嵌合中国分离株基因的丙型肝炎病毒（1b／2a）细胞培养模型的建立

目的：建立嵌合中国分离株基因的丙型肝炎病毒（HCV）细胞培养模型。方法：利用3片段融合PCR的方法将中国HCV河北分离株（1b）的全长包膜蛋白基因引入JFH1（2a）株基因骨架,构建包膜蛋白基因区相互置换的嵌合HCV（1b／2a）全长基因组,经线性化后体外转录获得全长RNA,转染Huh7．5．1细胞系,用免疫荧光及蛋白印迹实验检测。结果：该RNA可以产生具有体外感染活性的嵌合HCV,且感染性可在共同培养的细胞间传播。结论：首次在国内建立了嵌合中国HCV分离株基因的HCV细胞培养体系。     

Generation of a recombinant reporter hepatitis C virus useful for the analyses of virus entry, intra-cellular replication and virion production

The lack of a culture system that efficiently produces progeny virus has hampered hepatitis C virus (HCV) research. Recently, the discovery of a novel HCV isolate JFH1 and its chimeric derivative J6/JFH1 has led to the development of an efficient virus productive culture system. To construct an easy monitoring system for the viral life cycle of HCV, we generated bicistronic luciferase reporter virus genomes based on the JFH1 and J6/JFH1 isolates, respectively. Transfection of the J6/JFH1-based reporter genome to Huh7.5 cells produced significantly greater levels of progeny virus than transfection of the JFH1 genome. Furthermore, the expression of dominant-negative Vps4, a key molecule of the endosomal sorting complex required for transport machinery, inhibited the virus production of JFH1, but not that of J6/JFH1. These results may account for the different abilities to produce progeny virus between JFH1 and J6/JFH1. Using the J6/JFH1/Luc system, we showed that the two polyanions heparin and polyvinyl sulfate decreased the infectivity of J6/JFH1/Luc virus in a dose-dependent manner. We also analyzed the function of microRNA on HCV replication and found that miR-34b could affect the replication of HCV. The reporter virus generated in this study will be useful for investigating the nature of the HCV life cycle and for identification of HCV inhibitors.     

Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses

The lack of an efficient system to produce hepatitis C virus (HCV) particles has impeded the analysis of the HCV life cycle. Recently, we along with others demonstrated that transfection of Huh7 hepatoma cells with a novel HCV isolate (JFH1) yields infectious viruses. To facilitate studies of HCV replication, we generated JFH1-based bicistronic luciferase reporter virus genomes. We found that RNA replication of the reporter construct was only slightly attenuated and that virus titers produced were only three- to fivefold lower compared to the parental virus, making these reporter viruses an ideal tool for quantitative analyses of HCV infections. To expand the scope of the system, we created two chimeric JFH1 luciferase reporter viruses with structural proteins from the Con1 (genotype 1b) and J6CF (genotype 2a) strains. Using these and the authentic JFH1 reporter viruses, we analyzed the early steps of the HCV life cycle. Our data show that the mode of virus entry is conserved between these isolates and involves CD81 as a key receptor for pH-dependent virus entry. Competition studies and time course experiments suggest that interactions of HCV with cell surface-resident glycosaminoglycans aid in efficient infection of Huh7 cells and that CD81 acts during a postattachment step. The reporter viruses described here should be instrumental for investigating the viral life cycle and for the development of HCV inhibitors.     

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.     

A recombinant replication-competent hepatitis C virus expressing Azami-Green, a bright green-emitting fluorescent protein, suitable for visualization of infected cells

The hepatitis C virus (HCV) production system consists of transfecting the human hepatoma cell line Huh7 with genomic HCV RNA (JFH1). To monitor HCV replication by fluorescence microscopy, we constructed a recombinant HCV clone expressing Azami-Green (mAG), a bright green fluorescent protein, by inserting the mAG gene into the nonstructural protein 5A (NS5A) gene; the resultant clone was designated JFH1-hmAG. The Huh-7.5.1 (a subclone of Huh7) cells transfected with JFH1-hmAG RNA were found to produce cytoplasmic NS5A-mAG, as readily visualized by fluorescence microscopy, and infectious virus, as assayed with the culture supernatant, indicating that JFH1-hmAG is infectious and replication-competent. Furthermore, the replication of this virus was inhibited by interferon alpha in a dose-dependent manner. These results suggest that JFH1-hmAG is useful for studying HCV life cycle and the mechanism of interferon’s anti-HCV action and for screening and testing new anti-HCV drugs.     

