【作者】 于士颜；

【导师】 袁正宏；

【作者基本信息】 复旦大学 ， 病原生物学， 2011， 博士

【摘要】 乙型肝炎病毒目前依然是影响广大发展中国家人民健康的重要病原体。据统计,全球仍然有3亿5千万人感染乙型肝炎病毒,其中我国携带乙型肝炎病毒人数约占23%。尽管大规模的计划内疫苗接种已经大大降低了新生儿感染乙型肝炎病毒的风险,但是对已经感染乙型肝炎病毒的广大人群目前依然缺乏十分有效的药物以清除病毒。目前临床上使用干扰素a可以有效抑制乙型肝炎病毒复制,但是仅对不到30%的患者有效。遗憾的是,造成大部分乙型肝炎患者干扰素抵抗的分子机制至今仍不是十分清楚。I型干扰素(包括干扰素α[与干扰素β)是天然免疫的重要有机组成部分,在对抗并清除病毒性感染中发挥不可替代的作用。研究表明I型干扰素的诱生依赖于宿主细胞模式识别受体识别相关病毒成分并启动相关信号通路。然而,长期的生存压力促使以寄生为唯一生存方式的病毒进化出相应的对抗机制以克服I型干扰素的诱生从而建立并维持长期感染状态。研究发现慢性乙型肝炎患者体内pDC细胞合成干扰素α能力明显减弱。已有研究报道肝细胞在病毒或有关分子刺激下可产生I型干扰素,但乙肝病毒感染肝细胞后可在细胞内建立持续复制,提示病毒也能拮抗肝细胞干扰素的诱生或作用。而且有报道的确证实HBsAg、HBeAg以及病毒颗粒可以抑制Toll样受体介导的I型干扰素以及炎性因子的诱生。但是对于乙型肝炎病毒对肝细胞内RIG-I介导I型干扰素诱生途径是否存在抑制作用及机制目前了解不多。为此,本研究通过双荧光报告体系筛查定位于胞浆的病毒蛋白成分(Core蛋白,X蛋白以及Pol蛋白)对干扰素β启动子活性的影响,发现Pol蛋白对干扰素p启动子存在抑制现象。在此基础上,通过实时定量PCR技术以及ELISA技术检测了Pol蛋白对内源性干扰素p诱生的影响。结果发现Pol可以剂量依赖形式抑制内源性干扰素p的诱生。同时,病毒攻击保护实验证实Pol蛋白可以通过抑制PH5CH8诱生干扰素p从而消弱其保护Vero细胞抵抗NDV-GFP的感染能力。上述研究结果确认了乙型肝炎病毒Pol蛋白对原代肝细胞系PH5CH8干扰素β诱生的抑制效应。为了揭示Pol蛋白抑制干扰素β诱生的分子机制,将Pol与I型干扰素诱生通路中一系列关键分子共转染293T细胞,通过监测干扰素β的启动子活性变化来锁定Pol蛋白抑制I型干扰素诱生的环节。结果显示Pol可以抑制蛋白激酶TBK1/IKKε及其上游所有关键分子对干扰素β启动子的活化能力,但对干扰素调节因子3(IRF3)永久活化突变体没有抑制效应。为此,将Pol抑制I型干扰素诱生的环节锁定在TBK1/IKKε水平。进一步检测发现Pol蛋白可以抑制内源性IRF3活化,但是并未检测到Pol与IRF3存在直接相互作用,由此推测Pol可能通过某种间接途径抑制内源性IRF3的活化。由于有文献报道DDX3与TBK1/IKKε存在相互作用并参与TBK1/IKKε诱生I型干扰素,而且DDX3亦可以结合Pol抑制乙型肝炎病毒逆转录,故通过免疫共沉淀技术比较Pol表达对DDX3与TBK1/IKKε相互作用的影响。结果发现Pol表达可以抑制DDX3与TBK1/IKKε相互作用,同时还可以降低TBK1/IKKε对DDX3磷酸化效应。而过表达DDX3可以恢复Pol对I型干扰素的抑制能力,这提示Pol可能通过竞争性结合DDX3、降低TBK1/IKKε与DDX3相互作用机会从而发挥抑制I型干扰素诱生能力。本研究首次揭示了乙型肝炎病毒多聚酶Pol参与病毒对I型干扰素诱生的抑制作用,锁定其抑制环节发生在TBK1/IKKε水平,并且证实DDX3可能是Pol抑制I型干扰素诱生的靶点。上述结果有助于推动乙型肝炎病毒感染慢性化机制的研究以及新的靶点药物开发。更多还原

