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目的 构建丙型肝炎病毒(HCV)单顺反子复制子,研究其在Huh7.5和Huh7.1细胞中的复制功能,为研究HCV复制规律和抗病毒药物的筛选建立模型. 方法 用Quick change点突变方法删除pJ6JFH 1B1aRL质粒上的Core-E1-E2-p7-NS2片段(约3090 bp),得到△pJ6JFH1B1aRL,然后测序,选择序列正确的克隆,用AgeⅠ和AvrⅡ酶切回收目的片段约2280bp,同时用AgeⅠ和AvrⅡ酶切pSGRJFH1和其突变体质粒,回收载体片段.将目的片段和载体片段连接,构建由HCV-IRES启动的单顺反子复制子pSGRm-JFH IblaRL以及其变异突变体pSGRm-JFH 1b1aRLGND,用其RNA转染Huh7.5和Huh7.1细胞,研究复制子在细胞中的复制情况.结果 经过Quick change和多步克隆方法成功构建HCV-IRES启动的单顺反子复制子,复制子RNA转染Huh7.5和Huh7.1细胞72h后复制达高峰,96h复制开始下降,而对照的突变体从24h至96h均没有复制.结论 成功构建了HCV-IRES单顺反子复制子,转染Huh7.5和Huh7.1细胞后在不同时间均有复制.  相似文献   

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AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon,and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model.All sequences were computer-predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA.Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were trans...  相似文献   

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Multidrug resistance associated with the overexpression of ATP‐dependent binding cassette (ABC) proteins is widely accepted as an important cause of treatment failure in patients with neoplastic or infectious diseases. Some of them play also a pivotal role in detoxification processes. Herein, we investigated the effect of hepatitis C virus (HCV) replication and nonstructural 5A (NS5A) protein on the expression and functional activity of two ABC transport proteins: MDR1 and BCRP. RT‐quantitative real‐time polymerase chain reaction (qPCR) was carried out for mdr1 and bcrp mRNAs in both Huh7 cells expressing NS5A and Huh7.5 cells containing either full‐length‐ or subgenomic‐HCV replicon systems. The functional activity of these pumps was studied by performing a dye efflux assay with DiOC2 and Rhodamine 123. A dose‐dependent down‐regulation of mdr1 expression was documented in Huh7 cells expressing the NS5A protein, as well as in both replicon systems. In contrast, a significant increase of bcrp expression in both systems was recorded, which were in full agreement with the dye efflux assay results. These results warrant further in vivo studies in HCV patients with cholestasis and/or patients that are refractive to the pharmacotherapy due to the activity of these pumps.  相似文献   

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There is limited information about the role of hepatic stellate cells (HSC) in liver innate immunity against hepatitis C virus (HCV). We thus examined whether HSC can produce antiviral factors that inhibit HCV replication in human hepatocytes. HSC expressed functional Toll‐like receptor 3 (TLR‐3), which could be activated by its ligand, polyinosine‐polycytidylic acid (poly I:C), leading to the induction of interferon‐λ (IFN‐λ) at both mRNA and protein levels. TLR‐3 signalling of HSC also induced the expression of IFN regulatory factor 7 (IRF‐7), a key regulator of IFN signalling pathway. When HCV JFH‐1‐infected Huh7 cells were co‐cultured with HSC activated with poly I:C or incubated in media conditioned with supernatant (SN) from poly I:C‐activated HSC, HCV replication was significantly suppressed. This HSC SN action on HCV inhibition was mediated through IFN‐λ, which was evidenced by the observation that antibody to IFN‐λ receptors could neutralize the HSC‐mediated anti‐HCV effect. The role of IFN‐λ in HSC‐mediated anti‐HCV activity is further supported by the observation that HSC SN treatment induced the expression of IRF‐7 and IFN‐stimulated genes (ISGs), OAS‐1 and MxA in HCV‐infected Huh7 cells. These observations indicate that HSC may be a key regulatory bystander, participating in liver innate immunity against HCV infection using an IFN‐λ‐dependent mechanism.  相似文献   

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Hepatitis C virus (HCV) is a major human pathogen that causes serious illness, including acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Using a mass spectrometry-based proteomics approach, we have identified 175 proteins from a cell culture supernatant fraction containing the HCV genotype 2a (JFH1) virus, among which fatty acid synthase (FASN), the multifunctional enzyme catalyzing the de novo synthesis of fatty acids, was confirmed to be highly enriched. Subsequent studies showed that FASN expression increased in the human hepatoma cell line, Huh7, or its derivative, upon HCV infection. Blocking FASN activity by C75, a pharmacological FASN inhibitor, led to decreased HCV production. Reduction of FASN by RNA interference suppressed viral replication in both replicon and infection systems. Remarkably, FASN appeared to be selectively required for the expression of claudin-1, a tight junction protein that was recently identified as an entry coreceptor for HCV, but not for the expression of another HCV coreceptor, CD81. The decrease in Claudin-1 expression resulting from FASN inhibition was accompanied by a decrease in transepithelial electric resistance of Huh7 cells, implying a reduction in the relative tightness of the cell monolayer. Consequently, the entry of human immunodeficiency virus-HCV pseudotypes was significantly inhibited in C75-treated Huh7 cells. CONCLUSION: As far as we know, this is the first line of evidence that demonstrates that HCV infection directly induces FASN expression, and thus suggests a possible mechanism by which HCV infection alters the cellular lipid profile and causes diseases such as steatosis.  相似文献   

