AP1复合体在HCV感染过程中作用的机制研究

发布时间：2018-04-03 08:39

本文选题：适配蛋白复合体1　切入点：丙型肝炎病毒　出处：《北京协和医学院》2015年博士论文

【摘要】：丙型肝炎病毒(Hepatitis C virus, HCV)目前已导致全球约1亿5千万人感染,并可能进一步发展为肝硬化或肝癌等疾病。病毒的生命周期依赖于病毒与宿主因子的相互作用。目前,关于HCV与宿主相互作用的研究主要集中于病毒入侵和复制阶段,主要针对病毒受体和复制必须因子等,而对病毒生命周期晚期阶段的研究则相对较少。随着人类功能基因组筛选技术的成熟,越来越多的病毒相关宿主依赖因子通过此技术被发现并确认,为了进一步研究HCV生命周期所必须的宿主因子,本研究利用人类50k shRNA库,对HCV感染相关宿主基因进行了系统化筛选,并获得了206个候选基因。通过对候选基因的功能学鉴定,我们发现干扰AP1复合体西格玛3亚基(adaptor-related protein complex 1 sigma 3 subunit, AP1S3)的抗病毒效果明显并且稳定。AP1属于AP家族,是一个异四聚体蛋白复合体(两个大亚基、一个中亚基和一个小亚基),在细胞中识别分选信号,并组装成转运小泡,在细胞内各细胞器之间扮演着重要的物质运输作用。不同的AP复合体负责不同的转运路径,AP1主要在反式高尔基体(trans Golgi network, TGN)和内体之间运输,而AP2则主要在细胞内吞作用时发挥作用。许多病毒会劫持AP1复合体的胞内运输功能,以利于自身的组装和出芽,包括人免疫缺陷病毒(human immunodeficiency virus, HIV)和非洲猪瘟病毒(african swine fever virus, ASFV)。为了进一步验证AP1S3调节HCV生命周期的作用,我们使用了Jcl-Luc HCVcc和JFH-1 HCVcc对其进行了评价,发现干扰AP1S3具有良好的抗病毒效果。进而我们使用了HCV假病毒系统(HCVpp)、HCV复制子系统(2-3+)和HCV组装释放系统对AP1S3参与病毒生命周期的具体环节进行了鉴定。研究结果显示,AP1S3主要在HCV生命周期晚期阶段发挥作用,敲低AP1S3能够显著降低HCV子代病毒的产生,并且发现敲低AP1复合体的其他亚基也具有类似的作用。为了揭示AP1参与病毒调控的机制,本研究通过免疫共沉淀实验发现AP1能够通过两个经典的模体,YXXΦ和(D/E)XXXL(L/I),与HCV的两个结构蛋白core和E2相互作用。通过这两组模体所介导的相互作用,AP1能够保护core和E2免于蛋白酶体的降解。干扰AP1会导致core和E2蛋白泛素化程度的增加。进一步的体内外泛素化实验显示,作为E3泛素连接酶,E6相关蛋白(E6 associated protein, E6AP)能够介导core和E2的泛素化过程。为了鉴定干扰AP1是否能够引起其他病毒蛋白表达量的下降。我们选取了4种病毒包膜蛋白进行AP1相关的包膜蛋白降解实验,包括日本脑炎病毒(Japanese encephalitis virus, JEV)、西尼罗病毒(West Nile virus, WNV)、埃博拉病毒(Ebolavirus, EBOV)和流感病毒(influenza virus)。研究结果发现敲低AP1复合体能够抑制EBOV和流感病毒包膜蛋白的表达量,可能与糖基化修饰程度相关。同时,我们根据AP1-core/E2相互作用,设计合成了一个融合短肽,将TAT穿透肽、core的YXXΦ模体序列和E2的[D/E]XXXL[L/I]模体序列串联起来。实验结果显示,其具有良好的抗病毒效果。综上所述,AP1复合体是HCV生命过程中必须的宿主因子,这些发现对阐明AP1复合体参与HCV感染过程的具体机制提供了有力的实验数据,为HCV抗病毒药物的研发提供了新的靶点。
[Abstract]:Hepatitis C virus (Hepatitis C, virus, HCV) has killed about 150 million people worldwide infected, and may develop into liver cirrhosis or liver cancer. The interaction of the viral life cycle depends on the viral and host factors. At present, the research on HCV and host interaction focused on virus invasion and replication stage. Mainly for replication and virus receptor must factor, and the study on the late stage of the virus life cycle is relatively small. With the function of human genome screening technology matures, more and more dependent on host virus related factor was found and confirmed by this technology, the host factors in order to further study the HCV life cycle must, this research using human 50K shRNA Library of host gene HCV infection were systematically screened and obtained 206 candidate genes. Based on the function of candidate gene The identification, we found that the interference of AP1 complex subunit 3 sigma (adaptor-related protein complex 1 sigma 3 subunit, AP1S3) the antiviral effect is obvious and stable.AP1 belongs to the AP family, is an ISO four dimeric protein complex (two subunit, a subunit and a small subunit), signal sorting and recognition in the cell, and then assembled into transport vesicles, plays an important role in