in the competitive binding space, and based on these results, It has been found that the level of p50 and SREBP1 is up-regulated in the process of infection of SRE and SREBP1, and HCV NS3/4A specifically enhances the binding of SRE and SREBP1 on CYP4F12 promoter, while p7 protein promotes the binding of p50, thus revealing the induced regulation of CYP4F12 in HCV infection. The effects of HCV on other members of CYP4F subfamily were preliminarily explored, and it was found that CYP4F11 was regulated by CYP4F12 to a much greater extent. In this study, cytochrome P450 and HCV were first linked together. It provides new information for further understanding the process of HCV replication in cells and points out another way for HCV to affect lipid metabolism of cells.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R512.63

【相似文献】