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目的 构建能产生较高效价重组病毒的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系统.方法 利用PCR技术在HCV NS5A C端插入海肾萤光素酶( Renilla Luciferase,Rluc)报告基因,并引入能提高HCV效价的突变,酶切鉴定重组基因序列构建成功后,转染入...  相似文献   

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Recently, microdomains of organelle membranes rich in sphingomyelin and cholesterol (called "lipid rafts") have been considered to act as a scaffold for the hepatitis C virus (HCV) replication complex. Using the HCV cell culture system, we investigated the effect of myriocin, a sphingomyelin synthesis inhibitor, on HCV replication. We also investigated the combined effect of myriocin with interferon (IFN) and myriocin with simvastatin. Myriocin suppressed replication of both a genotype 1b subgenomic HCV replicon (Huh7/Rep-Feo) and genotype 2a infectious HCV (JFH-1 HCV) in a dose-dependent manner (for subgenomic HCV-1b, maximum of 79% at 1000 nmol/L; for genomic HCV-2a, maximum of 40% at 1000 nmol/L). Combination treatment with myriocin and IFN or myriocin and simvastatin attenuated HCV RNA replication synergistically in Huh7/Rep-Feo cells. Our data demonstrate that the sphingomyelin synthesis inhibitor strongly suppresses replication of both the subgenomic HCV-1b replicon and the JFH-1 strain of genotype 2a infectious HCV, indicating that lipid metabolism could be a novel target for HCV therapy.  相似文献   

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Aim: Hepatitis C virus (HCV), which infects an estimated 170 million people worldwide, is a major cause of chronic liver disease. The current standard therapy for chronic hepatitis C is based on pegylated interferon (IFN)alpha in combination with ribavirin. However, the success rate remains at approximately 50%. Therefore, alternative agents are needed for the treatment of HCV infection. Methods: Using an HCV-1b subgenomic replicon cell culture system (Huh7/Rep-Feo), we found that griseofulvin, an oral antifungal agent, suppressed HCV-RNA replication and protein expression in a dose-dependent manner. We also found that griseofulvin suppressed the replication of infectious HCV JFH-1. A combination of IFNalpha and griseofulvin exhibited a synergistic inhibitory effect in Huh7/Rep-Feo cells. Results: We found that griseofulvin blocked the cell cycle at the G(2)/M phase in the HCV subgenomic replicon cells, but did not inhibit HCV internal ribosome entry site-dependent translation. Conclusion: Our results suggest that griseofulvin may represent a new approach to the development of a novel therapy for HCV infection.  相似文献   

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Advances in hepatitis C: What is coming in the next 5 years?   总被引:3,自引:0,他引:3  
Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Numerous advances have been made in the understanding of HCV replication, including detailed molecular characterization of its viral proteins and genomic RNA. The inability to grow HCV in cell culture had impeded the development of antiviral agents against this virus. To overcome this obstacle, a number of unique tools have been prepared, such as molecular clones that are infectious in the chimpanzee animal model of infection, and the development of a subgenomic replicon system in Huh7 cells. In addition, the major non-structural proteins have been crystallized, thus enabling rational drug design directed to these targets. Current developments in antiviral agents are reviewed in the context of these potential new viral targets for the future treatment of HCV in chronically infected individuals.  相似文献   

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Background and Aim:  We have reported previously that synthetic small interfering RNA (siRNA) and DNA-based siRNA expression vectors efficiently and specifically suppress hepatitis C virus (HCV) replication in vitro . In this study, we investigated the effects of the siRNA targeting HCV-RNA in vivo .
Methods:  We constructed recombinant retrovirus and adenovirus expressing short hairpin RNA (shRNA), and transfected into replicon-expressing cells in vitro and transgenic mice in vivo .
Results:  Retroviral transduction of Huh7 cells to express shRNA and subsequent transfection of an HCV replicon into the cells showed that the cells had acquired resistance to HCV replication. Infection of cells expressing the HCV replicon with an adenovirus expressing shRNA resulted in efficient vector delivery and expression of shRNA, leading to suppression of the replicon in the cells by ∼10−3. Intravenous delivery of the adenovirus expressing shRNA into transgenic mice that can be induced to express HCV structural proteins by the Cre/ lox P switching system resulted in specific suppression of virus protein synthesis in the liver.
Conclusion:  Taken together, our results support the feasibility of utilizing gene targeting therapy based on siRNA and/or shRNA expression to counteract HCV replication, which might prove valuable in the treatment of hepatitis C.  相似文献   

