表达外源基因的HCV微型基因组的构建及初步应用研究
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摘要
病毒基因组复制是其生活周期中的重要环节。已知正链RNA病毒的复制分为两个步骤,一是正链RNA作为模板生成复制中间体负链RNA;二是负链RNA又作为模板去合成子代正链RNA。复制酶是依赖RNA的RNA聚合酶(RdRP),非编码区(UTR)是病毒基因组复制的主要调控区域,其中3′UTR是复制酶结合的主要区域,介导基因组的从头合成。在深入理解病毒复制机制及复制所需作用元件的基础上,构建RNA病毒微型基因组是近年来分子病毒学研究的一个热点方向。它有利于阐明病毒复制的分子机制、成熟与包装中所需的蛋白因子,并为抗病毒药物的设计提供新策略。
丙型肝炎病毒(HCV)属黄病毒科,为单股正链RNA病毒。HCV基因组全长约9.6Kb,含有单一开放读框(ORF),两侧为非编码区(5′UTR和3′UTR)。HCV基因组具有高度变异性,存在“准株”现象,主要有六个基因型和多个亚型。而HCV基因组序列分析表明,与复制密切相关的顺式作用元件高度保守,它们主要存在于5′UTR和3′UTR,以及core和ns5b基因的部分序列。NS5B蛋白具有RdRP活性,是HCV复制的关键酶。它与其它非结构区蛋白(如NS3、NS4A、NS4B等)形成复制酶复合物,锚定于细胞中内质网膜上发挥其生物功能。这一系列研究为构建HCV微型基因组提供了条件。
新型HCV治疗药物是各国学者研究的热点。目前关于HCV的治疗是主要以干扰素为基础的治疗方案,但效果有限而且副作用大,患者依从性差。具有很大潜力的小分子药物,如针对NS3/4A蛋白酶、解旋酶及RNA聚合酶NS5B的抑制剂,临床试验显示常常引起耐药突变体。因此,迫切需要寻找新的抗HCV策略,其中靶向治疗HCV感染细胞的方法是有潜力的方向之一。
本研究首先通过基因重组技术用顺式作用元件构建了HCV微型基因组,中间为反向插入的萤火虫荧光素酶报告基因(firefly luciferase,flu)与EMCV的IRES基因。该微型基因组RNA在病毒复制酶的作用下,从3′末端起始合成负链RNA,反向插入的flu及IRES基因即转换为正向,flu得以表达。然后,设计了几种不同截短的微型基因组,由它们生成的负链RNA的3′末端具有不同茎环截短的突变形式,以报告基因flu的表达水平来比较这一系列微型基因组。在此基础上,利用该微型基因组表达IFN-β,靶向抑制HCV感染细胞。本研究分以下三个部分:
1. HCV微型基因组的构建与功能鉴定
根据Genbank登录的HCV 1b型基因序列,设计引物分别扩增HCV 5UTR1-377、3UTR,其中扩增5UTR1-377的引物上游含有T7启动子核心序列;分别设计并扩增IRES(来自EMCV)、flu基因;合成HDV核酶Rz(ribozyme)的正义与反义序列,两者退火;将PCR产物各片段克隆至pMD18T载体,交由公司测序。序列鉴定正确后依次将5UTR、IRES、3UTR、HDV Rz片段克隆入pUC18载体,形成质粒pUC18-T7/5UTR-rIRES-3UTR-HDV Rz,缩写为pT7/5UrI3URz。然后通过Xba I单酶切位点将flu基因插入上述载体,PCR鉴定正反向后,命名反向插入flu的载体为pT7/5UrFI3URz,即为HCV微型基因组。HCV复制子质粒BB7经Sca I酶切线性化,在T7 RNA聚合酶作用下体外转录制备RNA,通过电穿法转染Huh7.5细胞,经G418压力筛选获得稳定细胞克隆,RT-PCR和Western blot证实成功构建了该复制子细胞系,命名为Huh7.5BB7。将pT7/5UrFI3URz质粒经EcoR ?酶切线性化,体外转录制备微型基因组RNA,通过电穿转染方法将其导入Huh7.5BB7细胞和Huh7.5细胞,48hr后处理细胞进行荧光素酶活性测定。实验结果表明,荧光素酶选择性表达于Huh7.5BB7细胞,提示已成功构建HCV微型基因组。
2.不同HCV微型基因组截短体的比较
从3′末端起始的从头合成是HCV复制的形式。负链RNA 3′UTR的二级结构已经解析,除了3′末端的最短的SL-A1茎环外,其它茎环与正链RNA 5′UTR均不同。目前,对于负链RNA 3′UTR的各个茎环在正链RNA的从头合成中作用尚不清楚。本研究依次构建了几种截短的微型基因组(?45、?124、?227、?131-315),由它们生成的负链RNA的3′末端具有不同茎环截短的突变形式。将这几种微型基因组体外转录制备微型基因组RNA,转染Huh7.5BB7细胞。48 hr后测定荧光素酶活性,结果显示:与全长微型基因组相比,?131-315微型基因组表达荧光素酶活性增强(p<0.01),而?45微型基因组表达荧光素酶活性降低(p<0.01),?124、?227微型基因组基本不表达荧光素酶。
3.利用微型基因组特异表达IFN-β靶向抑制HCV感染细胞
长效干扰素(PEG-IFN)联合利巴韦林是目前HCV最为标准的治疗方案。但各个基因型的持续病毒学应答率(SVR)不同,1型和4型仅为38~52%。其中一个重要原因是HCV编码蛋白通过多种途径抵抗了IFN的抗病毒反应。本研究将IFN-β基因反向插入微型基因组,当其进入细胞内,在病毒复制酶作用下合成负链微型基因组RNA后启动IFN-β表达,从而发挥抗病毒作用。首先从外周血中分离淋巴细胞,用poly I:C刺激其分泌IFN。用Trizol提取细胞总RNA,逆转录合成cDNA,PCR扩增IFN-β基因。序列鉴定正确后,将其克隆入pT7/5U?131-315rI3URz,PCR鉴定插入的正反向,将反向插入的质粒命名为pT7/5U?131-315rIFNI3URz。该重组质粒用EcoR I酶切后纯化,体外转录制备RNA,转染Huh7.5BB7细胞,48hr后收集细胞并提取蛋白。抗IFN-β抗体作为一抗,Western-blot分析显示:IFN-β特异性表达于复制子细胞中。用Xba I线性化HCV全长基因组质粒JFH-1后,体外转录制备RNA并转染Huh7.5细胞,In cell Western-blot和荧光实时定量PCR证实建立了Huh7.5JFH-1细胞系。为了利用HCV微型基因组进行靶向基因治疗,设计引物扩增微型基因组5U?131-315rIFNI3URz后克隆入pcDNA3.1载体,获得pCMV-5U?131-315rIFNI3URz。将其转染复制子细胞系,Western-blot显示IFN-β能够特异的表达于复制子细胞系中。pCMV-5U?131-315rIFNI3URz转染huh7.5JFH-1细胞系后,荧光实时定量PCR显示,它可以降低HCV RNA的拷贝水平,具有一定的剂量依赖效应。
综上所述,本研究成功构建了HCV微型基因组,对其表达外源基因的构建方式进行了优化;携带IFN-β的微型基因组能够靶向表达于HCV感染细胞,并可降低病毒RNA复制水平。
Virus genome replication plays an important role in its life cycle. It is known that the replication of plus-stranded RNA virus divides into two steps: genomic RNA is firstly transcribed into a minus-strand intermediate, which in turn serves as the template for producing progeny plus-strand RNA. During this process, RNA dependent RNA polymerase(RdRP)is the key replicase; Untranslated regions(UTRs) are major regulatory sequences in which the 3′UTR is regarded as a main binding region of replicase to direct the de novo synthesis of viral genome. Based on deep understanding of virus replication mechanism and its details, the construction of RNA virus minigenome has been a focus in molecular virology research recently. It can be used to elucidate the molecular mechanism of viral replication and identify the trans-acting proteins involved in virus maturation and packaging. Furthermore, it can provide new strategy against HCV infection.
Hepatitis C virus (HCV) is a member of the Flaviviridae family with a positive-sense RNA genome of about 9600 nucleotides in length. It contains a single open-reading frame (ORF) flanked by two untranslated regions (UTR). HCV genome has a high degree of sequence variation which leads to the production of viral quasispecies. There are six different genotypes with more than 100 subtypes worldwide. The analysis of HCV genome sequence indicates that cis-acting elements functioned in the RNA replication are highly conserved, including 5′UTR, 3′UTR, and part sequence of core and ns5b. NS5B is RdRP of HCV and playes a key role in RNA replication. Replicase complex formed by NS5B along with other NS protein (such as NS3, NS4A, NS4B) are always anchored on endocytoplasmic reticulum. These progresses on understanding HCV RNA replication make it possible to construct HCV minigenome.
The discovery of new anti-HCV drugs are main aim for the scientists in the worldwide. Potential small molecular inhibitors of NS3/4A protease, helicase, and RdRP NS5B always lead to drug resistant mutants in clinical trial. The present treatment for HCV infection is IFN-αin combination with ribavirin (RBV), having limited effectiveness and great side effects. Therefore, the targeting HCV infected cells’therapy is promising.
In this study we firstly use cis-acting elements to construct HCV minigenome, in which the antisense sequence of firefly luciferase (flu) and IRES gene from EMCV were inserted. RdRP replicase complex bind 3’UTR to synthesize minus strand RNA, in which flu and IRES gene were in the sense direction and expressed. Then several different truncated minigenomes were designed. The transcribed minus strand RNAs from them had different mutant stem loops at 3′end. The levels of luciferase activity of different truncated minigenomes were compared. Finally, HCV minigenomes were used to express IFN-βfor targeting gene therapy against HCV. This study can be divided into three parts:
1. Construction and functional identification of HCV minigenome
Designed and amplified HCV 5UTR1-377 , 3UTR sequences from HCV 1b gene sequence of Genbank,with the T7 promoter directly coupled at the 5′-end; Designed and amplified IRES sequence (from EMCV), firefly luciferase gene; Synthesized HDV Ribozyme sense, antisense sequence and annealed; Cloned these fragments into pMD18T vector separately to identify sequences, then cloned them into pUC18 vector to construct pUC18-T7/5UTR-rIRES-3UTR-HDV Rz, labeled to pT7/5UrI3URz. Then the luciferase gene was inserted into Xba I digested pT7/5UrI3URz, generating pT7/5UrFI3URz,which was the HCV minigenome. HCV replicon RNA was synthesized in vitro by T7 RNA polymerase after the plamid BB7 was linearized by Sca I. Huh7.5BB7 stable cell line was obtained by eletroperation of BB7 RNA,and selected with G418, then confirmed by RT-PCR and Western blot. Plasmid pT7/5UrFI3URz was linearized with EcoR ?, and transcripted into minigenome RNA which was transfected into Huh7.5BB7 cell and Huh7.5 cell via eletroperation. After 48 hrs, cells were harvested and luciferase activities were assessed. The results showed luciferase was expressed selectively in Huh7.5BB7 cell but not in Huh7.5 cell, which suggested the minigenome was constructed successfully.
