PCBP2增强IFN-α对HCV抗病毒活性分子机制的研究

发布时间：2018-01-01 09:15

本文关键词：PCBP2增强IFN-α对HCV抗病毒活性分子机制的研究　出处：《北京协和医学院》2011年博士论文　论文类型：学位论文

【摘要】：丙型肝炎病毒(Hepatitis C Virus, HCV)是一种正链小RNA病毒,由HCV感染引起的丙型肝炎是一种常见的慢性肝脏疾病,60-80%左右的持续感染者最终会发展成肝硬化或肝癌。干扰素α(IFN-α)可用于慢性丙型肝炎治疗,IFN-α的临床应用可使得病人血清和肝脏中的HCV RNA持续清除,达到对慢性感染的治愈效果。宿主细胞是HCV生命过程的载体,已经发现很多宿主细胞因子能够参与到HCV的生活周期中去。由于HCV是一种RNA病毒,这些细胞因子中很大一部分是RNA结合蛋白(RNA Binding Protein, RBP),这些RBP能与病毒基因组上的顺势元件结合而发挥对病毒的效应。Poly(C) binding protein2(PCBP2, hnRNPE2或α-CP2)是属于hnRNPE家族的一种RBP,可以特异性地与单链polyC富含区结合。在正常的细胞功能中,PCBP2可参与转录后调控、蛋白相互作用及miRNA的诱骗等机制中,发挥其细胞中对mRNA的稳定性影响和调控翻译的作用。PCBP2还可参与到许多RNA病毒的复制和翻译过程,包括脊髓灰质炎病毒(Poliovirus)、柯萨奇病毒(Coxsackievirus)、鼻病毒(Rhinovirus)等,这些RNA病毒都是与HCV结构相似的微小核糖核酸病毒。研究证实PCBP2能结合HCV基因组的5'-UTR和3’-UTR区域,提示PCBP2和HCV之间也有着某种联系,然而PCBP2对HCV生命过程所起的作用还并不确切。 HCV复制子(Replicon)细胞模型的建立对HCV的体内分子机制研究起到了重要的推动作用,该系统中遗传修饰后的HCV RNA (Replicon)可在人肝癌细胞系中有效地扩增,从而可反映病毒在细胞中的生命活动。在我们的初期研究中发现PCBP2不同于其它与HCV有相互作用的RBP,在HCV复制子细胞Rlb中表达下调而不是上调。通过对HCV NS蛋白在野生型Huh7.5.1细胞中的异位表达发现,HCV NS4B和NS5A蛋白的表达能显著地抑制PCBP2的表达水平,Realtime PCR结果表明NS4B对PCBP2的抑制可能发生在转录后水平,而NS5A则可能在转录水平抑制PCBP2的表达。R1b细胞中对PCBP2的抑制可以通过IFN-α的作用而回复。我们用IFN-α处理R1b细胞后发现HCV的RNA和蛋白表达都受到抑制,而PCBP2的表达则逐渐回复,具有剂量依赖性,并且在IFN-α治愈的R1b细胞中PCBP2的表达不再回调。这种回复作用在其它肝脏细胞系中没有发生,进一步说明PCBP2在R1b细胞中的抑制是由于HCV NS蛋白造成的。为了研究PCBP2对HCV的真正作用机制,我们在R1b细胞中分别过表达和敲低了PCBP2,结果表明过表达和敲低PCBP2对HCV的复制都没有显著影响。有意思的是,我们在过表达PCBP2的细胞中发现IFN-α对HCV的抑制活性显著增强,同时对IFN-a和利巴韦林联合作用也有相同的效应,而对利巴韦林单独作用没有影响。在PCBP2敲低的细胞中也有相应的效应。这些结果证实PCBP2能够增强IFN-a的作用而对利巴韦林无效。 PCBP2对IFN-a的作用机制研究中,我们利用RNA免疫沉淀(RNAImmunoprecipitation)技术来研究IFN-a信号通路分子中是否有PCBP2的作用底物。结果发现STAT1和STAT2的mRNA能富集在PCBP2的蛋白-RNA复合体中,与阳性底物a-globin相当,表明STAT1和STAT2的mRNA可能是PCBP2的靶mRNA,且PCBP2对STATI和STAT2mRNA的结合可导致其总蛋白及其磷酸化水平的上调。进一步通过RNA Pull Down技术,在STAT1和STAT2mRNA的3’-UTR中,分别找到了PCBP2的结合位点,其结合区域中都含有富含polyC的片段以及PCBP2识别的核心序列。通过Realtime PCR和RNA酶保护试验(RNase Protection Assay, RPA),我们进一步证实PCBP2能上调STAT1和STAT2的mRNA水平。其机制是结合mRNA后,可增强两者mRNA的稳定性,大大延长其半衰期。STAT1和STAT2mRNA的稳定性延长使得两者总蛋白的表达上调,从而增强IFN-α对HCV的抑制活性。本研究揭示了PCBP2对IFN-a的抗HCV活性中起着重要的作用,并有可能指导临床上使用IFN-α对HCV感染的个体治疗。同时我们揭示了RNA结合蛋白与HCV病毒之间一种相互作用的新机制,即通过增强IFN-α的效应而不是直接作用于病毒基因组,这些对于我们更好地理解HCV在体内的分子机制有着重要的启示。
[Abstract]:Hepatitis C virus (Hepatitis C, Virus, HCV) is a chain of small RNA virus, caused by HCV infection of hepatitis C is a common chronic liver disease, persistent infection about 60-80% people will eventually develop into cirrhosis or liver cancer. Interferon alpha (IFN- alpha) can be used for the treatment of chronic hepatitis, clinical application IFN- alpha can make HCV RNA in serum and liver in patients of chronic infection continue to clear, cure effect.
The host cell is the carrier of HCV life process, have found a lot of host cell factors can be involved in the life cycle of HCV to go. Because the HCV is a RNA virus, a large part of these cytokines is RNA binding protein (RNA Binding, Protein, RBP), the RBP and the virus genome homeopathic element binding play the effect of.Poly on virus (C) binding protein2 (PCBP2, hnRNPE2 or a -CP2) is a kind of RBP belongs to the hnRNPE family, can combine specifically with single stranded polyC rich region. In normal cell function, PCBP2 may be involved in transcription regulation, decoy mechanisms and miRNA protein interaction in the play the role of.PCBP2 mRNA influence on the stability and regulation of translation in the cells can also be involved in the replication and translation of many RNA viruses, including polio virus (Poliovirus), Coxsackie virus (Coxsackievirus), nose Virus (Rhinovirus), the RNA virus is a picornavirus with similar HCV structure. The study confirmed that PCBP2 can bind to HCV genome 5'-UTR and 3 '-UTR region, suggesting that between PCBP2 and HCV also have some connection, but PCBP2 plays the role of the HCV life is not exactly.
