干扰素刺激基因ISG20真核表达载体的构建及其抗丙型肝炎病毒复制作用的研究

目的：研究IFN—α抗HCV的作用机理及了解ISG20是否参与介导IFN-α对HCV的抑制作用。方法：用RT—PCR法及融合PCR法分别获得野生型及突变型ISG20cDNA，并将其克隆到真该表达载体pcDNA3．1上，转染含HCV复制子的Huh7细胞进行瞬时表达，通过Northern blot及Western blot分别检测HCVRNA及NS5A蛋白水平，研究表达ISG20对HCV复制子的影响。结果：构建的野生型及突变型ISG20真核表达载体在mRNA水平及蛋白水平的表达均得到证实，并且发现表达野生型ISG20对HCV复制子RNA有抑制作用。结论：成功克隆及表达了ISG20，并对其抗病毒作用进行了初步研究，提示ISG20可能介导IFN-α对HCV的抑制作用。     

病毒基因组耦联报告基因用于筛选适宜丙型肝炎病毒培养的亚细胞克隆株

目的 快捷筛选到适合丙型肝炎病毒(HCV)培养的细胞系.方法 将荧光素酶报告基因耦联到JFHlHCV基因组中,检测了多个人肝细胞系对HCV的易感性,进一步利用耦联荧光素酶的HCV复制子模型筛选并经过IFN-α处理更适宜HCV培养的Huh7亚细胞克隆株.结果 实验结果显示BEL7402、BEL7404、QSG7701、SMMC7721、QGY7701、QGY7703和HepG等肝细胞均不易感染HCV,仅限于Huh7细胞能被HCV感染.同时实验获得了更适宜HCV培养的Huh7亚细胞克隆株——Huh7G3.结论 在利用IFN-α处理细胞的过程中,通过检测荧光素酶活性,显示IFN-α对HCV亚基因组的复制抑制效果具有剂量依赖效应,研究同时显示:HCV复制子细胞能否抵抗HCV的超感染依赖于细胞内是否存在HCV RNA复制而不是其复制水平的高低.     

增强型绿色荧光蛋白标记的重组丙型肝炎病毒体外细胞培养系统的建立

目的 构建一个易检测且灵敏度高的丙型肝炎病毒(HCV)细胞感染模型,为HCV致病机制的研究和抗病毒药物的筛选提供一个有效的体外细胞培养体系.方法 利用重组PCR技术在HCV的非结构蛋白NS5A的C端引入优势突变点V2440L,然后在其RsrⅡ酶切位点插入增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因,基因测序及酶切鉴定重组基因序列构建成功后,体外转录获得RNA,然后转染入肝癌细胞系Huh7.5,用Western blot检测EGFP与NS5A融合蛋白,用细胞免疫荧光(IFA)检测病毒复制水平和EGFP表达情况.用RT-PCR检测转染细胞培养液不同时间点的HCV RNA水平.用IFN-α鉴定该系统用于抗丙型肝炎药物筛选的可行性.结果 在重组病毒JFH1-2440-EGFP RNA转染的细胞内可检测到EGFP的表达,EGFP表达水平与病毒复制水平一致.转染细胞的培养上清液能感染新的Huh7.5细胞,IFA结果显示转染后第9天培养上清液的病毒滴度为104 FFU/ml,提示JFH1-2440-EGFP RNA转染的细胞能释放有传染性的HCV病毒颗粒.RT-PCR检测结果显示转染细胞上清液中的HCV RNA在转染72 h后可达到3.06×105拷贝/ml,第9天可达到7.96×106拷贝/ml.EGFP的表达呈干扰素浓度依赖性.结论 构建的重组病毒HCVJFH1-2440-EGFP体外细胞培养系统具有经济、快速、敏感等优点,为HCV致病机制的研究和抗病毒药物的筛选提供了一个有效的工具.     

鸟苷结合蛋白1对柯萨奇病毒B3及乙型肝炎病毒体外介导的不同作用

目的构建人鸟苷结合蛋白1（hGBP-1）真核表达质粒，观察hGBP-1体外对柯萨奇病毒B3（CVB3）和乙型肝炎病毒（HBV）的抑制作用。方法长链RT-PCR扩增全长hGBP-1编码区基因，克隆到pCR2．1TA克隆载体，再亚克隆到pcDNA3．1（-）真核表达载体。体外转染HepG2细胞和Hela细胞，Western blot检测hGBP-1的表达。然后分别观察转染细胞中hGBP-1对HBV体外复制子pHBV1．3和CVB3的抑制作用。ELISA检测共转染HepG2细胞培养L清HBsAg、HBeAg水平；Southern blot检测细胞HBVDNA复制中间体。TCID50试验检测Hela细胞培养物中CVB3感染量。结果成功构建hGBP-1真核表达质粒，能在HepG2细胞和HeLa细胞进行高效表达。该质粒与pHBV1．3共转染HepG2细胞，不能抑制HBV复制，HBsAg、HBeAg及HBV DNA复制中间体水平与对照相比都无明显变化。转染质粒在HeLa细胞上对CVB3复制有明显的抑制作用，CVB3感染量显著降低，尤其在低剂量病毒攻击时能完全抑制CVB3复制。结论hGBP-1可能在IFN介导的抗CVB3中起重要作用，但不能抑制HBV的复制。     