2. Comparison of different truncated HCV minigenomes HCV replication is the de novo synthesis from the 3′-end of plus or minus strand. The secondary structure of the 3′-end at the minus strand HCV RNA has been determined. With the exception of the short SL-A1 stem loop located at the very 3′-end, other structures differe from its antisense sequence corresponding to 5′UTR. Therefore, their respective roles in the RNA synthesis initiation also differe from 5′UTR. In this study, we constructed several truncated minigenomes (?45、?124、?227、?131-315) which produced the minus strand RNAs with deletion of different stem loops at the 3′UTR. These minigenomes were transcribed into the corresponding RNAs in vitro, then transfected into Huh7.5BB7 cell. After 48 hrs, luciferase activities were assessed. The result shows that ?131-315 exhibited the higher luciferase activity compared with that of the full minigenome (p<0.01),whereas that of ?45 minigenome exhibited lower (p<0.01). ?125, ?227 minigenomes didn’t show any luciferase activity. RT-PCR also suggested the same results.
3. Exploiting HCV minigenome to express IFN-βfor HCV targeting gene therapy
PEG-IFN in combination with ribavirin has been the standard treatment for HCV infection currently. But sustained virological response (SVR) varies between different genotypes. The SVR rates ranged from 38% to 52% among genotype 1 and 4. One important reason is that HCV proteins could block IFN inducing anti-virus response. In this study, IFN-βgene was invertedly inserted into minigenome and transfected into the cells harboring HCV replicase in which the minus strand minigenome RNA was synthesized and IFN-βcould be expressed and inhibit HCV RNA replication. First, we extract lymphocytes from peripheral blood and stimulated by poly I:C to express the IFN gene. Total RNA extracted from lymphocytes was reversely transcripted into cDNA. IFN-βgene was amplified by PCR and was sequenced, and then the correct gene was cloned into pT7/5UrI3URz in antisense direction to obtain pT7/5UrIFNI3URz. The plasmid was linearized by EcoR I and transcripted into RNA in vitro, then transfected into Huh7.5BB7. After 48hrs, cell was harvested and total proteins were extracted to do Western-blot, IFN-βantibody as the first antibody. The result showed that IFN-βwas specifically expressed in replicon cell. To apply the targeting strategy to gene therapy which require expression from polymerase II RNA polymerase rather than T7-directed transcription. So we clone the minigenome 5UrIFNI3URz into the plasmid pcDNA3.1 by positioning the minigenome immediately adjacent to the transcription start site of the cytomegalovirus (CMV) promoter. Then the pCMV-5UrIFNI3URz was constructed. The results of Western-blot analysis showed that IFN-βcould be specifically expressed after the pCMV-5UrIFNI3URz was trasnfected into Huh7.5BB7 cell lines. By the same way as the establishment of Huh7.5BB7 cell, the Huh7.5JFH-1 cell line was constructed in which virus RNA can replicate autonomously and virus particle can be packed successfully. When the pCMV-5UrIFNI3URz was transfected into Huh7.5JFH-1 cell,the real-time PCR assay showed that the level of HCV RNA replication was distinctly decreased.
In conclusion, the HCV minigenome was successfully constructed and optimized for expression of the foreign gene. The targeting minigenome carrying IFN-βcould specifically expressed in HCV infected cell lines,the replication level was inhibited in a certain extent.
丙型肝炎病毒(HCV)属黄病毒科,为单股正链RNA病毒。HCV基因组全长约9.6Kb,含有单一开放读框(ORF),两侧为非编码区(5′UTR和3′UTR)。HCV基因组具有高度变异性,存在“准株”现象,主要有六个基因型和多个亚型。而HCV基因组序列分析表明,与复制密切相关的顺式作用元件高度保守,它们主要存在于5′UTR和3′UTR,以及core和ns5b基因的部分序列。NS5B蛋白具有RdRP活性,是HCV复制的关键酶。它与其它非结构区蛋白(如NS3、NS4A、NS4B等)形成复制酶复合物,锚定于细胞中内质网膜上发挥其生物功能。这一系列研究为构建HCV微型基因组提供了条件。
新型HCV治疗药物是各国学者研究的热点。目前关于HCV的治疗是主要以干扰素为基础的治疗方案,但效果有限而且副作用大,患者依从性差。具有很大潜力的小分子药物,如针对NS3/4A蛋白酶、解旋酶及RNA聚合酶NS5B的抑制剂,临床试验显示常常引起耐药突变体。因此,迫切需要寻找新的抗HCV策略,其中靶向治疗HCV感染细胞的方法是有潜力的方向之一。
本研究首先通过基因重组技术用顺式作用元件构建了HCV微型基因组,中间为反向插入的萤火虫荧光素酶报告基因(firefly luciferase,flu)与EMCV的IRES基因。该微型基因组RNA在病毒复制酶的作用下,从3′末端起始合成负链RNA,反向插入的flu及IRES基因即转换为正向,flu得以表达。然后,设计了几种不同截短的微型基因组,由它们生成的负链RNA的3′末端具有不同茎环截短的突变形式,以报告基因flu的表达水平来比较这一系列微型基因组。在此基础上,利用该微型基因组表达IFN-β,靶向抑制HCV感染细胞。本研究分以下三个部分:
1. HCV微型基因组的构建与功能鉴定
根据Genbank登录的HCV 1b型基因序列,设计引物分别扩增HCV 5UTR1-377、3UTR,其中扩增5UTR1-377的引物上游含有T7启动子核心序列;分别设计并扩增IRES(来自EMCV)、flu基因;合成HDV核酶Rz(ribozyme)的正义与反义序列,两者退火;将PCR产物各片段克隆至pMD18T载体,交由公司测序。序列鉴定正确后依次将5UTR、IRES、3UTR、HDV Rz片段克隆入pUC18载体,形成质粒pUC18-T7/5UTR-rIRES-3UTR-HDV Rz,缩写为pT7/5UrI3URz。然后通过Xba I单酶切位点将flu基因插入上述载体,PCR鉴定正反向后,命名反向插入flu的载体为pT7/5UrFI3URz,即为HCV微型基因组。HCV复制子质粒BB7经Sca I酶切线性化,在T7 RNA聚合酶作用下体外转录制备RNA,通过电穿法转染Huh7.5细胞,经G418压力筛选获得稳定细胞克隆,RT-PCR和Western blot证实成功构建了该复制子细胞系,命名为Huh7.5BB7。将pT7/5UrFI3URz质粒经EcoR ?酶切线性化,体外转录制备微型基因组RNA,通过电穿转染方法将其导入Huh7.5BB7细胞和Huh7.5细胞,48hr后处理细胞进行荧光素酶活性测定。实验结果表明,荧光素酶选择性表达于Huh7.5BB7细胞,提示已成功构建HCV微型基因组。
2.不同HCV微型基因组截短体的比较
从3′末端起始的从头合成是HCV复制的形式。负链RNA 3′UTR的二级结构已经解析,除了3′末端的最短的SL-A1茎环外,其它茎环与正链RNA 5′UTR均不同。目前,对于负链RNA 3′UTR的各个茎环在正链RNA的从头合成中作用尚不清楚。本研究依次构建了几种截短的微型基因组(?45、?124、?227、?131-315),由它们生成的负链RNA的3′末端具有不同茎环截短的突变形式。将这几种微型基因组体外转录制备微型基因组RNA,转染Huh7.5BB7细胞。48 hr后测定荧光素酶活性,结果显示:与全长微型基因组相比,?131-315微型基因组表达荧光素酶活性增强(p<0.01),而?45微型基因组表达荧光素酶活性降低(p<0.01),?124、?227微型基因组基本不表达荧光素酶。
3.利用微型基因组特异表达IFN-β靶向抑制HCV感染细胞
长效干扰素(PEG-IFN)联合利巴韦林是目前HCV最为标准的治疗方案。但各个基因型的持续病毒学应答率(SVR)不同,1型和4型仅为38~52%。其中一个重要原因是HCV编码蛋白通过多种途径抵抗了IFN的抗病毒反应。本研究将IFN-β基因反向插入微型基因组,当其进入细胞内,在病毒复制酶作用下合成负链微型基因组RNA后启动IFN-β表达,从而发挥抗病毒作用。首先从外周血中分离淋巴细胞,用poly I:C刺激其分泌IFN。用Trizol提取细胞总RNA,逆转录合成cDNA,PCR扩增IFN-β基因。序列鉴定正确后,将其克隆入pT7/5U?131-315rI3URz,PCR鉴定插入的正反向,将反向插入的质粒命名为pT7/5U?131-315rIFNI3URz。该重组质粒用EcoR I酶切后纯化,体外转录制备RNA,转染Huh7.5BB7细胞,48hr后收集细胞并提取蛋白。抗IFN-β抗体作为一抗,Western-blot分析显示:IFN-β特异性表达于复制子细胞中。用Xba I线性化HCV全长基因组质粒JFH-1后,体外转录制备RNA并转染Huh7.5细胞,In cell Western-blot和荧光实时定量PCR证实建立了Huh7.5JFH-1细胞系。为了利用HCV微型基因组进行靶向基因治疗,设计引物扩增微型基因组5U?131-315rIFNI3URz后克隆入pcDNA3.1载体,获得pCMV-5U?131-315rIFNI3URz。将其转染复制子细胞系,Western-blot显示IFN-β能够特异的表达于复制子细胞系中。pCMV-5U?131-315rIFNI3URz转染huh7.5JFH-1细胞系后,荧光实时定量PCR显示,它可以降低HCV RNA的拷贝水平,具有一定的剂量依赖效应。
综上所述,本研究成功构建了HCV微型基因组,对其表达外源基因的构建方式进行了优化;携带IFN-β的微型基因组能够靶向表达于HCV感染细胞,并可降低病毒RNA复制水平。
Virus genome replication plays an important role in its life cycle. It is known that the replication of plus-stranded RNA virus divides into two steps: genomic RNA is firstly transcribed into a minus-strand intermediate, which in turn serves as the template for producing progeny plus-strand RNA. During this process, RNA dependent RNA polymerase(RdRP)is the key replicase; Untranslated regions(UTRs) are major regulatory sequences in which the 3′UTR is regarded as a main binding region of replicase to direct the de novo synthesis of viral genome. Based on deep understanding of virus replication mechanism and its details, the construction of RNA virus minigenome has been a focus in molecular virology research recently. It can be used to elucidate the molecular mechanism of viral replication and identify the trans-acting proteins involved in virus maturation and packaging. Furthermore, it can provide new strategy against HCV infection.