A Cell Culture Adapted HCV JFH1 Variant That Increases Viral Titers and Permits the Production of High Titer Infectious Chimeric Reporter Viruses

The unique properties of the hepatitis C virus (HCV) JFH1 isolate have made it possible to produce and study HCV in an infectious cell culture system. However, relatively low virus titers restrict some of the uses of this system and preparing infectious chimeric reporter viruses have been difficult. In this study, we report cell culture-adapted mutations in wild-type JFH1 yielding higher titers of infectious particles of both JFH1 and chimeric JFH1 viruses carrying reporter genes. Sequencing analyses determined that ten of the sixteen nonsynonymous mutations were in the NS5A region. Individual viruses harboring specific adaptive mutations were prepared and studied. The mutations in the NS5A region, which included all three domains, were most effective in increasing infectious virus production. Insertion of two reporter genes in JFH1 without the adaptive mutations ablated the production of infectious HCV particles. However, the introduction of specific adaptive mutations in the NS5A region permitted reporter genes, Renilla luciferase (Rluc) and EGFP, to be introduced into JHF1 to produce chimeric HCV-NS5A-EGFP and HCV-NS5A-Rluc reporter viruses at relatively high titers of infectious virus. The quantity of hyperphosphorylated NS5A (p58) was decreased in the adapted JFH1 compared wild type JFH1 and is likely be involved in increased production of infectious virus based on previous studies of p58. The JFH1-derived mutant viruses and chimeric reporter viruses described here provide new tools for studying HCV biology, identifying HCV antivirals, and enable new ways of engineering additional infectious chimeric viruses.     

Cell culture replication of a genotype 1b hepatitis C virus isolate cloned from a patient who underwent liver transplantation

The introduction of the genotype 2a isolate JFH1 was a major breakthrough in the field of hepatitis C virus (HCV), allowing researchers to study the complete life cycle of the virus in cell culture. However, fully competent culture systems encompassing the most therapeutically relevant HCV genotypes are still lacking, especially for the highly drug-resistant genotype 1b. For most isolated HCV clones, efficient replication in cultured hepatoma cells requires the introduction of replication-enhancing mutations. However, such mutations may interfere with viral assembly, as occurs in the case of the genotype 1b isolate Con1. In this study, we show that a clinical serum carrying a genotype 1b virus with an exceptionally high viral load was able to infect Huh7.5 cells. Similar to previous reports, inoculation of Huh7.5 cells by natural virus is very inefficient compared to infection by cell culture HCV. A consensus sequence of a new genotype 1b HCV isolate was cloned from the clinical serum (designated Barcelona HCV1), and then subjected to replication studies. This virus replicated poorly in a transient fashion in Huh7.5 cells after electroporation with in vitro transcribed RNA. Nonetheless, approximately 3 weeks post electroporation and thereafter, core protein-positive cells were detected by immunofluorescence. Surprisingly, small amounts of core protein were also measurable in the supernatant of electroporated cells, suggesting that HCV particles might be assembled and released. Our findings not only enhance the current method of cloning in vitro HCV replication-competent isolates, but also offer valuable insights for the realization of fully competent culture systems for HCV.     

A concerted action of hepatitis C virus p7 and nonstructural protein 2 regulates core localization at the endoplasmic reticulum and virus assembly

Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly.     

Replication-competent recombinant vesicular stomatitis virus encoding hepatitis C virus envelope proteins

Although in vitro replication of the hepatitis C virus (HCV) JFH1 clone of genotype 2a (HCVcc) has been developed, a robust cell culture system for the 1a and 1b genotypes, which are the most prevalent viruses in the world and resistant to interferon therapy, has not yet been established. As a surrogate virus system, pseudotype viruses transiently bearing HCV envelope proteins based on the vesicular stomatitis virus (VSV) and retrovirus have been developed. Here, we have developed a replication-competent recombinant VSV with a genome encoding unmodified HCV E1 and E2 proteins in place of the VSV envelope protein (HCVrv) in human cell lines. HCVrv and a pseudotype VSV bearing the unmodified HCV envelope proteins (HCVpv) generated in 293T or Huh7 cells exhibited high infectivity in Huh7 cells. Generation of infectious HCVrv was limited in some cell lines examined. Furthermore, HCVrv but not HCVpv was able to propagate and form foci in Huh7 cells. The infection of Huh7 cells with HCVpv and HCVrv was neutralized by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. The infectivity of HCVrv was inhibited by an endoplasmic reticulum alpha-glucosidase inhibitor, N-(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin. Focus formation of HCVrv in Huh7 cells was impaired by Nn-DNJ treatment. These results indicate that the HCVrv developed in this study can be used to study HCV envelope proteins with respect to not only the biological functions in the entry process but also their maturation step.     