丙型肝炎病毒阳性血清体外感染人肝细胞的研究

目的 研究丙型肝炎病毒(HCV)抗体(Ab)阴性,HCV-RNA阳性血清建立体外感染肝细胞模型.方法 HCV Ab阴性,HCV-RNA阳性的窗口期血清与人肝细胞共同培养,用反转录-聚合酶链反应(RT-PCR)、免疫荧光染色、Western blot、共聚焦显微镜和透射电镜等方法检测细胞内HCV核酸复制、蛋白质表达及超微结构改变.结果 细胞与病毒共同培养7～45 d,细胞内和/或培养上清中可间断检出HCV正、负链RNA;细胞浆内有HCV 核心和NS3抗原的表达;细胞超微结构有改变,并于感染后第24天时观察到类似病毒样颗粒.结论 窗口期血清中的HCV能在人肝细胞7701中复制一段时间.     

丙型肝炎病毒JFH-1株在细胞培养中的复制

1989年确认丙型肝炎病毒(HCV)为丙型肝炎病原体后,因其宿主范围仅限于黑猩猩和人,一直不能在常规培养细胞中复制,1999年Lohmann等[1]开发出HCV复制子技术,使HCV非结构区亚基因组RNA在Huh-7细胞中能够复制,成为研究HCV复制机制的有力工具.     

丙型肝炎病毒体外感染人肝癌细胞株HepG2的研究

目的：研究人肝癌细胞株HepG2体外对丙型肝炎病毒（hepatitis C virus,HCV)的易感性。方法：慢性丙型肝炎患者的血清与HepG2共同孵育后，以逆转录－聚合酶链反应、免疫组化法、原位杂交法分别检测细胞和（或）培养上清中的HCV正、负链RNA、HCV抗原表达和HCV RNA在细胞内的定位。结果：感染血清和细胞共同孵育后的第2－40天，从细胞内和培养上清中均可间断地检测出HCV正、负链HCV RNA，HCV NS3抗原在细胞内能稳定表达，原位杂交证实HCV RNA阳性物质多位于细胞质中。结论：HepG2细胞在体外不但对HCV易感，而且可以支持HCV体外复制。     

HCV NS5A基因在hepG2细胞中的表达以及对PI3K的影响

目的：探讨HCV—NS5A对PI3K表达的影响。方法：应用PCR技术从含有HCV全长开放阅读框的质粒中获得NS5A全长基因片段，利用基因重组技术将其克隆至真核表达载体pcDNA3．0（-）中。通过酶切、PCR及测序鉴定，NS5A基因已正确插入到pcDNA3．0（-）中，再利用脂质体转染HepG2细胞。结果：经RT—PCR及Western blot检测，HCV的NS5A基因在HepG2细胞中获得表达，而且在表达重组NS5A的转染HepG2细胞中，检测到PI3K蛋白的表达。结论：NS5A可在体外激活PI3K及其信号通路。     

HBV X基因对成人肝细胞HL-7702线粒体功能的影响

目的:研究HBV X基因与线粒体COXⅢ基因相互作用以及对线粒体功能的影响. 方法:将HBV X基因真核表达载体pCDNA3-X及空载体pCDNA3转染成人肝细胞HL-7702细胞, 经过2周G418筛选, 获得稳定表达的新细胞株, 分别命名为HL-7702/HBx和H-7702/pcDNA3, RT-PCR和Western blot方法证实HBV X基因在HL-7702/HBx细胞内的稳定表达.抽提细胞总RNA进行半定量RT-PCR及分离细胞线粒体进行Western blot检测COX Ⅲ表达水平变化, 并通过酶动力学方法检测线粒体细胞色素氧化酶活性.结果:重组质粒pcDNA3-X转染后经G418筛选, RT-PCR和Western blot分析结果显示HL-7702-HBx细胞能够稳定表达HBV X基因.RT-PCR和Western blot结果发现HBV X基因下调COX Ⅲ蛋白表达而不影响mRNA水平表达.酶动力学检测发现线粒体细胞色素C氧化酶活性降低.结论:HBV X基因通过转录后水平调节COX Ⅲ表达, HBx通过与COXⅢ相互作用抑制线粒体细胞色素氧化酶活性.     