Hepatitis C virus (HCV) is a member of the Flaviviridae family with a positive-sense RNA genome of about 9600 nucleotides in length. It contains a single open-reading frame (ORF) flanked by two untranslated regions (UTR). HCV genome has a high degree of sequence variation which leads to the production of viral quasispecies. There are six different genotypes with more than 100 subtypes worldwide. The analysis of HCV genome sequence indicates that cis-acting elements functioned in the RNA replication are highly conserved, including 5′UTR, 3′UTR, and part sequence of core and ns5b. NS5B is RdRP of HCV and playes a key role in RNA replication. Replicase complex formed by NS5B along with other NS protein (such as NS3, NS4A, NS4B) are always anchored on endocytoplasmic reticulum. These progresses on understanding HCV RNA replication make it possible to construct HCV minigenome.
The discovery of new anti-HCV drugs are main aim for the scientists in the worldwide. Potential small molecular inhibitors of NS3/4A protease, helicase, and RdRP NS5B always lead to drug resistant mutants in clinical trial. The present treatment for HCV infection is IFN-αin combination with ribavirin (RBV), having limited effectiveness and great side effects. Therefore, the targeting HCV infected cells’therapy is promising.
In this study we firstly use cis-acting elements to construct HCV minigenome, in which the antisense sequence of firefly luciferase (flu) and IRES gene from EMCV were inserted. RdRP replicase complex bind 3’UTR to synthesize minus strand RNA, in which flu and IRES gene were in the sense direction and expressed. Then several different truncated minigenomes were designed. The transcribed minus strand RNAs from them had different mutant stem loops at 3′end. The levels of luciferase activity of different truncated minigenomes were compared. Finally, HCV minigenomes were used to express IFN-βfor targeting gene therapy against HCV. This study can be divided into three parts:
1. Construction and functional identification of HCV minigenome
Designed and amplified HCV 5UTR1-377 , 3UTR sequences from HCV 1b gene sequence of Genbank,with the T7 promoter directly coupled at the 5′-end; Designed and amplified IRES sequence (from EMCV), firefly luciferase gene; Synthesized HDV Ribozyme sense, antisense sequence and annealed; Cloned these fragments into pMD18T vector separately to identify sequences, then cloned them into pUC18 vector to construct pUC18-T7/5UTR-rIRES-3UTR-HDV Rz, labeled to pT7/5UrI3URz. Then the luciferase gene was inserted into Xba I digested pT7/5UrI3URz, generating pT7/5UrFI3URz,which was the HCV minigenome. HCV replicon RNA was synthesized in vitro by T7 RNA polymerase after the plamid BB7 was linearized by Sca I. Huh7.5BB7 stable cell line was obtained by eletroperation of BB7 RNA,and selected with G418, then confirmed by RT-PCR and Western blot. Plasmid pT7/5UrFI3URz was linearized with EcoR ?, and transcripted into minigenome RNA which was transfected into Huh7.5BB7 cell and Huh7.5 cell via eletroperation. After 48 hrs, cells were harvested and luciferase activities were assessed. The results showed luciferase was expressed selectively in Huh7.5BB7 cell but not in Huh7.5 cell, which suggested the minigenome was constructed successfully.
2. Comparison of different truncated HCV minigenomes HCV replication is the de novo synthesis from the 3′-end of plus or minus strand. The secondary structure of the 3′-end at the minus strand HCV RNA has been determined. With the exception of the short SL-A1 stem loop located at the very 3′-end, other structures differe from its antisense sequence corresponding to 5′UTR. Therefore, their respective roles in the RNA synthesis initiation also differe from 5′UTR. In this study, we constructed several truncated minigenomes (?45、?124、?227、?131-315) which produced the minus strand RNAs with deletion of different stem loops at the 3′UTR. These minigenomes were transcribed into the corresponding RNAs in vitro, then transfected into Huh7.5BB7 cell. After 48 hrs, luciferase activities were assessed. The result shows that ?131-315 exhibited the higher luciferase activity compared with that of the full minigenome (p<0.01),whereas that of ?45 minigenome exhibited lower (p<0.01). ?125, ?227 minigenomes didn’t show any luciferase activity. RT-PCR also suggested the same results.
3. Exploiting HCV minigenome to express IFN-βfor HCV targeting gene therapy
PEG-IFN in combination with ribavirin has been the standard treatment for HCV infection currently. But sustained virological response (SVR) varies between different genotypes. The SVR rates ranged from 38% to 52% among genotype 1 and 4. One important reason is that HCV proteins could block IFN inducing anti-virus response. In this study, IFN-βgene was invertedly inserted into minigenome and transfected into the cells harboring HCV replicase in which the minus strand minigenome RNA was synthesized and IFN-βcould be expressed and inhibit HCV RNA replication. First, we extract lymphocytes from peripheral blood and stimulated by poly I:C to express the IFN gene. Total RNA extracted from lymphocytes was reversely transcripted into cDNA. IFN-βgene was amplified by PCR and was sequenced, and then the correct gene was cloned into pT7/5UrI3URz in antisense direction to obtain pT7/5UrIFNI3URz. The plasmid was linearized by EcoR I and transcripted into RNA in vitro, then transfected into Huh7.5BB7. After 48hrs, cell was harvested and total proteins were extracted to do Western-blot, IFN-βantibody as the first antibody. The result showed that IFN-βwas specifically expressed in replicon cell. To apply the targeting strategy to gene therapy which require expression from polymerase II RNA polymerase rather than T7-directed transcription. So we clone the minigenome 5UrIFNI3URz into the plasmid pcDNA3.1 by positioning the minigenome immediately adjacent to the transcription start site of the cytomegalovirus (CMV) promoter. Then the pCMV-5UrIFNI3URz was constructed. The results of Western-blot analysis showed that IFN-βcould be specifically expressed after the pCMV-5UrIFNI3URz was trasnfected into Huh7.5BB7 cell lines. By the same way as the establishment of Huh7.5BB7 cell, the Huh7.5JFH-1 cell line was constructed in which virus RNA can replicate autonomously and virus particle can be packed successfully. When the pCMV-5UrIFNI3URz was transfected into Huh7.5JFH-1 cell,the real-time PCR assay showed that the level of HCV RNA replication was distinctly decreased.
In conclusion, the HCV minigenome was successfully constructed and optimized for expression of the foreign gene. The targeting minigenome carrying IFN-βcould specifically expressed in HCV infected cell lines,the replication level was inhibited in a certain extent.
引文
1 Farci P, Purcell RH. Clinical significance of hepatitis C virus genotypes and quasispecies[J]. Semin Liver Dis JT- Seminars in liver disease, 2000; 20(1):103-26.
2 You S, Rice CM. 3' RNA elements in hepatitis C virus replication: kissing partners and long poly(U)[J]. J Virol, 2008; 82(1):184-95.
3 刘丽君, 魏来. 丙型肝炎病毒的流行病学[J]. 传染病信息. 2007; 20(5):261-264.
4 Zekri AR, El-Din HM, Bahnassy AA, Khaled MM, Omar A, Fouad I, El-Hefnewi M, Thakeb F,El-Awady M. Genetic distance and heterogenecity between quasispecies is a critical predictor to IFN response in Egyptian patients with HCV genotype-4[J]. Virol J JT- Virology journal, 2007; 4:16.
5 De Francesco R, Carfi A. Advances in the development of new therapeutic agents targeting the NS3-4A serine protease or the NS5B RNA-dependent RNA polymerase of the hepatitis C virus[J]. Adv Drug Deliv Rev JT- Advanced drug delivery reviews, 2007; 59(12):1242-62.
6 Lohmann V, Korner F, Koch J, Herian U, Theilmann L,Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line[J]. Science, 1999; 285(5424):110-3.
7 Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, Habermann A, Krausslich HG, Mizokami M, Bartenschlager R,Liang TJ. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome[J]. Nat Med, 2005; 11(7):791-6.
8 Lindenbach BD, Rice CM. Unravelling hepatitis C virus replication from genome to function[J]. Nature, 2005; 436(7053):933-8.
9 Moradpour D, Penin F,Rice CM. Replication of hepatitis C virus[J]. Nat Rev Microbiol, 2007; 5(6):453-63.
10 Hong Z, Cameron CE, Walker MP, Castro C, Yao N, Lau JY, Zhong W. A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase[J]. Virology, 2001; 285(1):6-11.
11 Suzuki K, Shinzawa H, Kuboki M, Ishibashi M, Yoshii E, Saito T,Takahashi T. Secondary structure of the hepatitis C virus 5' untranslated region and efficacy ofinterferon therapy for chronic hepatitis C[J]. Liver, 1998; 18(5):331-6.
12 Cheng JC, Chang MF, Chang SC. Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3' end of the viral RNA[J]. J Virol, 1999; 73(8):7044-9.
13 You S, Stump DD, Branch AD, Rice CM. A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication[J]. J Virol, 2004; 78(3):1352-66.
14 Halpin K, Bankamp B, Harcourt BH, Bellini WJ,Rota PA. Nipah virus conforms to the rule of six in a minigenome replication assay[J]. J Gen Virol, 2004; 85(Pt 3):701-7.
15 Zhang J, Yamada O, Sakamoto T, Yoshida H, Araki H,Shimotohno K. Exploiting cis-acting replication elements to direct hepatitis C virus-dependent transgene expression[J]. J Virol, 2005; 79(10):5923-32.
16 Astier-Gin T, Bellecave P, Litvak S, Ventura M. Template requirements and binding of hepatitis C virus NS5B polymerase during in vitro RNA synthesis from the 3'-end of virus minus-strand RNA[J]. FEBS J, 2005; 272(15):3872-86.
17 Dansako H, Ikeda M,Kato N. Limited suppression of the interferon-beta production by hepatitis C virus serine protease in cultured human hepatocytes[J]. FEBS J, 2007; 274(16):4161-76.
18 Diamantis I, Vafiadis I, Voskaridou E, Dellatetsima J, Jaggi N, Gyr K,Battegay M. Genotype distribution of hepatitis C virus infection in Greece: correlation with different risk factors and response to interferon therapy[J]. Eur J Gastroenterol Hepatol, 1998; 10(1):75-9.
19 中华医学会肝病学分会、传染病与寄生虫病学分会. 丙型肝炎防治指南. 中华医学杂志. 2004; 84(9): 775-780.
20 Glenn JS. Molecular virology of the hepatitis C virus: implication for novel therapies[J]. Clin Liver Dis, 2005; 9(3):353-69, v.
21 Shao SW, Wu WB, Yu JG, Zhao P,Qi ZT. [Antigenicity of hepatitis C virus F protein and serum prevalence of anti-F in HCV-infected patients][J]. Zhonghua Gan Zang Bing Za Zhi, 2006; 14(12):890-3.
