【作者】 杨敬；

【导师】 徐志凯； 尹文；

【作者基本信息】 第四军医大学 ， 病原生物学， 2008， 博士

【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。更多还原

【Abstract】 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 therapyPEG-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.更多还原