Activation of the interferon-beta promoter during hepatitis C virus RNA replication

In this study we examined the impact of hepatitis C virus (HCV) RNA replication on the innate antiviral response of the host cell. Replication of an HCV subgenomic replicon stimulated the activation of the interferon (IFN)-beta promoter and the production of IFN in human hepatoma cells. Using a variety of functional assays, we found that HCV RNA replication induced the activation and DNA-binding activity of NFkappaB and interferon regulatory factor (IRF)-1. In addition, microscopy experiments revealed a higher frequency of cells containing the nuclear-localized, active form of IRF-3 in HCV replicon cultures versus control cultures. Consistent with these observations, cells harboring the HCV replicon exhibited high basal level expression of a subset of IFN-stimulated antiviral genes. Our results indicate that HCV RNA replication can stimulate cellular antiviral programs that contribute to the assembly and activation of the IFN-beta enhanceosome complex and initiation of the antiviral state. Stable HCV RNA replication in the face of the host antiviral response suggests that HCV may encode one or more proteins capable of overcoming specific antiviral processes, thereby supporting persistent infection.     

Replication of a novel subgenomic HCV genotype 1a replicon expressing a puromycin resistance gene in Huh-7 cells

Genotype 1a is a most prevalent genotype of hepatitis C virus in North America yet HCV replication has been studied predominantly with genotype 1b subgenomic replicons under neomycin selection in Huh-7 cells. Development of 1a-related dicistronic replicons under neo selection proved difficult and required either "conditioned" Huh-7 cells and/or chimeric genomes harboring pre-engineered adaptive mutations. We report the construction of a novel dicistronic genotype 1a(H77C) replicon expressing the puromycin N-acetyltransferase (PAC) gene as a selectable marker that, without prior introduction of adaptive mutations, allows establishment of puromycin-resistant Huh-7 colonies after transfection of naive Huh-7 cells. The large majority of HCV1a/PAC replicons did not reveal any adaptive mutations on short-term passage of Huh-7 cells. Continued passage led to mutations in the non-structural genes although these mutations did not significantly enhance replication of the original replicon. Transfection with total cellular RNA isolated from HCV1a/PAC replicon-containing cells led to a significant increase in colony-forming ability. The data identify PAC as an efficient selectable marker for studies of HCV replication, which may be useful with different genotypes in different host cell systems.     

Nonstructural protein 5A does not contribute to the resistance of hepatitis C virus replication to interferon alpha in cell culture

The hepatitis C virus (HCV) subgenomic replicon system was used to study a possible involvement of nonstructural protein 5A (NS5A) in the mechanisms of HCV resistance to interferon alpha (IFN-alpha). A series of chimeric HCV replicons was constructed. In these replicons, the NS5A gene in the backbone of the Con1 replicon was swapped by corresponding fragments obtained from four IFN-alpha responder and four IFN-alpha nonresponder patients that had been infected with the same HCV AD78 strain. Experiments with transfected Huh7 cells did not reveal significant differences in sensitivity of HCV RNA replication to IFN-alpha in cell clones, bearing chimeric Con1/AD78 replicons with NS5A sequences from IFN responders and nonresponders. Thus, these data provide no evidence that the NS5A protein contributes to the resistance of HCV replication to IFN-alpha.     

SYNCRIP (synaptotagmin-binding, cytoplasmic RNA-interacting protein) is a host factor involved in hepatitis C virus RNA replication

Hepatitis C virus (HCV) RNA replication requires viral nonstructural proteins as well as cellular factors. Recently, a cellular protein, synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP), also known as NSAP1, was found to bind HCV RNA and enhance HCV IRES-dependent translation. We investigate whether this protein is also involved in the HCV RNA replication. We found that SYNCRIP was associated with detergent-resistant membrane fractions and colocalized with newly-synthesized HCV RNA. Knock-down of SYNCRIP by siRNA significantly decreased the amount of HCV RNA in the cells containing a subgenomic replicon or a full-length viral RNA. Lastly, an in vitro replication assay after immunodepletion of SYNCRIP showed that SYNCRIP was directly involved in HCV RNA replication. These findings indicate that SYNCRIP has dual functions, participating in both RNA replication and translation in HCV life cycle.     

Cell culture-adaptive NS3 mutations required for the robust replication of genome-length hepatitis C virus RNA

We recently established a genome-length HCV RNA-replicating cell line (O strain of genotype 1b; here called O cells) using cured cells derived from sO cells, in which HCV subgenomic replicon RNA with an adaptive NS5A mutation (S2200R) is replicated. Characterization of the O cells revealed a second adaptive NS3 mutation (K1609E) required for genome-length HCV RNA replication. To clarify the role of adaptive mutation in genome-length HCV RNA replication, we newly established one and three kinds of genome-length HCV RNA-replicating cell lines possessing the cell background of sO and O cells, respectively, and found additional adaptive NS3 mutations (Q1112R, P1115L, and E1202G) required for the robust replication of genome-length HCV RNA. We further found that specific combinations of adaptive NS3 mutations drastically enhanced HCV RNA replication, regardless of the cell lines examined. These findings suggest that specific viral factors may affect the replication level of genome-length HCV RNA.     