22 Bartenschlager R, Sparacio S. Hepatitis C virus molecular clones and their replication capacity in vivo and in cell culture[J]. Virus Res, 2007; 127(2):195-207.
23 Zhang J, Yamada O, Ito T, Akiyama M, Hashimoto Y, Yoshida H, Makino R, Masago A, Uemura H,Araki H. A single nucleotide insertion in the 5'-untranslated region of hepatitis C virus leads to enhanced cap-independent translation[J]. Virology, 1999; 261(2):263-70.
24 Luo G, Xin S, Cai Z. Role of the 5'-proximal stem-loop structure of the 5' untranslated region in replication and translation of hepatitis C virus RNA[J]. J Virol, 2003; 77(5):3312-8.
25 Collier AJ, Gallego J, Klinck R, Cole PT, Harris SJ, Harrison GP, Aboul-Ela F, Varani G,Walker S. A conserved RNA structure within the HCV IRES eIF3-binding site[J]. Nat Struct Biol, 2002; 9(5):375-80.
26 Masao Honda, Michael R. Beard, Li-Hua Ping, Stanley M. Lemon. A Phylogenetically Conserved Stem-Loop Structure at the 5' Border of the Internal Ribosome Entry Site of Hepatitis C Virus Is Required for Cap-Independent Viral Translation [J]. J. Virol, 1999; 73:1165-1174.
27 Jubin R. Hepatitis C IRES: translating translation into a therapeutic target[J]. Curr Opin Mol Ther, 2001; 3(3):278-87.
28 Kim CS, Seol SK, Song OK, Park JH,Jang SK. An RNA-binding protein, hnRNP A1, and a scaffold protein, septin 6, facilitate hepatitis C virus replication[J]. J Virol, 2007; 81(8):3852-65.
29 Tischendorf JJ, Beger C, Korf M, Manns MP,Kruger M. Polypyrimidine tract-binding protein (PTB) inhibits Hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation, but does not affect HCV replication[J]. Arch Virol, 2004; 149(10):1955-70.
30 Honda M, Shimazaki T,Kaneko S. La protein is a potent regulator of replication of hepatitis C virus in patients with chronic hepatitis C through internal ribosomal entry site-directed translation[J]. Gastroenterology, 2005; 128(2):449-62.
31 Yanagi M, St Claire M, Emerson SU, Purcell RH, Bukh J. In vivo analysis of the 3' untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone[J]. Proc Natl Acad Sci U S A, 1999; 96(5):2291-5.
32 Scholle F, Li K, Bodola F, Ikeda M, Luxon BA, Lemon SM. Virus-host cell interactions during hepatitis C virus RNA replication: impact of polyprotein expression on the cellular transcriptome and cell cycle association with viral RNA synthesis[J]. J Virol, 2004; 78(3):1513-24.
33 Friebe P, Bartenschlager R. Genetic analysis of sequences in the 3' nontranslated region of hepatitis C virus that are important for RNA replication[J]. J Virol, 2002; 76(11):5326-38.
34 Lee H, Shin H, Wimmer E, Paul AV. cis-acting RNA signals in the NS5B C-terminal coding sequence of the hepatitis C virus genome[J]. J Virol, 2004;
78(20):10865-77.
35 Tuplin A, Evans DJ, Simmonds P. Detailed mapping of RNA secondary structures in core and NS5B-encoding region sequences of hepatitis C virus by RNase cleavage and novel bioinformatic prediction methods[J]. J Gen Virol, 2004; 85(Pt 10):3037-47.
36 Zhang J, Yamada O, Yoshida H, Sakamoto T, Araki H,Shimotohno K. Helper virus-independent trans-replication of hepatitis C virus-derived minigenome[J]. Biochem Biophys Res Commun, 2007; 352(1):170-6.
37 Bartenschlager R, Lohmann V. Replication of hepatitis C virus[J]. J Gen Virol, 2000; 81(Pt 7):1631-48.
38 Gontarek RR, Gutshall LL, Herold KM, Tsai J, Sathe GM, Mao J, Prescott C,Del Vecchio AM. hnRNP C and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'NTR of the HCV RNA genome[J]. Nucleic Acids Res, 1999; 27(6):1457-63.
39 Chung RT, Kaplan LM. Heterogeneous nuclear ribonucleoprotein I (hnRNP-I/PTB) selectively binds the conserved 3' terminus of hepatitis C viral RNA[J]. Biochem Biophys Res Commun, 1999; 254(2):351-62.
40 Lesburg CA, Cable MB, Ferrari E, Hong Z, Mannarino AF, Weber PC. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site[J]. Nat Struct Biol, 1999; 6(10):937-43.
41 Dutartre H, Boretto J, Guillemot JC, Canard B. A relaxed discrimination of
2'-O-methyl-GTP relative to GTP between de novo and Elongative RNA synthesis by the hepatitis C RNA-dependent RNA polymerase NS5B[J]. J Biol Chem, 2005; 280(8):6359-68.
42 Lee KJ, Choi J, Ou JH, Lai MM. The C-terminal transmembrane domain of hepatitis C virus (HCV) RNA polymerase is essential for HCV replication in vivo[J]. J Virol, 2004; 78(7):3797-802.
43 Kim JL, Morgenstern KA, Lin C, Fox T, Dwyer MD, Landro JA, Chambers SP,Markland W, Lepre CA, O'Malley ET, Harbeson SL, Rice CM, Murcko MA, Caron PR,Thomson JA. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide[J]. Cell, 1996; 87(2):343-55.
44 Zhang T, Lin RT, Li Y, Douglas SD, Maxcey C, Ho C, Lai JP, Wang YJ, Wan Q,Ho WZ. Hepatitis C virus inhibits intracellular interferon alpha expression in human hepatic cell lines[J]. Hepatology, 2005; 42(4):819-27.
45 Kasprzak A, Adamek A. Role of hepatitis C virus proteins (C, NS3, NS5A) in hepatic oncogenesis[J]. Hepatol Res, 2008; 38(1):1-26.
46 Zhang C, Cai Z, Kim YC, Kumar R, Yuan F, Shi PY, Kao C,Luo G. Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase[J]. J Virol, 2005; 79(14):8687-97.
47 Lindenbach BD, Pragai BM, Montserret R, Beran RK, Pyle AM, Penin F,Rice CM. The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication[J]. J Virol, 2007; 81(17):8905-18.
48 Elazar M, Liu P, Rice CM, Glenn JS. An N-terminal amphipathic helix in hepatitis C virus (HCV) NS4B mediates membrane association, correct localization of replication complex proteins, and HCV RNA replication[J]. J Virol, 2004; 78(20):11393-400.
49 Lundin M, Lindstrom H, Gronwall C, Persson MA. Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein[J]. J Gen Virol, 2006; 87(Pt 11):3263-72.
50 Neddermann P, Quintavalle M, Di Pietro C, Clementi A, Cerretani M, Altamura S, Bartholomew L,De Francesco R. Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture[J]. J Virol, 2004; 78(23):13306-14.
51 Szabo G. Hepatitis C virus NS5A protein--a master regulator?[J]. Gastroenterology, 2006; 130(3):995-9.
52 Liang Y, Ye H, Kang CB,Yoon HS. Domain 2 of nonstructural protein 5A (NS5A) of hepatitis C virus is natively unfolded[J]. Biochemistry, 2007; 46(41):11550-8.
53 Tellinghuisen TL, Foss KL, Treadaway JC, Rice CM. Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5Aprotein[J]. J Virol, 2008; 82(3):1073-83.
54 Xue Q, Ding H, Liu M, Zhao P, Gao J, Ren H, Liu Y,Qi ZT. Inhibition of hepatitis C virus replication and expression by small interfering RNA targeting host cellular genes[J]. Arch Virol, 2007; 152(5):955-62.
55 Wang C, Gale M Jr, Keller BC, Huang H, Brown MS, Goldstein JL, Ye J. Identification of FBL2 as a geranylgeranylated cellular protein required for hepatitis C virus RNA replication[J]. Mol Cell, 2005; 18(4):425-34.
56 Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y,Shimotohno K. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase[J]. Mol Cell, 2005; 19(1):111-22.
57 Yi M, Lemon SM. Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells[J]. J Virol, 2004; 78(15):7904-15.
58 Evans MJ, Rice CM,Goff SP. Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication[J]. Proc Natl Acad Sci U S A, 2004; 101(35):13038-43.
59 Salonen A, Ahola T, Kaariainen L. Viral RNA replication in association with cellular membranes[J]. Curr Top Microbiol Immunol, 2005; 285:139-73.
60 Zheng Y, Gao B, Ye L, Kong L, Jing W, Yang X, Wu Z,Ye L. Hepatitis C virus non-structural protein NS4B can modulate an unfolded protein response[J]. J Microbiol, 2005; 43(6):529-36.
61 Moradpour D, Gosert R, Egger D, Penin F, Blum HE,Bienz K. Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex[J]. Antiviral Res, 2003; 60(2):103-9.
62 Qadri I, Iwahashi M, Capasso JM, Hopken MW, Flores S, Schaack J,Simon FR. Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP-1[J]. Biochem J, 2004; 378(Pt 3):919-28.
63 Bartenschlager R, Lohmann V. Replication of the hepatitis C virus[J]. Baillieres Best Pract Res Clin Gastroenterol, 2000; 14(2):241-54.
64 Yi M, Lemon SM. 3' nontranslated RNA signals required for replication of hepatitis C virus RNA[J]. J Virol, 2003; 77(6):3557-68.
65 Luo G, Hamatake RK, Mathis DM, Racela J, Rigat KL, Lemm J,Colonno RJ. Denovo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus[J]. J Virol, 2000; 74(2):851-63.
66 Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW,Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome[J]. Science, 1989; 244(4902):359-62.
67 Yoo BJ, Selby MJ, Choe J, Suh BS, Choi SH, Joh JS, Nuovo GJ, Lee HS, Houghton M,Han JH. Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV[J]. J Virol, 1995; 69(1):32-8.
68 Ishii N, Watashi K, Hishiki T, Goto K, Inoue D, Hijikata M, Wakita T, Kato N,Shimotohno K. Diverse effects of cyclosporine on hepatitis C virus strain replication[J]. J Virol, 2006; 80(9):4510-20.
69 Cornu TI, de la Torre JC. RING finger Z protein of lymphocytic choriomeningitis virus (LCMV) inhibits transcription and RNA replication of an LCMV S-segment minigenome[J]. J Virol, 2001; 75(19):9415-26.
70 Ikegami T, Peters CJ, Makino S. Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system[J]. J Virol, 2005; 79(9):5606-15.