material transport between organelles in cells. AP complex responsible for the different transport paths, AP1 mainly in the trans Golgi network (trans Golgi network, TGN) and transport within the body, and the AP2 is mainly in endocytosis when the transportation function play a role. Many viruses hijack AP1 complex in the cell, in order to facilitate their assembly and budding, including human immunodeficiency virus (human immunodeficiency, virus, HIV) and African swine plague virus (African swine FEVE R virus, ASFV). In order to verify the AP1S3 regulation of the HCV life cycle, we use Jcl-Luc HCVcc and JFH-1 HCVcc were used to evaluate the interference, found AP1S3 has good antiviral effect. Then we use the HCV pseudovirion system (HCVpp), HCV replicon system (2-3+) and HCV specific link assembly the release system of AP1S3 involved in the life cycle of the virus were identified. The results showed that AP1S3 mainly play a role in the late stage of the life cycle of HCV, knockdown of AP1S3 could significantly reduce HCV progeny virus production, and found that knockdown of AP1 complex of other subunits may have a similar effect. In order to reveal the mechanism involved in the regulation of AP1 virus in this study, the co immunoprecipitation experiments demonstrated that AP1 can through the die body two classic, with YXX (D/E) and XXXL (L/I), each with two structural proteins core and E2 HCV. By the two groups The interaction motif mediated degradation, AP1 can protect core and E2 from the proteasome. AP1 interference could lead to the increase of core and E2 protein ubiquitination level. In vitro and in vivo ubiquitination experiments further show that, as an E3 ubiquitin ligase, E6 related protein (E6 associated protein, E6AP) can mediate core and the ubiquitination of E2. In order to identify whether AP1 interference can cause the decrease of expression of other viral proteins. We selected 4 kinds of virus envelope protein of AP1 envelope protein degradation experiment, including Japanese encephalitis virus (Japanese encephalitis, virus, JEV), Siniro (West Nile virus, WNV virus, Ebola virus (Ebolavirus) EBOV, influenza virus (influenza) and virus). Results showed that knockdown of AP1 expression and EBOV complex can inhibit the influenza virus envelope protein, may related with carbohydrate modification. At the same time, I According to the interaction of AP1-core/E2, a fusion peptide was synthesized, TAT penetrating peptides, [D/E]XXXL[L/I] sequence core sequence and YXX phi E2 series. Experimental results show that it has good antiviral effect. In summary, the AP1 complex is a host factor HCV must be in the process of life, these findings provide strong experimental data to explain the mechanism of AP1 specific complex involved in the course of HCV infection, and provides a new target for the development of HCV antiviral drugs.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R373.21

【共引文献】