【作者】 陈奇英；

【导师】 袁正宏；

【作者基本信息】 复旦大学 ， 病原生物学， 2009， 硕士

【摘要】 干扰素诱导蛋白DAI抑制乙型肝炎病毒复制的初步研究硕士学位论文中文摘要乙型肝炎病毒(HBV)是危害人类健康的主要病原体之一,在全球范围内,有近四亿感染者,由此引发的肝炎肝硬化以及肝癌每年导致近百万人的死亡。干扰素是一类具有广谱抗病毒作用的细胞因子,自其1957年被发现以来已经受到大量的关注并被广泛应用到病毒感染的临床治疗中,但是其抑制HBV复制的机理还不甚明了。先前的研究表明干扰素可以通过诱导抗病毒蛋白(MxA,APOBE3,MyD88)的表达来发挥其抑制HBV复制的作用,但是这些蛋白并不能揭示干扰素抑制HBV复制的全部机理,提示还有其他抗乙型肝炎病毒的干扰素诱生蛋白的存在,值得进一步寻找新的受干扰素诱导的抑制HBV复制的蛋白并揭示其抑制HBV复制的机理。基因微点阵芯片技术(Micro array)的发展和应用使得有可能从新的角度研究干扰素抗病毒的机制及寻找新的抗病毒蛋白,已经有不少利用cDNA基因芯片技术检测经干扰素处理后的人类肝细胞或者试验动物的肝细胞的基因表达谱的变化,Chisari,F.V等发现,对有HBV复制的小鼠肝细胞(HBVMet4)用干扰素处理六小时后,IFN-γ和IFN-β可以使DAI (DNA-dependent activator of IFN-regulatory factor,也叫Z-DNA binding protein (ZBP1)或者DLM-1)的表达量分别升高426和577倍。DAI最初作为一种RNA结合蛋白被发现,其可以与β-actin mRNA的zipcode结构高特异性结合来决定β-actin mRNA的细胞内定位从而来维持细胞的极性。而后Yineng Fu等发现在巨噬细胞DAI可以被IFN-γ或者LPS刺激后上调,被称为肿瘤基质和激活巨噬细胞蛋白(Tumor stroma and activated macrophage protein),提示在宿主天然防御中发挥重要的作用。2007年Tadatsugu Taniguchi等证明DAI作为DNA识别蛋白可以与IRF3和TBK1相互作用并能强烈地诱导Ⅰ干扰素的产生。由此可以推测DAI可能在干扰素介导的抗HBV复制中发挥着重要的作用。本研究首先证实了IFN-α可以在肝癌来源的Huh7细胞内诱导DAI的mRNA水平的上调。随即将DAI蛋白的表达质粒与HBV的复制型质粒HBV1.3共转Huh7细胞后,发现DAI可以明显地下调乙肝表面抗原(HBsAg)以及e抗原(HBeAg),细胞内HBV RNA及HBV核心颗粒DNA的水平。鉴于poly(dA-dT).poly(dT-dA) (B-DNA)是一种可以强烈诱导DAI表达的人工合成DNA,在HBV的复制的Huh7细胞中加入B-DNA可以诱导DAI的表达并抑制HBV的复制。由此提示外源性及内源性的DAI都可以抑制HBV的复制。DAI作为一种诱导干扰素产生的重要分子,其对HBV的抑制作用是否与诱导干扰素的产生相关?研究表明DAI可以与TBK1相互作用后通过招募干扰素调节因子3(IRF-3)进而诱导干扰素的产生,其抑制HBV的作用是否与此相关,为此我们首先在Huh7细胞内过表达TBK1,结果发现其并不能增强DAI对HBV的抑制作用。进而我们将DAI与IRF-3共转细胞,检测IRF-3的活化程度,结果显示IRF-3的入核和磷酸化都无明显变化,我们将DAI与IRF-3的永久失活体质粒(失去N端的DNA结合区域的截短体(IRF-3△N))共转细胞,发现IRF-3△N并不影响DAI对HBV的抑制作用,由此提示Huh7细胞内DAI不能激活IRF-3,DAI抑制HBV复制不依赖于TBK1/IRF-3介导的干扰素产生。NF-κB是固有抗病毒免疫中的一个重要的转录因子,研究发现B-DNA诱导的NF-κB的DNA结合活性中DAI有重要的作用,本研究的实验也证明DAI能以剂量依赖的方式激活NF-kB转录启动活性。我们进一步应用NF-κB的超抑制剂I kBα-SR(superrepressor)发现可以回复DAI对HBV的抑制作用,这提示DAI抑制HBV的活性可能与其激活NF-KB的相关。NF-κB活化后可以启动包括干扰素在内的抗病毒细胞因子的转录,为探索DAI抑制HBV是否依赖于其对干扰素的诱生,我们首先在RNA和蛋白水平检测DAI在Huh7细胞内能否诱导干扰素产生,结果发现DAI不能诱导干扰素的产生。为了进一步排除干扰素或者其他分泌到细胞外的细胞因子是DAI发挥抑制HBV的效应因子,我们做了上清置换试验,将转染了DAI的细胞上清加入到HBV复制的细胞中,结果并未发现DAI表达的细胞上清具有抑制HBV的复制的效应。综上所述,本文的结果证明DAI (ZBP1/DLM-1)是一种具有抗乙型肝炎病毒活性的干扰素诱生蛋白,过表达DAI可以下调HBV RNA,复制中间体DNA以及e、s抗原水平,这种下调可能与激活NF-κB的活性相关但不依赖于它对干扰素产生的诱导。以上发现丰富了对干扰素抗病毒作用机制的认识,为HBV感染的治疗提供了新的思路和实验基础。更多还原

【Abstract】 Interferon-inducible DAI protein inhibits Hepatitis B virus replicationHepatitis B virus infection is a global public health problem, with over 300 million people worldwide being chronic HBV carriers and millions of deaths annually attributable to HBV related liver failure (cirrhosis or hepatocellular carcinoma). As a secreted cytokine that elicits distinct antiviral effect, interferon (IFN) has been used for treatment of chronic HBV infection for more than two decades, yet the mechanism of action of this antiviral remains poorly understood. Previous studies have proved that anti-virus action of interferon is mediated by the induction of anti-virus protein, plenty of