71 You S, Padmanabhan R. A novel in vitro replication system for Dengue virus. Initiation of RNA synthesis at the 3'-end of exogenous viral RNA templates requires 5'- and 3'-terminal complementary sequence motifs of the viral RNA[J]. J Biol Chem, 1999; 274(47):33714-22.
72 Strauss DM, Glustrom LW, Wuttke DS. Towards an understanding of the poliovirus replication complex: the solution structure of the soluble domain of the poliovirus 3A protein[J]. J Mol Biol, 2003; 330(2):225-34.
73 Fujimura T, Esteban R. Interactions of the RNA polymerase with the viral genome at the 5'- and 3'-ends contribute to 20S RNA narnavirus persistence in yeast[J]. J Biol Chem, 2007; 282(26):19011-9.
74 Dumas E, Masante C, Astier-Gin T, Lapaillerie D, Ventura M. The hepatitis C virus minigenome: a new cellular model for studying viral replication[J]. J Virol Methods, 2007; 142(1-2):59-66.
75 曲建慧, 张玲霞. 干扰素的分子生物学机制研究进展[J]. 国际流行病学传染病学杂志.2006;33(1):38-41.
76 徐 春 娥 , 陈 薇 . 干 扰 素 系 统 与 病 毒 的 相 互 作 用 [J]. 中 国 生 物 工 程 杂志.2006;26(4):95-100.
77 Pestka S. Purification and cloning of interferon alpha[J]. Curr Top Microbiol Immunol, 2007; 316:23-37.
78 Markowitz CE. Interferon-beta: mechanism of action and dosing issues[J]. Neurology, 2007; 68(24 Suppl 4):S8-11.
79 Powell HJ, Cong Y, Yu JJ, Guentzel MN, Berton MT, Klose KE, Murthy AK,Arulanandam BP. CD4(+) T cells are required during priming but not the effector phase of antibody-mediated IFN-gamma-dependent protective immunity against pulmonary Francisella novicida infection[J]. Immunol Cell Biol, 2008.
80 宋仕玲.干扰素抵抗与 JAK/STAT 信号通道系统[J].国外医学.病毒学分册.2004; 11(2):40-43.
81 卢年芳 . 干扰素抗病毒作用的机制 [J]. 国外医学 . 流行病学 . 传染病学分册.2003;30(4):226-228.
82 Iacopino F, Ferrandina G, Scambia G, Benedetti-Panici P, Mancuso S,Sica G. Interferons inhibit EGF-stimulated cell growth and reduce EGF binding in human breast cancer cells[J]. Anticancer Res, 1996; 16(4A):1919-24.
83 Ferrantini M, Capone I, Belardelli F. Interferon-alpha and cancer: mechanisms of action and new perspectives of clinical use[J]. Biochimie, 2007; 89(6-7):884-93.
84 Tong MJ, Reddy KR, Lee WM, Pockros PJ, Hoefs JC, Keeffe EB, Hollinger FB, Hathcote EJ, White H, Foust RT, Jensen DM, Krawitt EL, Fromm H, Black M, Blatt LM, Klein M,Lubina J. Treatment of chronic hepatitis C with consensus interferon: a multicenter, randomized, controlled trial. Consensus Interferon Study Group[J]. Hepatology, 1997; 26(3):747-54.
85 El-Zayadi AR, Attia M, Barakat EM, Badran HM, Hamdy H, El-Tawil A, El-Nakeeb A, Selim O,Saied A. Response of hepatitis C genotype-4 naive patients to 24 weeks of Peg-interferon-alpha2b/ribavirin or induction-dose interferon-alpha2b/ribavirin/amantadine: a non-randomized controlled study[J]. Am J Gastroenterol, 2005; 100(11):2447-52.
86 Cornberg M, Huppe D, Wiegand J, Felten G, Wedemeyer H, Manns MP. [Treatment of chronic hepatitis C with PEG-interferon alpha-2b and ribavirin: 24 weeks oftherapy are sufficient for HCV genotype 2 and 3][J]. Z Gastroenterol, 2003; 41(6):517-22.
87 Cornberg M, Wedemeyer H, Manns MP. Treatment of chronic hepatitis C with PEGylated interferon and ribavirin[J]. Curr Gastroenterol Rep, 2002; 4(1):23-30.
88 Legrand-Abravanel F, Nicot F, Boulestin A, Sandres-Saune K, Vinel JP, Alric L,Izopet J. Pegylated interferon and ribavirin therapy for chronic hepatitis C virus genotype 4 infection[J]. J Med Virol, 2005; 77(1):66-9.
89 Moreno-Otero R, Trapero-Marugan M, Gomez-Dominguez E, Garcia-Buey L,Moreno-Monteagudo JA. Is interferon-beta an alternative treatment for chronic hepatitis C?[J]. World J Gastroenterol, 2006; 12(17):2730-6.
90 Chevaliez S, Pawlotsky JM. Interferon-based therapy of hepatitis C[J]. Adv Drug Deliv Rev, 2007; 59(12):1222-41.
91 程勇前, 聂青和. 丙型肝炎病毒的干扰素抵抗机制研究进展[J]. 肝脏.2007; 12(4):303-305.
92 Miller K, McArdle S, Gale MJ Jr, Geller DA, Tenoever B, Hiscott J, Gretch DR,Polyak SJ. Effects of the hepatitis C virus core protein on innate cellular defense pathways[J]. J Interferon Cytokine Res, 2004; 24(7):391-402.
93 Goyal A, Hofmann WP, Hermann E, Traver S, Hissar SS, Arora N, Blum HE, Zeuzem S, Sarrazin C,Sarin SK. The hepatitis C virus NS5A protein and response to interferon alpha: mutational analyses in patients with chronic HCV genotype 3a infection from India[J]. Med Microbiol Immunol, 2007; 196(1):11-21.
94 Hung CH, Lee CM, Lu SN, Lee JF, Wang JH, Tung HD, Chen TM, Hu TH, Chen WJ,Changchien CS. Mutations in the NS5A and E2-PePHD region of hepatitis C virus type 1b and correlation with the response to combination therapy with interferon and ribavirin[J]. J Viral Hepat, 2003; 10(2):87-94.
95 Breiman A, Grandvaux N, Lin R, Ottone C, Akira S, Yoneyama M, Fujita T, Hiscott J,Meurs EF. Inhibition of RIG-I-dependent signaling to the interferon pathway during hepatitis C virus expression and restoration of signaling by IKKepsilon[J]. J Virol, 2005; 79(7):3969-78.
96 Li XD, Sun L, Seth RB, Pineda G, Chen ZJ. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity[J]. Proc Natl Acad Sci U S A, 2005; 102(49):17717-22.
97 Bode JG, Brenndorfer ED, Haussinger D. Subversion of innate host antiviral strategies by the hepatitis C virus[J]. Arch Biochem Biophys, 2007; 462(2):254-65.
98 杨敬. HCV NS3 蛋白的表达、纯化、单克隆抗体制备及其结合区域的分析[D]. 徐志凯 第四军医大学; 2005:28-46.
99 J.萨姆布鲁克 , D .W.拉塞尔. 分子克隆实验指南. 第三版 [M]. 北京:科学出版社, 2002:1296-1298.
100 Frolov I, McBride MS,Rice CM. cis-acting RNA elements required for replication of bovine viral diarrhea virus-hepatitis C virus 5' nontranslated region chimeras[J]. RNA, 1998; 4(11):1418-35.
101 Jang SJ, Wang LF, Radkowski M, Rakela J,Laskus T. Differences between hepatitis C virus 5' untranslated region quasispecies in serum and liver[J]. J Gen Virol, 1999; 80 (Pt 3):711-6.
102 Rijnbrand RC, Lemon SM. Internal ribosome entry site-mediated translation in hepatitis C virus replication[J]. Curr Top Microbiol Immunol, 2000; 242:85-116.
103 Wood J, Frederickson RM, Fields S,Patel AH. Hepatitis C virus 3'X region interacts with human ribosomal proteins[J]. J Virol, 2001; 75(3):1348-58.
104 Freiberg A, Dolores LK, Enterlein S,Flick R. Establishment and characterization of plasmid-driven minigenome rescue systems for Nipah virus: RNA polymerase I- and T7-catalyzed generation of functional paramyxoviral RNA[J]. Virology, 2008; 370(1):33-44.
105 Groseth A, Feldmann H, Theriault S, Mehmetoglu G,Flick R. RNA polymerase I-driven minigenome system for Ebola viruses[J]. J Virol, 2005; 79(7):4425-33.
106 Johnson RF, McCarthy SE, Godlewski PJ, Harty RN. Ebola virus VP35-VP40 interaction is sufficient for packaging 3E-5E minigenome RNA into virus-like particles[J]. J Virol, 2006; 80(11):5135-44.
107 Yi M, Lemon SM. Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication[J]. RNA, 2003; 9(3):331-45.
108 Schuster C, Isel C, Imbert I, Ehresmann C, Marquet R,Kieny MP. Secondary structure of the 3' terminus of hepatitis C virus minus-strand RNA[J]. J Virol, 2002; 76(16):8058-68.
109 Smith RM, Walton CM, Wu CH,Wu GY. Secondary structure and hybridizationaccessibility of hepatitis C virus 3'-terminal sequences[J]. J Virol, 2002; 76(19):9563-74.
110 金伯泉. 细胞和分子免疫学实验技术[M]. 西安:第四军医大学出版社,2002: 61-64.
111 Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, Liu CC, Maruyama T, Hynes RO, Burton DR, McKeating JA,Rice CM. Complete replication of hepatitis C virus in cell culture[J]. Science, 2005; 309(5734):623-6.
112 Craxi A, Laffi G, Zignego AL. Hepatitis C virus (HCV) infection: A systemic disease[J]. Mol Aspects Med, 2008; 29(1-2):85-95.
113 Holland-Staley CA, Kovari LC, Golenberg EM, Pobursky KJ, Mayers DL. Genetic diversity and response to IFN of the NS3 protease gene from clinical strains of the hepatitis C virus[J]. Arch Virol, 2002; 147(7):1385-406.
114 De Francesco R, Tomei L, Altamura S, Summa V,Migliaccio G. Approaching a new era for hepatitis C virus therapy: inhibitors of the NS3-4A serine protease and the NS5B RNA-dependent RNA polymerase[J]. Antiviral Res, 2003; 58(1):1-16.
115 Thomas A, Laxton C, Rodman J, Myangar N, Horscroft N,Parkinson T. Investigating Toll-like receptor agonists for potential to treat hepatitis C virus infection[J]. Antimicrob Agents Chemother, 2007; 51(8):2969-78.
116 Weber F, Kochs G, Haller O. Inverse interference: how viruses fight the interferon system[J]. Viral Immunol, 2004; 17(4):498-515.