Activity-based Protein Profiling Identifies a Host Enzyme,Carboxylesterase 1, Which Is Differentially Active during Hepatitis C Virus Replication

Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation.     

Development of mouse hepatocyte lines permissive for hepatitis C virus (HCV)

The lack of a suitable small animal model for the analysis of hepatitis C virus (HCV) infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. Human and mouse harbor a comparable system for antiviral type I interferon (IFN) induction and amplification, which regulates viral infection and replication. Using hepatocytes from knockout (ko) mice, we determined the critical step of the IFN-inducing/amplification pathways regulating HCV replication in mouse. The results infer that interferon-beta promoter stimulator (IPS-1) or interferon A receptor (IFNAR) were a crucial barrier to HCV replication in mouse hepatocytes. Although both IFNARko and IPS-1ko hepatocytes showed a reduced induction of type I interferons in response to viral infection, only IPS-1-/- cells circumvented cell death from HCV cytopathic effect and significantly improved J6JFH1 replication, suggesting IPS-1 to be a key player regulating HCV replication in mouse hepatocytes. We then established mouse hepatocyte lines lacking IPS-1 or IFNAR through immortalization with SV40T antigen. Expression of human (h)CD81 on these hepatocyte lines rendered both lines HCVcc-permissive. We also found that the chimeric J6JFH1 construct, having the structure region from J6 isolate enhanced HCV replication in mouse hepatocytes rather than the full length original JFH1 construct, a new finding that suggests the possible role of the HCV structural region in HCV replication. This is the first report on the entry and replication of HCV infectious particles in mouse hepatocytes. These mouse hepatocyte lines will facilitate establishing a mouse HCV infection model with multifarious applications.     

Baculovirus Mediated Production of Infectious Hepatitis C Virus in Human Hepatoma Cells Stably Expressing T7 RNA Polymerase

Although much has been learned about Hepatitis C virus (HCV), research progress has been hindered by the lack of a suitable cell culture system supporting its replication. Recently, a unique HCV strain JFH1 has been found to replicate efficiently in cell culture with production of infectious HCV (HCVcc). Baculovirus vectors were found to be efficient delivery vehicles and a HBV recombinant baculovirus/HepG2 system efficiently delivered the HBV genome into HepG2 resulting in HBV replication. In this study, we developed a recombinant baculovirus expression system to generate infectious HCV particles in hepatoma cell line Huh7-lunetT7 by using cDNA from the HCV JFH1 genotype. Results show that HCV positive, negative RNA strands and proteins were produced in this system. Furthermore, HCV particles were produced and secreted into the culture medium. Sucrose density gradient centrifugation of the culture medium revealed co-localization of HCV RNA and structural proteins in the fraction with a density of 1.08–1.13 g/ml. Electron microscopy (EM) showed viral particles approximately 55 nm in diameter, which could be recognized by anti-HCV E2 antibodies. Real-time RT-PCR detected that the level of HCV vRNA in the supernatant was 10⁷ copies/ml at 72 h post-transduction (hpt). In addition, the JFH1 virus produced by the recombinant baculovirus was confirmed to be infectious in vitro. In summary, this system provides a novel tool not only for the analysis of the replication and pathogenesis of HCV but also to screen for potential therapeutic targets.     

Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient

Subgenomic replicons of hepatitis C virus (HCV) have been widely used for studying HCV replication. Here, we report a new subgenomic replicon based on a strain isolated from a chronically infected patient. The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers. A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a. The subgenomic replicon of CCH was replication-deficient in cell culture, due to dysfunctions in NS3 and NS5B. Various JFH1/CCH chimeric replicons were constructed, and specific mutations were introduced. The introduction of mutations could partially restore the replication of chimeric replicons. A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.     