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Soon after the discovery of the hepatitis C virus (HCV), attention turned to the development of models whereby replication of the virus could be investigated. Among the HCV replication models developed, the HCV RNA replicon model and the newly discovered infectious cell culture systems have had an immediate impact on the study of HCV replication, and will continue to lead to important advances in our understanding of HCV replication. The aim of this study is to deal with developments in HCV replication models in a chronological order from the early 1990s to the recent infectious HCV cell culture systems.  相似文献   

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AIM: To address the effect of heat-shock protein 90(HSP90) inhibitors on the release of the hepatitis C virus(HCV), a cell culture-derived HCV(JFH1/HCVcc) from Huh-7 cells was examined.METHODS: We quantified both the intracellular and extracellular(culture medium) levels of the components(RNA and core) of JFH-1/HCVcc. The intracellular HCV RNA and core levels were determined after the JFH1/HCVcc-infected Huh-7 cells were treated with radicicol for 36 h. The extracellular HCV RNA and core protein levels were determined from the medium of the last 24 h of radicicol treatment. To determine the possible role of the HSP90 inhibitor in HCV release, we examined the effect of a combined application of low doses of the HSP90 inhibitor radicicol and the RNA replication inhibitors cyclosporin A(Cs A) or interferon. Finally, we statistically examined the combined effect of radicicoland Cs A using the combination index(CI) and graphical representation proposed by Chou and Talalay.RESULTS: We found that the HSP90 inhibitors had greater inhibitory effects on the HCV RNA and core protein levels measured in the medium than inside the cells. This inhibitory effect was observed in the presence of a low level of a known RNA replication inhibitor(Cs A or interferon-α). Treating the cells with a combination of radicicol and cyclosporin A for 24 h resulted in significant synergy(CI 1) that affected the release of both the viral RNA and the core protein. CONCLUSION: In addition to having an inhibitory effect on RNA replication, HSP90 inhibitors may interfere with an HCV replication step that occurs after the synthesis of viral RNA, such as assembly and release.  相似文献   

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Neuroinvasion of hepatitis C virus (HCV) is evidenced by recent clinical studies. In this study, serum‐derived HCV infection of astrocytes was analysed. Astrocytes were infected with HCV‐positive serum, and viral replication was assessed on different days postinfection. RT‐PCR was positive for HCV‐negative strand on 5th and 7th day postinfection in the HCV‐positive serum‐infected astrocytes. Real‐time RNA count in the cell culture supernatant was steadily increasing from day 3 to day 7. To reconfirm the viral replication, astrocytes were treated with an antiviral before the serum infection, and the antiviral treatment significantly reduced the viral RNA count. Further, the virus‐infected cells stained positive for the presence of viral core protein. Electron microscopy revealed the presence of HCV‐like particles in the astrocyte cell culture supernatant. In conclusion, serum‐derived HCV replicates in human astrocyte cell line SVG.  相似文献   

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Summary. Hepatitis C virus (HCV) infection results in several changes in mitochondrial function including increased reactive oxygen species (ROS) production and greater sensitivity to oxidant, Ca2+ and cytokine‐induced cell death. Prior studies in protein over‐expression systems have shown that this effect can be induced by the core protein, but other viral proteins and replication events may contribute as well. To evaluate the specific role of core protein in the context of viral replication and infection, we compared mitochondrial sensitivity in Huh7‐derived HCV replicon bearing cells with or without core protein expression with that of cells infected with the JFH1 virus strain. JFH1 infection increased hydrogen peroxide production and sensitized cells to oxidant‐induced loss of mitochondrial membrane potential and cell death. An identical phenomenon occurred in genome‐length replicons‐bearing cells but not in cells bearing the subgenomic replicons lacking core protein. Both cell death and mitochondrial depolarization were Ca2+ dependent and could be prevented by Ca2+ chelation. The difference in the mitochondrial response of the two replicon systems could be demonstrated even in isolated mitochondria derived from the two cell lines with the ‘genome‐length’ mitochondria displaying greater sensitivity to Ca2+‐induced cytochrome c release. In vitro incubation of ‘subgenomic’ mitochondria with core protein increased oxidant sensitivity to a level similar to that of mitochondria derived from cells bearing genome‐length replicons. These results indicate that increased mitochondrial ROS production and a reduced threshold for Ca2+ and ROS‐induced permeability transition is a characteristic of HCV infection. This phenomenon is a direct consequence of core protein interactions with mitochondria and is present whenever core is expressed, either in infection, full‐length replicon‐bearing cells, or in over‐expression systems.  相似文献   

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AIM: To develop a hepatocellular carcinoma (HCC) xenograft model for studying hepatitis C virus (HCV) replication in a mice, and antiviral treatment.METHODS: We developed a stable S3-green fluorescence protein (GFP) cell line that replicated the GFP-tagged HCV sub-genomic RNA derived from a highly efficient JFH1 virus. S3-GFP replicon cell line was injected subcutaneously into γ-irradiated SCID mice. We showed that the S3-GFP replicon cell line formed human HCC xenografts in SCID mice. Cells were isolated f...  相似文献   

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