117 Dupuis S, Jouanguy E, Al-Hajjar S, Fieschi C, Al-Mohsen IZ, Al-Jumaah S, Yang K, Chapgier A, Eidenschenk C, Eid P, Al Ghonaium A, Tufenkeji H, Frayha H, Al-Gazlan S, Al-Rayes H, Schreiber RD, Gresser I,Casanova JL. Impaired response to interferon-alpha/beta and lethal viral disease in human STAT1 deficiency[J]. Nat Genet, 2003; 33(3):388-91.
118 Ferreon JC, Ferreon AC, Li K, Lemon SM. Molecular determinants of TRIF proteolysis mediated by the hepatitis C virus NS3/4A protease[J]. J Biol Chem, 2005; 280(21):20483-92.
119 Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, Bartenschlager R,Tschopp J. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus[J]. Nature, 2005; 437(7062):1167-72.
120 Hiscott J. Triggering the innate antiviral response through IRF-3 activation[J]. JBiol Chem, 2007; 282(21):15325-9.
121 Sato M, Suemori H, Hata N, Asagiri M, Ogasawara K, Nakao K, Nakaya T, Katsuki M, Noguchi S, Tanaka N,Taniguchi T. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction[J]. Immunity, 2000; 13(4):539-48.
122 Marie I, Durbin JE, Levy DE. Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7[J]. EMBO J, 1998; 17(22):6660-9.
123 Erlandsson L, Blumenthal R, Eloranta ML, Engel H, Alm G, Weiss S,Leanderson T. Interferon-beta is required for interferon-alpha production in mouse fibroblasts[J]. Curr Biol, 1998; 8(4):223-6.
124 Bode JG, Ludwig S, Ehrhardt C, Albrecht U, Erhardt A, Schaper F, Heinrich PC,Haussinger D. IFN-alpha antagonistic activity of HCV core protein involves induction of suppressor of cytokine signaling-3[J]. FASEB J, 2003; 17(3):488-90.
125 Meurs EF, Breiman A. The interferon inducing pathways and the hepatitis C virus[J]. World J Gastroenterol, 2007; 13(17):2446-54.
126 Otsuka M, Kato N, Moriyama M, Taniguchi H, Wang Y, Dharel N, Kawabe T,Omata M. Interaction between the HCV NS3 protein and the host TBK1 protein leads to inhibition of cellular antiviral responses[J]. Hepatology, 2005; 41(5):1004-12.
127 Chan HL, Ren H, Chow WC,Wee T. Randomized trial of interferon beta-1a with or without ribavirin in Asian patients with chronic hepatitis C[J]. Hepatology, 2007; 46(2):315-23.
128 Festi D, Sandri L, Mazzella G, Roda E, Sacco T, Staniscia T, Capodicasa S, Vestito A,Colecchia A. Safety of interferon beta treatment for chronic HCV hepatitis[J]. World J Gastroenterol, 2004; 10(1):12-6.
129 Han Q, Liu Z, Kang W, Li H, Zhang L,Zhang N. Interferon Beta 1a versus Interferon Beta 1a plus Ribavirin for the Treatment of Chronic Hepatitis C in Chinese Patients: A Randomized, Placebo-Controlled Trial[J]. Dig Dis Sci, 2007.
130 Cai Z, Zhang C, Chang KS, Jiang J, Ahn BC, Wakita T, Liang TJ,Luo G. Robust production of infectious hepatitis C virus (HCV) from stably HCV cDNA-transfected human hepatoma cells[J]. J Virol, 2005; 79(22):13963-73.
2 You S, Rice CM. 3' RNA elements in hepatitis C virus replication: kissing partners and long poly(U)[J]. J Virol, 2008; 82(1):184-95.
3 刘丽君, 魏来. 丙型肝炎病毒的流行病学[J]. 传染病信息. 2007; 20(5):261-264.
4 Zekri AR, El-Din HM, Bahnassy AA, Khaled MM, Omar A, Fouad I, El-Hefnewi M, Thakeb F,El-Awady M. Genetic distance and heterogenecity between quasispecies is a critical predictor to IFN response in Egyptian patients with HCV genotype-4[J]. Virol J JT- Virology journal, 2007; 4:16.
5 De Francesco R, Carfi A. Advances in the development of new therapeutic agents targeting the NS3-4A serine protease or the NS5B RNA-dependent RNA polymerase of the hepatitis C virus[J]. Adv Drug Deliv Rev JT- Advanced drug delivery reviews, 2007; 59(12):1242-62.
6 Lohmann V, Korner F, Koch J, Herian U, Theilmann L,Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line[J]. Science, 1999; 285(5424):110-3.
7 Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, Habermann A, Krausslich HG, Mizokami M, Bartenschlager R,Liang TJ. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome[J]. Nat Med, 2005; 11(7):791-6.
8 Lindenbach BD, Rice CM. Unravelling hepatitis C virus replication from genome to function[J]. Nature, 2005; 436(7053):933-8.
9 Moradpour D, Penin F,Rice CM. Replication of hepatitis C virus[J]. Nat Rev Microbiol, 2007; 5(6):453-63.
10 Hong Z, Cameron CE, Walker MP, Castro C, Yao N, Lau JY, Zhong W. A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase[J]. Virology, 2001; 285(1):6-11.
11 Suzuki K, Shinzawa H, Kuboki M, Ishibashi M, Yoshii E, Saito T,Takahashi T. Secondary structure of the hepatitis C virus 5' untranslated region and efficacy ofinterferon therapy for chronic hepatitis C[J]. Liver, 1998; 18(5):331-6.
12 Cheng JC, Chang MF, Chang SC. Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3' end of the viral RNA[J]. J Virol, 1999; 73(8):7044-9.
13 You S, Stump DD, Branch AD, Rice CM. A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication[J]. J Virol, 2004; 78(3):1352-66.
14 Halpin K, Bankamp B, Harcourt BH, Bellini WJ,Rota PA. Nipah virus conforms to the rule of six in a minigenome replication assay[J]. J Gen Virol, 2004; 85(Pt 3):701-7.
15 Zhang J, Yamada O, Sakamoto T, Yoshida H, Araki H,Shimotohno K. Exploiting cis-acting replication elements to direct hepatitis C virus-dependent transgene expression[J]. J Virol, 2005; 79(10):5923-32.
16 Astier-Gin T, Bellecave P, Litvak S, Ventura M. Template requirements and binding of hepatitis C virus NS5B polymerase during in vitro RNA synthesis from the 3'-end of virus minus-strand RNA[J]. FEBS J, 2005; 272(15):3872-86.
17 Dansako H, Ikeda M,Kato N. Limited suppression of the interferon-beta production by hepatitis C virus serine protease in cultured human hepatocytes[J]. FEBS J, 2007; 274(16):4161-76.
18 Diamantis I, Vafiadis I, Voskaridou E, Dellatetsima J, Jaggi N, Gyr K,Battegay M. Genotype distribution of hepatitis C virus infection in Greece: correlation with different risk factors and response to interferon therapy[J]. Eur J Gastroenterol Hepatol, 1998; 10(1):75-9.
19 中华医学会肝病学分会、传染病与寄生虫病学分会. 丙型肝炎防治指南. 中华医学杂志. 2004; 84(9): 775-780.
20 Glenn JS. Molecular virology of the hepatitis C virus: implication for novel therapies[J]. Clin Liver Dis, 2005; 9(3):353-69, v.
21 Shao SW, Wu WB, Yu JG, Zhao P,Qi ZT. [Antigenicity of hepatitis C virus F protein and serum prevalence of anti-F in HCV-infected patients][J]. Zhonghua Gan Zang Bing Za Zhi, 2006; 14(12):890-3.
22 Bartenschlager R, Sparacio S. Hepatitis C virus molecular clones and their replication capacity in vivo and in cell culture[J]. Virus Res, 2007; 127(2):195-207.
23 Zhang J, Yamada O, Ito T, Akiyama M, Hashimoto Y, Yoshida H, Makino R, Masago A, Uemura H,Araki H. A single nucleotide insertion in the 5'-untranslated region of hepatitis C virus leads to enhanced cap-independent translation[J]. Virology, 1999; 261(2):263-70.
24 Luo G, Xin S, Cai Z. Role of the 5'-proximal stem-loop structure of the 5' untranslated region in replication and translation of hepatitis C virus RNA[J]. J Virol, 2003; 77(5):3312-8.
25 Collier AJ, Gallego J, Klinck R, Cole PT, Harris SJ, Harrison GP, Aboul-Ela F, Varani G,Walker S. A conserved RNA structure within the HCV IRES eIF3-binding site[J]. Nat Struct Biol, 2002; 9(5):375-80.
26 Masao Honda, Michael R. Beard, Li-Hua Ping, Stanley M. Lemon. A Phylogenetically Conserved Stem-Loop Structure at the 5' Border of the Internal Ribosome Entry Site of Hepatitis C Virus Is Required for Cap-Independent Viral Translation [J]. J. Virol, 1999; 73:1165-1174.
27 Jubin R. Hepatitis C IRES: translating translation into a therapeutic target[J]. Curr Opin Mol Ther, 2001; 3(3):278-87.
28 Kim CS, Seol SK, Song OK, Park JH,Jang SK. An RNA-binding protein, hnRNP A1, and a scaffold protein, septin 6, facilitate hepatitis C virus replication[J]. J Virol, 2007; 81(8):3852-65.
29 Tischendorf JJ, Beger C, Korf M, Manns MP,Kruger M. Polypyrimidine tract-binding protein (PTB) inhibits Hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation, but does not affect HCV replication[J]. Arch Virol, 2004; 149(10):1955-70.
30 Honda M, Shimazaki T,Kaneko S. La protein is a potent regulator of replication of hepatitis C virus in patients with chronic hepatitis C through internal ribosomal entry site-directed translation[J]. Gastroenterology, 2005; 128(2):449-62.
31 Yanagi M, St Claire M, Emerson SU, Purcell RH, Bukh J. In vivo analysis of the 3' untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone[J]. Proc Natl Acad Sci U S A, 1999; 96(5):2291-5.
32 Scholle F, Li K, Bodola F, Ikeda M, Luxon BA, Lemon SM. Virus-host cell interactions during hepatitis C virus RNA replication: impact of polyprotein expression on the cellular transcriptome and cell cycle association with viral RNA synthesis[J]. J Virol, 2004; 78(3):1513-24.
33 Friebe P, Bartenschlager R. Genetic analysis of sequences in the 3' nontranslated region of hepatitis C virus that are important for RNA replication[J]. J Virol, 2002; 76(11):5326-38.
34 Lee H, Shin H, Wimmer E, Paul AV. cis-acting RNA signals in the NS5B C-terminal coding sequence of the hepatitis C virus genome[J]. J Virol, 2004;
78(20):10865-77.