genes are regulated by IFN, for example,2’,5’-oligoadenylate synthetase, protein kinase R, and MxA and these products are the key mediators of IFN’s antiviral response. Although previous studies have been demonstrated that a few of IFN-inducible proteins could exert anti-HBV effect in vitro or in vivo, such as APOBEC3, MxA and MyD88, they still could not fully account for IFN’s effect on HBV replication. Hence, other IFN induced protein(s) might also be involved in the inhibition of HBV replication by IFN.DAI (DLM-1/ZBP1) was initially identified as a highly conserved RNA-binding protein, which could bind to the zipcode motif ofβ-actin mRNA and therefore be responsible for its intracellular localization and cell polarity. Francis V. Chisari et al have reported that the expression level of DAI’s mRNA could be up-regulated after IFN-α/βand IFN-γstimulation in the highly differentiated hepatocyte HBVMet-4 harboring HBV replication. In 2007, Tadadsugu Taniguchi et al have demonstrated that DAI could significantly induce the expression of typeⅠIFN through binding to IRF3 and TBK1 as a DNA sensor. Therefore, it is quite possible that DAI might role in the inhibition of HBV replication exerted by typeⅠandⅡIFN.To verify DAI can induced by interferon in Human Hepatoma Cell Line(Huh7), we treat Huh7 cells with different dose of interferon-α. We found that IFN-a treatment can greatly activate DAI transcription.To determine the anti-HBV effect of DAI protein, we co-transfected Huh7 human hepatoma cells with an HBV-producing plasmid HBV 1.3 and either a control vector or vectors encoding HA tagged forms of DAI protein. Transfect ion rates were controlled by the inclusion of a GFP-expressing plasmid. To examine the effect of DAI on the synthesis of HBV proteins,48h after transfection, supernatants were collected and assayed for HBV surface antigen (HBsAg) and e antigen (HBeAg) by ELASA (Enzyme-Linked ImmunoSorbent Assay). Compare with the vector, the secretion of both HBsAg and HBeAg was significantly reduced in DAI transfect cell. To investigate whether the reduction of HBV protein synthesis was associated with the reduction of viral RNA transcripts, Northern blot analysis and real-time PCR was used to examine the cytoplasmic HBV RNAs in Huh7 cell after transfection with pHBV1.3 for 48h. The results show that DAI can reduce the HBV RNA in a dose-dependent manner. To investigate whether the HBV DNA replication was inhibited by DAI protein, viral replicative intermediates DNA extracted from core particles 48h after DAI transfection and then quantified by real-time PCR and Southern blot. We found that DAI can significantly reduce the HBV DNA replication.Poly(dA-dT).poly(dT-dA) (B-DNA) was identified as a DAI stimulator, I want to known is if B-DNA can induce endogenous DAI transcription and inhibit HBV replication? We add B-DNA to