丙型肝炎病毒F蛋白缺失对病毒复制及感染性的影响

探索了F蛋白缺失及核心蛋白(Core)二级结构改变对丙型肝炎病毒(HCV)复制和感染性的影响．利用定点突变方法,将J6JFH1的核心基因引进5个终止密码子以中断F蛋白的表达,从而获得F蛋白缺失的病毒复制子J6JFH1/ΔF．体外制备RNA转录体,并电穿孔转染Huh7.5.1细胞,采用免疫荧光、实时荧光定量PCR方法以及病毒感染等方法,观察F蛋白缺失对病毒复制、蛋白质表达及转染细胞上清感染性病毒颗粒产生的影响．在此基础上,构建5个单一突变病毒体,对HCV核心蛋白进行二级结构分析,观察核心蛋白二级结构对HCV复制和翻译的影响．结果显示,转染48 h后,J6JFH1/ΔF与野生型J6JFH1相比,J6JFH1/ΔF转染阳性细胞数明显降低,细胞内HCV RNA 水平降低约95%,J6JFH1/ΔF转染后不同时间点细胞上清中HCV RNA拷贝数和病毒颗粒也明显降低．5个单一突变体不影响核心基因二级结构,病毒在细胞内复制和感染性与野生型水平一致．J6JFH1/ΔF所产生的改变可能是由于5处突变导致核心基因二级结构改变而造成的．结果说明,HCV F蛋白缺失不影响病毒的复制翻译及病毒颗粒的包装释放,核心蛋白二级结构的改变对病毒复制和翻译则产生较大影响．     

Focal Adhesion Kinase (FAK) Mediates the Induction of Pro-Oncogenic and Fibrogenic Phenotypes in Hepatitis C Virus (HCV)-Infected Cells

Hepatitis C Virus (HCV) infection is one of the most common etiological factors involved in fibrosis development and its progression to hepatocellular carcinoma (HCC). The pivotal role of hepatic stellate cells (HCSs) and extracellular matrix (ECM) in fibrogenesis is now certainly accepted, while the network of molecular interactions connecting HCV is emerging as a master regulator of several biological processes including proliferation, inflammation, cytoskeleton and ECM remodeling. In this study, the effects of HCV proteins expression on liver cancer cells, both pro-invasive and pro-fibrogenic phenotypes were explored. As a model of HCV infection, we used permissive Huh7.5.1 hepatoma cells infected with JFH1-derived ccHCV. Conditioned medium from these cells was used to stimulate LX-2 cells, a line of HSCs. We found that the HCV infection of Huh7.5.1 cells decreased adhesion, increased migration and caused the delocalization of alpha-actinin from plasma membrane to cytoplasm and increased expression levels of paxillin. The treatment of LX-2 cells, with conditioned medium from HCV-infected Huh7.5.1 cells, caused an increase in cell proliferation, expression of alpha-smooth muscle actin, hyaluronic acid release and apoptosis rate measured as cleaved poly ADP-ribose polymerase (PARP). These effects were accompanied in Huh7.5.1 cells by an HCV-dependent increasing of FAK activation that physically interacts with phosphorylated paxillin and alpha-actinin, and a rising of tumor necrosis factor alpha production/release. Silencing of FAK by siRNA reverted all effects of HCV infection, both those directed on Huh7.5.1 cells, and those indirect effects on the LX-2 cells. Moreover and interestingly, FAK inhibition enhances apoptosis in HCV-conditioned LX-2 cells. In conclusion, our findings demonstrate that HCV, through FAK activation, may promote cytoskeletal reorganization and a pro-oncogenic phenotype in hepatocyte-like cells, and a fibrogenic phenotype in HSCs.     

Efficient culture adaptation of hepatitis C virus recombinants with genotype-specific core-NS2 by using previously identified mutations