Detection of anti-hepatitis C virus effects of interferon and ribavirin by a sensitive replicon system

Although combination therapy with interferon and ribavirin has improved the treatment for chronic hepatitis C virus (HCV) infection, the detailed anti-HCV effect of ribavirin in clinical concentrations remains uncertain. To detect the anti-HCV effect of ribavirin in lower concentrations, a sensitive and accurate assay system was developed using the reporter replicon system with an HCV genotype 2a subgenomic replicon (clone JFH-1) that exhibits robust replication in various cell lines. This reporter replicon was generated by introducing the luciferase reporter gene (instead of the neomycin resistance gene) into the subgenomic JFH-1 replicon. To assess the replication of this reporter replicon, luciferase activity was measured serially up to day 3 after transient transfection of Huh7 cells. The luciferase activity increased exponentially over the time course of the experiment. After adjustment for transfection efficiency and transfected cell viability, the impacts of interferon and ribavirin were determined. The administration of interferon and ribavirin resulted in dose-dependent suppression of replicon RNA replications. The 50% inhibitory concentration of interferon and ribavirin was 1.80 IU/ml and 3.70 microg/ml, respectively. In clinical concentrations, replications were reduced to 0.09% and 53.74% by interferon (100 IU/ml) and ribavirin (3 microg/ml), respectively. Combination use of ribavirin and interferon enhanced the anti-HCV effect of interferon by 1.46- to 1.62-fold. In conclusion, we developed an accurate and sensitive replicon system, and the antivirus effect of interferon and ribavirin was easily detected within their clinical concentrations by this replicon system. This system will provide a powerful tool for screening new antiviral compounds against HCV.     

Interferon-alpha and ribavirin resistance of Huh7 cells transfected with HCV subgenomic replicon

To model HCV resistance to a treatment with interferon-alpha (IFN-alpha) and ribavirin, Huh7 cells, bearing HCV subgenomic replicons, were treated with these compounds for several weeks. Analysis of the cell clones, which were able to support replication of HCV RNA in the presence of high concentrations of these antivirals, demonstrated that the observed resistance was due to changes in the host cell phenotype but not to the emergence of resistant variants of the replicon. No changes in the type I IFN receptor mRNA levels or sequences were found in IFN-treated cells suggesting that the observed resistance of replicon-containing cells to IFN-alpha was caused by modifications of some other cellular factors. The resistance of cells to high concentrations of ribavirin was due to a single point mutation in the NS5A gene of the HCV replicon, and was not associated with a defect in a ribavirin uptake. This mutation, however, did not change the sensitivity of the replicon itself to this antiviral.     

Curcumin inhibits HCV replication by induction of heme oxygenase-1 and suppression of AKT

Although hepatitis C virus (HCV) affects approximately 130-170 million people worldwide, no vaccines are available. HCV is an important cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma, leading to the need for liver transplantation. In this study, curcumin, a constituent used in traditional Chinese medicine, has been evaluated for its anti-HCV activity and mechanism, using a human hepatoma cell line containing the HCV genotype?1b subgenomic replicon. Below the concentration of 20% cytotoxicity, curcumin dose-dependently inhibited HCV replication by luciferase reporter gene assay, HCV RNA detection and HCV protein analysis. Under the same conditions, curcumin also dose-dependently induced heme oxygenase-1 with the highest induction at 24?h. Hemin, a heme oxygenase-1 inducer, also inhibited HCV protein expression in a dose-dependent manner. The knockdown of heme oxygenase-1 partially reversed the curcumin-inhibited HCV protein expression. In addition to the heme oxygenase-1 induction, signaling molecule activities of AKT, extracellular signal-regulated kinases (ERK) and nuclear factor-κB (NF-κB) were inhibited by curcumin. Using specific inhibitors of PI3K-AKT, MEK-ERK and NF-κB, the results suggested that only PI3K-AKT inhibition is positively involved in curcumin-inhibited HCV replication. Inhibition of ERK and NF-κB was likely to promote HCV protein expression. In summary, curcumin inhibited HCV replication by heme oxygenase-1 induction and AKT pathway inhibition. Although curcumin also inhibits ERK and NF-κB activities, it slightly increased the HCV protein expression. This result may provide information when curcumin is used as an adjuvant in anti-HCV therapy.