cell culture supernatant after HBV plasmid was transfect to Huh7 cell. Our result shows that B-DNA can induce DAI transcription and inhibit HBV replication in Huh7, although not as significantly as DAI over-expression.We future tudy the mechanism of DAI inhibit HBV repliacation.DAI have been implicated as a DNA sensor and interferon stimulator. So we determine whether the anti-HBV effect of DAI protein is interferon dependent. Taniguchi et al have shown that DAI can interact with TBK1 then recruit and activate IRF-3 result in producing interferon. So we want to know whether over-expression TBK1 can improve the anti-HBV effect of DAI. The results show that TBK1 can not affect the anti-HBV effect of DAI. IRF-3 was downstream of TBK1 and Taniguchi et al have shown that DAI (DLM-1/ZBP1) is critical for B-DNA-mediated IRF3 activation.So we speculate that IRF3 maybe play a role in DAI mediated anti-HBV effect. To test this idea, we block the IRF-3 with an IRF-3 domain negative plasmid. The result shows that the anti-HBV effect of DAI was not affected by this IRF-3 inhibitor.NF-κB is the key transcription factor in innate inmmune system. It is report that that DAI is necessary in B-DNA-mediated NF-κB binding activity.We first detects if NF-κB activity can be activated by DAI. As expected, DAI showed increased activation of NF-κB in dose dependent manner. Then we block the NF-κB activation with a NF-κB inhibitory plasmid IκBα-SR.When the activity of NF-κB was block, the anti-HBV effect of DAI is completely disappearing. So we speculate that the anti-HBV effect of DAI is related to activation of NF-κB.NF-κB activation can produce many kinds of cytokines inclucing interferon. To test whether anti-viral effect of DAI is associated with producing interferon or other cytokines, we first detect the interferon secret in supernatants by ELISA. The result shows that DAI can not triggers the secretion of interferon. To confirm that DAI inhibit HBV replication is not dependent on producing interferon or other cytokines, supernatant transfer experiment was performed in which cultured media were harvested from Huh7 cells transfected with plasmids expressing DAI and applied onto pHBV1.3 transfected Huh7 cells. It is show that the viral RNA levels were not affected in Huh7 cells treated with this conditioned medium. This result suggesting that inhibition of HBV replication by DAI is an intracellular event and independent of secreted cytokines.In summary, our studies demonstrated that the interferon inducible protein DAI can inhibit HBV replication. We also document that the DAI inhibit HBV replication is associated with the activation of NF-κB signaling pathway which may contributes to a better understanding of the mechanisms of interferon-induced inhibition of HBV replication and provide a new approach in the control of HBV infection.更多还原