Hepatitis C virus (HCV) is an important cause of chronic liver disease, and interferon-based therapy cures only 40 to 80% of patients, depending on HCV genotype. Research was accelerated by genotype 2a (strain JFH1) infectious cell culture systems. We previously developed viable JFH1-based recombinants encoding the structural proteins (core, E1, E2), p7, and NS2 of prototype isolates of the seven major HCV genotypes; most recombinants required adaptive mutations. To enable genotype-, subtype-, and isolate-specific studies, we developed efficient core-NS2 recombinants from additional genotype 1a (HC-TN and DH6), 1b (DH1 and DH5), and 3a (DBN) isolates, using previously identified adaptive mutations. Introduction of mutations from isolates of the same subtype either led to immediate efficient virus production or accelerated culture adaptation. The DH6 and DH5 recombinants without introduced mutations did not adapt to culture. Universal adaptive effects of mutations in NS3 (Q1247L, I1312V, K1398Q, R1408W, and Q1496L) and NS5A (V2418L) were investigated for JFH1-based genotype 1 to 5 core-NS2 recombinants; several mutations conferred adaptation to H77C (1a), J4 (1b), S52 (3a), and SA13 (5a) but not to ED43 (4a). The mutations permitting robust virus production in Huh7.5 cells had no apparent effect on viral replication but allowed efficient assembly of intracellular infectious HCV for adapted novel or previously developed recombinants. In conclusion, previously identified mutations permitted development of novel HCV core-NS2 genotype recombinants. Mutations adapting several recombinants to culture were identified, but no mutations were universally adaptive across genotypes. This work provides tools for analysis of HCV genotype specificity and may promote the understanding of genotype-specific patterns in HCV disease and control.     

Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A

To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (Δ40) or 25-aa (Δ25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. Δ40 conferred efficient growth characteristics to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log(10) focus-forming units (FFU)/ml. 2a(J6) with Δ40 or Δ25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFPΔ40 acquired various deletions in EGFP, while 2a(J6)Δ40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5AΔ40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those of the nontagged recombinants, with peak infectivity titers of 4 to 5 log(10) FFU/ml. RLuc recombinants showed slightly less efficient growth characteristics, with peak infectivity titers up to 10-fold lower. Overall, the EGFP and RLuc recombinants were genetically stable after one viral passage. The usefulness of these reporter viruses for high-throughput fluorescence- and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa-2b) and that genotype 1 to 7 viruses all responded to treatment with p7 ion channel inhibitors.     

Superinfection exclusion in cells infected with hepatitis C virus

Superinfection exclusion is the ability of an established virus infection to interfere with infection by a second virus. In this study, we found that Huh-7.5 cells acutely infected with hepatitis C virus (HCV) genotype 2a (chimeric strain J6/JFH) and cells harboring HCV genotype 1a, 1b, or 2a full-length or subgenomic replicons were resistant to infection with cell culture-produced HCV (HCVcc). Replicon-containing cells became permissive for HCVcc infection after treatment with an HCV-specific protease inhibitor. With the exception of cells harboring a J6/JFH-FLneo replicon, infected or replicon-containing cells were permissive for HCV pseudoparticle (HCVpp) entry, demonstrating a postentry superinfection block downstream of primary translation. The surprising resistance of J6/JFH-FLneo replicon-containing cells to HCVpp infection suggested a defect in virus entry. This block was due to reduced expression of the HCV coreceptor CD81. Further analyses indicated that J6/JFH may be toxic for cells expressing high levels of CD81, thus selecting for a CD81(low) population. CD81 down regulation was not observed in acutely infected cells, suggesting that this may not be a general mechanism of HCV superinfection exclusion. Thus, HCV establishes superinfection exclusion at a postentry step, and this effect is reversible by treatment of infected cells with antiviral compounds.     

Hepatitis C virus infection induces apoptosis through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway

We previously reported that cells harboring the hepatitis C virus (HCV) RNA replicon as well as those expressing HCV NS3/4A exhibited increased sensitivity to suboptimal doses of apoptotic stimuli to undergo mitochondrion-mediated apoptosis (Y. Nomura-Takigawa, et al., J. Gen. Virol. 87:1935-1945, 2006). Little is known, however, about whether or not HCV infection induces apoptosis of the virus-infected cells. In this study, by using the chimeric J6/JFH1 strain of HCV genotype 2a, we demonstrated that HCV infection induced cell death in Huh7.5 cells. The cell death was associated with activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of DNA repair enzyme poly(ADP-ribose) polymerase, which is known to be an important substrate for activated caspase 3. These results suggest that HCV-induced cell death is, in fact, apoptosis. Moreover, HCV infection activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes. Concomitantly, HCV infection induced disruption of mitochondrial transmembrane potential, followed by mitochondrial swelling and release of cytochrome c from mitochondria. HCV infection also caused oxidative stress via increased production of mitochondrial superoxide. On the other hand, HCV infection did not mediate increased expression of glucose-regulated protein 78 (GRP78) or GRP94, which are known as endoplasmic reticulum (ER) stress-induced proteins; this result suggests that ER stress is not primarily involved in HCV-induced apoptosis in our experimental system. Taken together, our present results suggest that HCV infection induces apoptosis of the host cell through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway(s).