35 Tuplin A, Evans DJ, Simmonds P. Detailed mapping of RNA secondary structures in core and NS5B-encoding region sequences of hepatitis C virus by RNase cleavage and novel bioinformatic prediction methods[J]. J Gen Virol, 2004; 85(Pt 10):3037-47.
36 Zhang J, Yamada O, Yoshida H, Sakamoto T, Araki H,Shimotohno K. Helper virus-independent trans-replication of hepatitis C virus-derived minigenome[J]. Biochem Biophys Res Commun, 2007; 352(1):170-6.
37 Bartenschlager R, Lohmann V. Replication of hepatitis C virus[J]. J Gen Virol, 2000; 81(Pt 7):1631-48.
38 Gontarek RR, Gutshall LL, Herold KM, Tsai J, Sathe GM, Mao J, Prescott C,Del Vecchio AM. hnRNP C and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'NTR of the HCV RNA genome[J]. Nucleic Acids Res, 1999; 27(6):1457-63.
39 Chung RT, Kaplan LM. Heterogeneous nuclear ribonucleoprotein I (hnRNP-I/PTB) selectively binds the conserved 3' terminus of hepatitis C viral RNA[J]. Biochem Biophys Res Commun, 1999; 254(2):351-62.
40 Lesburg CA, Cable MB, Ferrari E, Hong Z, Mannarino AF, Weber PC. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site[J]. Nat Struct Biol, 1999; 6(10):937-43.
41 Dutartre H, Boretto J, Guillemot JC, Canard B. A relaxed discrimination of
2'-O-methyl-GTP relative to GTP between de novo and Elongative RNA synthesis by the hepatitis C RNA-dependent RNA polymerase NS5B[J]. J Biol Chem, 2005; 280(8):6359-68.
42 Lee KJ, Choi J, Ou JH, Lai MM. The C-terminal transmembrane domain of hepatitis C virus (HCV) RNA polymerase is essential for HCV replication in vivo[J]. J Virol, 2004; 78(7):3797-802.
43 Kim JL, Morgenstern KA, Lin C, Fox T, Dwyer MD, Landro JA, Chambers SP,Markland W, Lepre CA, O'Malley ET, Harbeson SL, Rice CM, Murcko MA, Caron PR,Thomson JA. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide[J]. Cell, 1996; 87(2):343-55.
44 Zhang T, Lin RT, Li Y, Douglas SD, Maxcey C, Ho C, Lai JP, Wang YJ, Wan Q,Ho WZ. Hepatitis C virus inhibits intracellular interferon alpha expression in human hepatic cell lines[J]. Hepatology, 2005; 42(4):819-27.
45 Kasprzak A, Adamek A. Role of hepatitis C virus proteins (C, NS3, NS5A) in hepatic oncogenesis[J]. Hepatol Res, 2008; 38(1):1-26.
46 Zhang C, Cai Z, Kim YC, Kumar R, Yuan F, Shi PY, Kao C,Luo G. Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase[J]. J Virol, 2005; 79(14):8687-97.
47 Lindenbach BD, Pragai BM, Montserret R, Beran RK, Pyle AM, Penin F,Rice CM. The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication[J]. J Virol, 2007; 81(17):8905-18.
48 Elazar M, Liu P, Rice CM, Glenn JS. An N-terminal amphipathic helix in hepatitis C virus (HCV) NS4B mediates membrane association, correct localization of replication complex proteins, and HCV RNA replication[J]. J Virol, 2004; 78(20):11393-400.
49 Lundin M, Lindstrom H, Gronwall C, Persson MA. Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein[J]. J Gen Virol, 2006; 87(Pt 11):3263-72.
50 Neddermann P, Quintavalle M, Di Pietro C, Clementi A, Cerretani M, Altamura S, Bartholomew L,De Francesco R. Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture[J]. J Virol, 2004; 78(23):13306-14.
51 Szabo G. Hepatitis C virus NS5A protein--a master regulator?[J]. Gastroenterology, 2006; 130(3):995-9.
52 Liang Y, Ye H, Kang CB,Yoon HS. Domain 2 of nonstructural protein 5A (NS5A) of hepatitis C virus is natively unfolded[J]. Biochemistry, 2007; 46(41):11550-8.
53 Tellinghuisen TL, Foss KL, Treadaway JC, Rice CM. Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5Aprotein[J]. J Virol, 2008; 82(3):1073-83.
54 Xue Q, Ding H, Liu M, Zhao P, Gao J, Ren H, Liu Y,Qi ZT. Inhibition of hepatitis C virus replication and expression by small interfering RNA targeting host cellular genes[J]. Arch Virol, 2007; 152(5):955-62.
55 Wang C, Gale M Jr, Keller BC, Huang H, Brown MS, Goldstein JL, Ye J. Identification of FBL2 as a geranylgeranylated cellular protein required for hepatitis C virus RNA replication[J]. Mol Cell, 2005; 18(4):425-34.
56 Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y,Shimotohno K. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase[J]. Mol Cell, 2005; 19(1):111-22.
57 Yi M, Lemon SM. Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells[J]. J Virol, 2004; 78(15):7904-15.
58 Evans MJ, Rice CM,Goff SP. Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication[J]. Proc Natl Acad Sci U S A, 2004; 101(35):13038-43.
59 Salonen A, Ahola T, Kaariainen L. Viral RNA replication in association with cellular membranes[J]. Curr Top Microbiol Immunol, 2005; 285:139-73.
60 Zheng Y, Gao B, Ye L, Kong L, Jing W, Yang X, Wu Z,Ye L. Hepatitis C virus non-structural protein NS4B can modulate an unfolded protein response[J]. J Microbiol, 2005; 43(6):529-36.
61 Moradpour D, Gosert R, Egger D, Penin F, Blum HE,Bienz K. Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex[J]. Antiviral Res, 2003; 60(2):103-9.
62 Qadri I, Iwahashi M, Capasso JM, Hopken MW, Flores S, Schaack J,Simon FR. Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP-1[J]. Biochem J, 2004; 378(Pt 3):919-28.
63 Bartenschlager R, Lohmann V. Replication of the hepatitis C virus[J]. Baillieres Best Pract Res Clin Gastroenterol, 2000; 14(2):241-54.
64 Yi M, Lemon SM. 3' nontranslated RNA signals required for replication of hepatitis C virus RNA[J]. J Virol, 2003; 77(6):3557-68.
65 Luo G, Hamatake RK, Mathis DM, Racela J, Rigat KL, Lemm J,Colonno RJ. Denovo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus[J]. J Virol, 2000; 74(2):851-63.
66 Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW,Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome[J]. Science, 1989; 244(4902):359-62.
67 Yoo BJ, Selby MJ, Choe J, Suh BS, Choi SH, Joh JS, Nuovo GJ, Lee HS, Houghton M,Han JH. Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV[J]. J Virol, 1995; 69(1):32-8.
68 Ishii N, Watashi K, Hishiki T, Goto K, Inoue D, Hijikata M, Wakita T, Kato N,Shimotohno K. Diverse effects of cyclosporine on hepatitis C virus strain replication[J]. J Virol, 2006; 80(9):4510-20.
69 Cornu TI, de la Torre JC. RING finger Z protein of lymphocytic choriomeningitis virus (LCMV) inhibits transcription and RNA replication of an LCMV S-segment minigenome[J]. J Virol, 2001; 75(19):9415-26.
70 Ikegami T, Peters CJ, Makino S. Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system[J]. J Virol, 2005; 79(9):5606-15.
71 You S, Padmanabhan R. A novel in vitro replication system for Dengue virus. Initiation of RNA synthesis at the 3'-end of exogenous viral RNA templates requires 5'- and 3'-terminal complementary sequence motifs of the viral RNA[J]. J Biol Chem, 1999; 274(47):33714-22.
72 Strauss DM, Glustrom LW, Wuttke DS. Towards an understanding of the poliovirus replication complex: the solution structure of the soluble domain of the poliovirus 3A protein[J]. J Mol Biol, 2003; 330(2):225-34.
73 Fujimura T, Esteban R. Interactions of the RNA polymerase with the viral genome at the 5'- and 3'-ends contribute to 20S RNA narnavirus persistence in yeast[J]. J Biol Chem, 2007; 282(26):19011-9.
74 Dumas E, Masante C, Astier-Gin T, Lapaillerie D, Ventura M. The hepatitis C virus minigenome: a new cellular model for studying viral replication[J]. J Virol Methods, 2007; 142(1-2):59-66.
75 曲建慧, 张玲霞. 干扰素的分子生物学机制研究进展[J]. 国际流行病学传染病学杂志.2006;33(1):38-41.
76 徐 春 娥 , 陈 薇 . 干 扰 素 系 统 与 病 毒 的 相 互 作 用 [J]. 中 国 生 物 工 程 杂志.2006;26(4):95-100.
77 Pestka S. Purification and cloning of interferon alpha[J]. Curr Top Microbiol Immunol, 2007; 316:23-37.
78 Markowitz CE. Interferon-beta: mechanism of action and dosing issues[J]. Neurology, 2007; 68(24 Suppl 4):S8-11.
79 Powell HJ, Cong Y, Yu JJ, Guentzel MN, Berton MT, Klose KE, Murthy AK,Arulanandam BP. CD4(+) T cells are required during priming but not the effector phase of antibody-mediated IFN-gamma-dependent protective immunity against pulmonary Francisella novicida infection[J]. Immunol Cell Biol, 2008.
80 宋仕玲.干扰素抵抗与 JAK/STAT 信号通道系统[J].国外医学.病毒学分册.2004; 11(2):40-43.
81 卢年芳 . 干扰素抗病毒作用的机制 [J]. 国外医学 . 流行病学 . 传染病学分册.2003;30(4):226-228.
82 Iacopino F, Ferrandina G, Scambia G, Benedetti-Panici P, Mancuso S,Sica G. Interferons inhibit EGF-stimulated cell growth and reduce EGF binding in human breast cancer cells[J]. Anticancer Res, 1996; 16(4A):1919-24.
83 Ferrantini M, Capone I, Belardelli F. Interferon-alpha and cancer: mechanisms of action and new perspectives of clinical use[J]. Biochimie, 2007; 89(6-7):884-93.
84 Tong MJ, Reddy KR, Lee WM, Pockros PJ, Hoefs JC, Keeffe EB, Hollinger FB, Hathcote EJ, White H, Foust RT, Jensen DM, Krawitt EL, Fromm H, Black M, Blatt LM, Klein M,Lubina J. Treatment of chronic hepatitis C with consensus interferon: a multicenter, randomized, controlled trial. Consensus Interferon Study Group[J]. Hepatology, 1997; 26(3):747-54.
85 El-Zayadi AR, Attia M, Barakat EM, Badran HM, Hamdy H, El-Tawil A, El-Nakeeb A, Selim O,Saied A. Response of hepatitis C genotype-4 naive patients to 24 weeks of Peg-interferon-alpha2b/ribavirin or induction-dose interferon-alpha2b/ribavirin/amantadine: a non-randomized controlled study[J]. Am J Gastroenterol, 2005; 100(11):2447-52.
