發(fā)布時間：2018-04-03 08:39

  本文選題：適配蛋白復(fù)合體1　切入點(diǎn)：丙型肝炎病毒　出處：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：丙型肝炎病毒(Hepatitis C virus, HCV)目前已導(dǎo)致全球約1億5千萬人感染,并可能進(jìn)一步發(fā)展為肝硬化或肝癌等疾病。病毒的生命周期依賴于病毒與宿主因子的相互作用。目前,關(guān)于HCV與宿主相互作用的研究主要集中于病毒入侵和復(fù)制階段,主要針對病毒受體和復(fù)制必須因子等,而對病毒生命周期晚期階段的研究則相對較少。隨著人類功能基因組篩選技術(shù)的成熟,越來越多的病毒相關(guān)宿主依賴因子通過此技術(shù)被發(fā)現(xiàn)并確認(rèn),為了進(jìn)一步研究HCV生命周期所必須的宿主因子,本研究利用人類50k shRNA庫,對HCV感染相關(guān)宿主基因進(jìn)行了系統(tǒng)化篩選,并獲得了206個候選基因。通過對候選基因的功能學(xué)鑒定,我們發(fā)現(xiàn)干擾AP1復(fù)合體西格瑪3亞基(adaptor-related protein complex 1 sigma 3 subunit, AP1S3)的抗病毒效果明顯并且穩(wěn)定。AP1屬于AP家族,是一個異四聚體蛋白復(fù)合體(兩個大亞基、一個中亞基和一個小亞基),在細(xì)胞中識別分選信號,并組裝成轉(zhuǎn)運(yùn)小泡,在細(xì)胞內(nèi)各細(xì)胞器之間扮演著重要的物質(zhì)運(yùn)輸作用。不同的AP復(fù)合體負(fù)責(zé)不同的轉(zhuǎn)運(yùn)路徑,AP1主要在反式高爾基體(trans Golgi network, TGN)和內(nèi)體之間運(yùn)輸,而AP2則主要在細(xì)胞內(nèi)吞作用時發(fā)揮作用。許多病毒會劫持AP1復(fù)合體的胞內(nèi)運(yùn)輸功能,以利于自身的組裝和出芽,包括人免疫缺陷病毒(human immunodeficiency virus, HIV)和非洲豬瘟病毒(african swine fever virus, ASFV)。為了進(jìn)一步驗(yàn)證AP1S3調(diào)節(jié)HCV生命周期的作用,我們使用了Jcl-Luc HCVcc和JFH-1 HCVcc對其進(jìn)行了評價,發(fā)現(xiàn)干擾AP1S3具有良好的抗病毒效果。進(jìn)而我們使用了HCV假病毒系統(tǒng)(HCVpp)、HCV復(fù)制子系統(tǒng)(2-3+)和HCV組裝釋放系統(tǒng)對AP1S3參與病毒生命周期的具體環(huán)節(jié)進(jìn)行了鑒定。研究結(jié)果顯示,AP1S3主要在HCV生命周期晚期階段發(fā)揮作用,敲低AP1S3能夠顯著降低HCV子代病毒的產(chǎn)生,并且發(fā)現(xiàn)敲低AP1復(fù)合體的其他亞基也具有類似的作用。為了揭示AP1參與病毒調(diào)控的機(jī)制,本研究通過免疫共沉淀實(shí)驗(yàn)發(fā)現(xiàn)AP1能夠通過兩個經(jīng)典的模體,YXXΦ和(D/E)XXXL(L/I),與HCV的兩個結(jié)構(gòu)蛋白core和E2相互作用。通過這兩組模體所介導(dǎo)的相互作用,AP1能夠保護(hù)core和E2免于蛋白酶體的降解。干擾AP1會導(dǎo)致core和E2蛋白泛素化程度的增加。進(jìn)一步的體內(nèi)外泛素化實(shí)驗(yàn)顯示,作為E3泛素連接酶,E6相關(guān)蛋白(E6 associated protein, E6AP)能夠介導(dǎo)core和E2的泛素化過程。為了鑒定干擾AP1是否能夠引起其他病毒蛋白表達(dá)量的下降。我們選取了4種病毒包膜蛋白進(jìn)行AP1相關(guān)的包膜蛋白降解實(shí)驗(yàn),包括日本腦炎病毒(Japanese encephalitis virus, JEV)、西尼羅病毒(West Nile virus, WNV)、埃博拉病毒(Ebolavirus, EBOV)和流感病毒(influenza virus)。研究結(jié)果發(fā)現(xiàn)敲低AP1復(fù)合體能夠抑制EBOV和流感病毒包膜蛋白的表達(dá)量,可能與糖基化修飾程度相關(guān)。同時,我們根據(jù)AP1-core/E2相互作用,設(shè)計合成了一個融合短肽,將TAT穿透肽、core的YXXΦ模體序列和E2的[D/E]XXXL[L/I]模體序列串聯(lián)起來。實(shí)驗(yàn)結(jié)果顯示,其具有良好的抗病毒效果。綜上所述,AP1復(fù)合體是HCV生命過程中必須的宿主因子,這些發(fā)現(xiàn)對闡明AP1復(fù)合體參與HCV感染過程的具體機(jī)制提供了有力的實(shí)驗(yàn)數(shù)據(jù),為HCV抗病毒藥物的研發(fā)提供了新的靶點(diǎn)。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R373.21

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張久聰;孫利;聶青和;;HCV受體及入胞相關(guān)分子的研究進(jìn)展[J];傳染病信息;2009年04期

2 李堯;代解杰;孫曉梅;夏雪山;;樹，

本文編號：1704445