【Abstract】 Molecular Mechanism of TypeⅠInterferon Induction Inhibited by Hepatitis B Virus PolymeraseHepatitis B virus (HBV) is still one of the most dangerous pathogens jeopardizing public health globally. True, the routine vaccination has significantly decreased the risk of HBV infection in the population of newborn. But few and rare proper approaches can be clinically used to control HBV proliferation in those who have been chronically infected. More sadly,70% of patients with chronic HBV infection have no response to interferon a (IFNa) which is proved to effectively inhibit viral genes expression and proliferation. However, the molecular mechanism remains hazy. Thus, more investigation is needed to undertake to uncover the interplay between HBV and typeⅠIFN.TypeⅠIFN (including IFNa and IFNβ), playing a critical role in control and clearance of viral infection, can be induced by cellular pattern recognition receptors, such as Toll-like receptors (TLRs), RIG-I like helicases. Once engagement with viral ligands, TLR and RIG-I will recruit specific adaptors, for instance, TRIF, IPS1 and further motivate kinases TBK1/IKKεwhich can phosphorylate and activate interferon regulatory factor 3(IRF3), a key transcriptional factor required for typeⅠIFN production. In addition, DEAD-box helicase DDX3 is also another crucial component in the program of typeⅠIFN induction by TBK1/IKKε.In the long trajectory of evolution, viruses have developed numerous strategies to avert typeⅠIFN induction. For example, hepatitis C virus encodes NS3A to cleave key adaptors IPS1 and TRIF to sabotage typeⅠIFN synthesis. Similarly, a growing body of evidence also suggests that HBV can abrogate typeⅠIFN. HBV surface antigen (HBsAg), e antigen (HBeAg) and virions have been reported to almost completely suppress TLR-mediated antiviral activity and cytokine induction in murine liver parenchymal cells, indicating that HBV can counteract the TLR-mediated innate immune response. However, little is known about whether and how HBV disturbs cytoplasmic RIG-I-mediated IFNβinduction in human hepatocytes:In this study, we performed a functional screen assay to determine whether cytoplasmic viral proteins (including core, X and Pol) interfere with IFNP induction triggered by Sendai virus (SeV) and Newcastle Disease virus (NDV) challenge in primary human hepatocytes PH5CH8. Our results showed that only Pol inhibited the activation of IFNβpromoter in response to SeV and NDV infection though the expression of Core or X was higher than Pol. Interestingly, Pol also inhibited the activation of TLR3 signaling when poly(I:C) was directly added into cell culture, suggesting that the site of inhibition by Pol could be at the lower level in typeⅠIFN induction axis. Furthermore, we excluded the possibility of non-specific effects on IFNβpromoter using the unrelated p53 promoter reporter system. To further characterize the inhibitory effect of Pol, the induction of endogenous IFNβin PH5CH8 cells with or without Pol expression was examined using real-time quantitative PCR and ELISA. The data showed Pol could impede endogenous IFNβinduction in a dose-dependent manner. In addition, ectopic expression of Pol impaired the transferrable antiviral capacity of primary hepatocytes PH5CH8 in response to NDV infection which can protect Vero cells from NDV-GFP infection via secretion of IFNβ.To uncover the level at which Pol interferes with typeⅠIFN induction, 293T cells were co-tranfected with key molecules in typeⅠIFN induction axis and Pol. We found that Pol blocked the activation of IFNβpromoter triggered by all key molecules tested except IRF3-5D mutant. Moreover, we also determined the effect of Pol on the activation of endogenous IRF3 using native-PAGE, western blot and immunofluorescence. Our results showed that Pol could abrogate endogenous IRF3 activation by SeV. Unfortunately, we didn’t detect the direct interaction between Pol and IRF3, implying that the inhibitory effect of Pol on IRF3 activation is indirect. Thus, we defined the inhibition at the level of TBK1/IKKε.Given that DDX3 is involved in TBK1/IKKεmediated typeⅠIFN induction and also participates in inhibition of HBV reverse transcription via interaction with Pol, we speculated that DDX3 could be the target in the inhibition of typeⅠIFN by Pol. Immunoprecipitation assay revealed that less of TBK1/IKKεwas probed in the DDX3 immunoprecipitation complex in the cells with pol expression than in those without pol expression. However, over-expression of DDX3 restored the inhibition of SeV or TBK1 mediated induction of typeⅠIFN by Pol. It indicates that Pol can dampen the interaction between TBK1/IKKεand DDX3 through the competitive binding to DDX3.Taken together, these findings reveal a novel role of HBV polymerase in HBV counteraction of IFNβproduction in human hepatocytes更多还原