HCV replicon (Replicon) to establish cell model study on the molecular mechanism of HCV in vivo has played an important role in promoting HCV, RNA genetic modified in the system (Replicon) can be effectively amplified in human hepatocellular carcinoma cell line, which can reflect the virus in the cell life activities. PCBP2 is different from the other HCV interaction of RBP in the early stage of our study, the HCV replicon cell Rlb expression down than up. The expression of ectopic HCV NS protein in wild-type Huh7.5.1 cells found in the expression of HCV, NS4B and NS5A protein expression level of PCBP2 was significantly inhibited, Realtime PCR results showed that the inhibition of NS4B on PCBP2 may occur at the post transcriptional level, while NS5A may inhibit PCBP2 in.R1b cells can be restored by IFN- alpha effect inhibition of PCBP2 expression at the transcriptional level. We use IFN- treated R1b cells After the discovery of the RNA and protein expression of HCV were inhibited, while the expression of PCBP2 gradually reply, in a dose dependent manner. The expression of PCBP2 and IFN- alpha cured in R1b cells in no callback. This response does not occur in other liver cells, further inhibition of PCBP2 in R1b cells is due to HCV the NS protein caused.
In order to study the effects of PCBP2 on the mechanism of HCV in R1b cells, we were overexpression and knockdown of PCBP2, the results showed that over expression and replication of HCV PCBP2 at low do not affect. Interestingly, we in the over expression of PCBP2 cells was found to inhibit the activity of IFN- alpha HCV increased significantly at the same time, the combined effects of IFN-a and Leigh Bhave Lin also have the same effect, but have no influence on Leigh Bhave Lin alone. In PCBP2 knockdown cells also have the corresponding effect. These results demonstrate that PCBP2 can enhance the effect of IFN-a on Leigh Bhave Lin is invalid.
Study on the action mechanism of PCBP2 on IFN-a, we use RNA immunoprecipitation (RNAImmunoprecipitation) technique to study the substrate is the PCBP2 IFN-a signaling pathway molecules. The results showed that STAT1 and STAT2 mRNA can be enriched in protein -RNA complexes in PCBP2, similar with the positive substrate a-globin, indicating that STAT1 and STAT2 may be mRNA the target mRNA PCBP2, and PCBP2 of STATI and STAT2mRNA combination can lead to the total protein and phosphorylation levels of Pull up-regulated. Further RNA Down technology, STAT1 and STAT2mRNA in the 3 '-UTR, were found in the PCBP2 binding sites, the binding region contains rich fragments of polyC and PCBP2 recognition the core sequence.
By Realtime PCR and RNA (RNase Protection Assay enzyme protection test, RPA), we further confirmed that PCBP2 can increase STAT1 and STAT2 level of mRNA. The mechanism is combined with mRNA, can enhance the stability of both mRNA and stability greatly prolong the half-life of.STAT1 and STAT2mRNA makes up the total extension of protein expression, thereby enhancing the inhibition the activity of IFN- alpha on HCV.
This study reveals that plays an important role in anti HCV activity of IFN-a in PCBP2, and may guide the clinical use of IFN- alpha on the individual treatment of HCV infection. At the same time, we reveal the new RNA binding mechanism between HCV protein and a virus interaction, namely by enhancing the IFN- alpha effect but not directly these effects on viral genome, for us to better understand HCV has important implications in the molecular mechanism of the body.

【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】：2011
【分类号】：R373.21

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