86 Cornberg M, Huppe D, Wiegand J, Felten G, Wedemeyer H, Manns MP. [Treatment of chronic hepatitis C with PEG-interferon alpha-2b and ribavirin: 24 weeks oftherapy are sufficient for HCV genotype 2 and 3][J]. Z Gastroenterol, 2003; 41(6):517-22.
87 Cornberg M, Wedemeyer H, Manns MP. Treatment of chronic hepatitis C with PEGylated interferon and ribavirin[J]. Curr Gastroenterol Rep, 2002; 4(1):23-30.
88 Legrand-Abravanel F, Nicot F, Boulestin A, Sandres-Saune K, Vinel JP, Alric L,Izopet J. Pegylated interferon and ribavirin therapy for chronic hepatitis C virus genotype 4 infection[J]. J Med Virol, 2005; 77(1):66-9.
89 Moreno-Otero R, Trapero-Marugan M, Gomez-Dominguez E, Garcia-Buey L,Moreno-Monteagudo JA. Is interferon-beta an alternative treatment for chronic hepatitis C?[J]. World J Gastroenterol, 2006; 12(17):2730-6.
90 Chevaliez S, Pawlotsky JM. Interferon-based therapy of hepatitis C[J]. Adv Drug Deliv Rev, 2007; 59(12):1222-41.
91 程勇前, 聂青和. 丙型肝炎病毒的干扰素抵抗机制研究进展[J]. 肝脏.2007; 12(4):303-305.
92 Miller K, McArdle S, Gale MJ Jr, Geller DA, Tenoever B, Hiscott J, Gretch DR,Polyak SJ. Effects of the hepatitis C virus core protein on innate cellular defense pathways[J]. J Interferon Cytokine Res, 2004; 24(7):391-402.
93 Goyal A, Hofmann WP, Hermann E, Traver S, Hissar SS, Arora N, Blum HE, Zeuzem S, Sarrazin C,Sarin SK. The hepatitis C virus NS5A protein and response to interferon alpha: mutational analyses in patients with chronic HCV genotype 3a infection from India[J]. Med Microbiol Immunol, 2007; 196(1):11-21.
94 Hung CH, Lee CM, Lu SN, Lee JF, Wang JH, Tung HD, Chen TM, Hu TH, Chen WJ,Changchien CS. Mutations in the NS5A and E2-PePHD region of hepatitis C virus type 1b and correlation with the response to combination therapy with interferon and ribavirin[J]. J Viral Hepat, 2003; 10(2):87-94.
95 Breiman A, Grandvaux N, Lin R, Ottone C, Akira S, Yoneyama M, Fujita T, Hiscott J,Meurs EF. Inhibition of RIG-I-dependent signaling to the interferon pathway during hepatitis C virus expression and restoration of signaling by IKKepsilon[J]. J Virol, 2005; 79(7):3969-78.
96 Li XD, Sun L, Seth RB, Pineda G, Chen ZJ. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity[J]. Proc Natl Acad Sci U S A, 2005; 102(49):17717-22.
97 Bode JG, Brenndorfer ED, Haussinger D. Subversion of innate host antiviral strategies by the hepatitis C virus[J]. Arch Biochem Biophys, 2007; 462(2):254-65.
98 杨敬. HCV NS3 蛋白的表达、纯化、单克隆抗体制备及其结合区域的分析[D]. 徐志凯 第四军医大学; 2005:28-46.
99 J.萨姆布鲁克 , D .W.拉塞尔. 分子克隆实验指南. 第三版 [M]. 北京:科学出版社, 2002:1296-1298.
100 Frolov I, McBride MS,Rice CM. cis-acting RNA elements required for replication of bovine viral diarrhea virus-hepatitis C virus 5' nontranslated region chimeras[J]. RNA, 1998; 4(11):1418-35.
101 Jang SJ, Wang LF, Radkowski M, Rakela J,Laskus T. Differences between hepatitis C virus 5' untranslated region quasispecies in serum and liver[J]. J Gen Virol, 1999; 80 (Pt 3):711-6.
102 Rijnbrand RC, Lemon SM. Internal ribosome entry site-mediated translation in hepatitis C virus replication[J]. Curr Top Microbiol Immunol, 2000; 242:85-116.
103 Wood J, Frederickson RM, Fields S,Patel AH. Hepatitis C virus 3'X region interacts with human ribosomal proteins[J]. J Virol, 2001; 75(3):1348-58.
104 Freiberg A, Dolores LK, Enterlein S,Flick R. Establishment and characterization of plasmid-driven minigenome rescue systems for Nipah virus: RNA polymerase I- and T7-catalyzed generation of functional paramyxoviral RNA[J]. Virology, 2008; 370(1):33-44.
105 Groseth A, Feldmann H, Theriault S, Mehmetoglu G,Flick R. RNA polymerase I-driven minigenome system for Ebola viruses[J]. J Virol, 2005; 79(7):4425-33.
106 Johnson RF, McCarthy SE, Godlewski PJ, Harty RN. Ebola virus VP35-VP40 interaction is sufficient for packaging 3E-5E minigenome RNA into virus-like particles[J]. J Virol, 2006; 80(11):5135-44.
107 Yi M, Lemon SM. Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication[J]. RNA, 2003; 9(3):331-45.
108 Schuster C, Isel C, Imbert I, Ehresmann C, Marquet R,Kieny MP. Secondary structure of the 3' terminus of hepatitis C virus minus-strand RNA[J]. J Virol, 2002; 76(16):8058-68.
109 Smith RM, Walton CM, Wu CH,Wu GY. Secondary structure and hybridizationaccessibility of hepatitis C virus 3'-terminal sequences[J]. J Virol, 2002; 76(19):9563-74.
110 金伯泉. 细胞和分子免疫学实验技术[M]. 西安:第四军医大学出版社,2002: 61-64.
111 Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, Liu CC, Maruyama T, Hynes RO, Burton DR, McKeating JA,Rice CM. Complete replication of hepatitis C virus in cell culture[J]. Science, 2005; 309(5734):623-6.
112 Craxi A, Laffi G, Zignego AL. Hepatitis C virus (HCV) infection: A systemic disease[J]. Mol Aspects Med, 2008; 29(1-2):85-95.
113 Holland-Staley CA, Kovari LC, Golenberg EM, Pobursky KJ, Mayers DL. Genetic diversity and response to IFN of the NS3 protease gene from clinical strains of the hepatitis C virus[J]. Arch Virol, 2002; 147(7):1385-406.
114 De Francesco R, Tomei L, Altamura S, Summa V,Migliaccio G. Approaching a new era for hepatitis C virus therapy: inhibitors of the NS3-4A serine protease and the NS5B RNA-dependent RNA polymerase[J]. Antiviral Res, 2003; 58(1):1-16.
115 Thomas A, Laxton C, Rodman J, Myangar N, Horscroft N,Parkinson T. Investigating Toll-like receptor agonists for potential to treat hepatitis C virus infection[J]. Antimicrob Agents Chemother, 2007; 51(8):2969-78.
116 Weber F, Kochs G, Haller O. Inverse interference: how viruses fight the interferon system[J]. Viral Immunol, 2004; 17(4):498-515.
117 Dupuis S, Jouanguy E, Al-Hajjar S, Fieschi C, Al-Mohsen IZ, Al-Jumaah S, Yang K, Chapgier A, Eidenschenk C, Eid P, Al Ghonaium A, Tufenkeji H, Frayha H, Al-Gazlan S, Al-Rayes H, Schreiber RD, Gresser I,Casanova JL. Impaired response to interferon-alpha/beta and lethal viral disease in human STAT1 deficiency[J]. Nat Genet, 2003; 33(3):388-91.
118 Ferreon JC, Ferreon AC, Li K, Lemon SM. Molecular determinants of TRIF proteolysis mediated by the hepatitis C virus NS3/4A protease[J]. J Biol Chem, 2005; 280(21):20483-92.
119 Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, Bartenschlager R,Tschopp J. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus[J]. Nature, 2005; 437(7062):1167-72.
120 Hiscott J. Triggering the innate antiviral response through IRF-3 activation[J]. JBiol Chem, 2007; 282(21):15325-9.
121 Sato M, Suemori H, Hata N, Asagiri M, Ogasawara K, Nakao K, Nakaya T, Katsuki M, Noguchi S, Tanaka N,Taniguchi T. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction[J]. Immunity, 2000; 13(4):539-48.
122 Marie I, Durbin JE, Levy DE. Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7[J]. EMBO J, 1998; 17(22):6660-9.
123 Erlandsson L, Blumenthal R, Eloranta ML, Engel H, Alm G, Weiss S,Leanderson T. Interferon-beta is required for interferon-alpha production in mouse fibroblasts[J]. Curr Biol, 1998; 8(4):223-6.
124 Bode JG, Ludwig S, Ehrhardt C, Albrecht U, Erhardt A, Schaper F, Heinrich PC,Haussinger D. IFN-alpha antagonistic activity of HCV core protein involves induction of suppressor of cytokine signaling-3[J]. FASEB J, 2003; 17(3):488-90.
125 Meurs EF, Breiman A. The interferon inducing pathways and the hepatitis C virus[J]. World J Gastroenterol, 2007; 13(17):2446-54.
126 Otsuka M, Kato N, Moriyama M, Taniguchi H, Wang Y, Dharel N, Kawabe T,Omata M. Interaction between the HCV NS3 protein and the host TBK1 protein leads to inhibition of cellular antiviral responses[J]. Hepatology, 2005; 41(5):1004-12.
127 Chan HL, Ren H, Chow WC,Wee T. Randomized trial of interferon beta-1a with or without ribavirin in Asian patients with chronic hepatitis C[J]. Hepatology, 2007; 46(2):315-23.
128 Festi D, Sandri L, Mazzella G, Roda E, Sacco T, Staniscia T, Capodicasa S, Vestito A,Colecchia A. Safety of interferon beta treatment for chronic HCV hepatitis[J]. World J Gastroenterol, 2004; 10(1):12-6.
129 Han Q, Liu Z, Kang W, Li H, Zhang L,Zhang N. Interferon Beta 1a versus Interferon Beta 1a plus Ribavirin for the Treatment of Chronic Hepatitis C in Chinese Patients: A Randomized, Placebo-Controlled Trial[J]. Dig Dis Sci, 2007.
130 Cai Z, Zhang C, Chang KS, Jiang J, Ahn BC, Wakita T, Liang TJ,Luo G. Robust production of infectious hepatitis C virus (HCV) from stably HCV cDNA-transfected human hepatoma cells[J]. J Virol, 2005; 79(22):13963-73.