本文關(guān)鍵詞：PCBP2增強IFN-α對HCV抗病毒活性分子機制的研究　出處：《北京協(xié)和醫(yī)學(xué)院》2011年博士論文　論文類型：學(xué)位論文

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【摘要】：丙型肝炎病毒(Hepatitis C Virus, HCV)是一種正鏈小RNA病毒,由HCV感染引起的丙型肝炎是一種常見的慢性肝臟疾病,60-80%左右的持續(xù)感染者最終會發(fā)展成肝硬化或肝癌。干擾素α(IFN-α)可用于慢性丙型肝炎治療,IFN-α的臨床應(yīng)用可使得病人血清和肝臟中的HCV RNA持續(xù)清除,達到對慢性感染的治愈效果。 宿主細胞是HCV生命過程的載體,已經(jīng)發(fā)現(xiàn)很多宿主細胞因子能夠參與到HCV的生活周期中去。由于HCV是一種RNA病毒,這些細胞因子中很大一部分是RNA結(jié)合蛋白(RNA Binding Protein, RBP),這些RBP能與病毒基因組上的順勢元件結(jié)合而發(fā)揮對病毒的效應(yīng)。Poly(C) binding protein2(PCBP2, hnRNPE2或α-CP2)是屬于hnRNPE家族的一種RBP,可以特異性地與單鏈polyC富含區(qū)結(jié)合。在正常的細胞功能中,PCBP2可參與轉(zhuǎn)錄后調(diào)控、蛋白相互作用及miRNA的誘騙等機制中,發(fā)揮其細胞中對mRNA的穩(wěn)定性影響和調(diào)控翻譯的作用。PCBP2還可參與到許多RNA病毒的復(fù)制和翻譯過程,包括脊髓灰質(zhì)炎病毒(Poliovirus)、柯薩奇病毒(Coxsackievirus)、鼻病毒(Rhinovirus)等,這些RNA病毒都是與HCV結(jié)構(gòu)相似的微小核糖核酸病毒。研究證實PCBP2能結(jié)合HCV基因組的5'-UTR和3’-UTR區(qū)域,提示PCBP2和HCV之間也有著某種聯(lián)系,然而PCBP2對HCV生命過程所起的作用還并不確切。 HCV復(fù)制子(Replicon)細胞模型的建立對HCV的體內(nèi)分子機制研究起到了重要的推動作用,該系統(tǒng)中遺傳修飾后的HCV RNA (Replicon)可在人肝癌細胞系中有效地擴增,從而可反映病毒在細胞中的生命活動。在我們的初期研究中發(fā)現(xiàn)PCBP2不同于其它與HCV有相互作用的RBP,在HCV復(fù)制子細胞Rlb中表達下調(diào)而不是上調(diào)。通過對HCV NS蛋白在野生型Huh7.5.1細胞中的異位表達發(fā)現(xiàn),HCV NS4B和NS5A蛋白的表達能顯著地抑制PCBP2的表達水平,Realtime PCR結(jié)果表明NS4B對PCBP2的抑制可能發(fā)生在轉(zhuǎn)錄后水平,而NS5A則可能在轉(zhuǎn)錄水平抑制PCBP2的表達。R1b細胞中對PCBP2的抑制可以通過IFN-α的作用而回復(fù)。我們用IFN-α處理R1b細胞后發(fā)現(xiàn)HCV的RNA和蛋白表達都受到抑制,而PCBP2的表達則逐漸回復(fù),具有劑量依賴性,并且在IFN-α治愈的R1b細胞中PCBP2的表達不再回調(diào)。這種回復(fù)作用在其它肝臟細胞系中沒有發(fā)生,進一步說明PCBP2在R1b細胞中的抑制是由于HCV NS蛋白造成的。 為了研究PCBP2對HCV的真正作用機制,我們在R1b細胞中分別過表達和敲低了PCBP2,結(jié)果表明過表達和敲低PCBP2對HCV的復(fù)制都沒有顯著影響。有意思的是,我們在過表達PCBP2的細胞中發(fā)現(xiàn)IFN-α對HCV的抑制活性顯著增強,同時對IFN-a和利巴韋林聯(lián)合作用也有相同的效應(yīng),而對利巴韋林單獨作用沒有影響。在PCBP2敲低的細胞中也有相應(yīng)的效應(yīng)。這些結(jié)果證實PCBP2能夠增強IFN-a的作用而對利巴韋林無效。 PCBP2對IFN-a的作用機制研究中,我們利用RNA免疫沉淀(RNAImmunoprecipitation)技術(shù)來研究IFN-a信號通路分子中是否有PCBP2的作用底物。結(jié)果發(fā)現(xiàn)STAT1和STAT2的mRNA能富集在PCBP2的蛋白-RNA復(fù)合體中,與陽性底物a-globin相當(dāng),表明STAT1和STAT2的mRNA可能是PCBP2的靶mRNA,且PCBP2對STATI和STAT2mRNA的結(jié)合可導(dǎo)致其總蛋白及其磷酸化水平的上調(diào)。進一步通過RNA Pull Down技術(shù),在STAT1和STAT2mRNA的3’-UTR中,分別找到了PCBP2的結(jié)合位點,其結(jié)合區(qū)域中都含有富含polyC的片段以及PCBP2識別的核心序列。 通過Realtime PCR和RNA酶保護試驗(RNase Protection Assay, RPA),我們進一步證實PCBP2能上調(diào)STAT1和STAT2的mRNA水平。其機制是結(jié)合mRNA后,可增強兩者mRNA的穩(wěn)定性,大大延長其半衰期。STAT1和STAT2mRNA的穩(wěn)定性延長使得兩者總蛋白的表達上調(diào),從而增強IFN-α對HCV的抑制活性。 本研究揭示了PCBP2對IFN-a的抗HCV活性中起著重要的作用,并有可能指導(dǎo)臨床上使用IFN-α對HCV感染的個體治療。同時我們揭示了RNA結(jié)合蛋白與HCV病毒之間一種相互作用的新機制,即通過增強IFN-α的效應(yīng)而不是直接作用于病毒基因組,這些對于我們更好地理解HCV在體內(nèi)的分子機制有著重要的啟示。
[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.

【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】：2011
【分類號】：R373.21

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本文編號：1363872