Correspondence Yoshiharu Matsuura, Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel: 81 6 6879 8340; fax: 81 6 6879 8269; email: firstname.lastname@example.org
Although a cell culture system for HCV JFH-1 strain has been developed, no robust cell culture system for serum-derived HCV is available. In this study, we have established systems capable of monitoring infection with JFH-1 virus based on specific reporter gene expression through proteolysis of chimeric transcription factors by HCV NS3/4A protease. We utilized a transcriptional factor Gal4-TBP that synergistically enhances transcription of the GAL4UAS and HIV-1 LTR tandem promoter with the Tat protein. We constructed chimeric Tat and Gal4-TBP transcription factors containing the HCV NS3/4A cleavage sequence of a mitochondria-resident IPS-1, but not those of the HCV polyprotein, and manipulated them to localize in the ER. Upon infection with JFH-1 virus, the transcription factors were efficiently cleaved by HCV protease, migrated into the nucleus and activated the reporter gene under the tandem promoter. Upon infection with JFH-1 virus, the Huh7OK1/TG-Luc cell line carrying the transcription factors and a luciferase gene under the promoter expressed luciferase in a dose-dependent manner in close correlation with HCV RNA replication. Huh7OK1/TG-LNGFR cells carrying the transcription factors and a cDNA of human low affinity nerve growth factor receptor under the promoter were selectively concentrated by immunomagnetic cell sorting upon infection with JFH-1 virus. These results indicate that the chimeric constructs bearing the ER-resident IPS-1 sequence are specifically recognized and efficiently cleaved by HCV protease and are harnessed for detection of HCV replication and for recovery of HCV-infected cells. This strategy may be applicable for the establishment of cell culture systems for the isolation of serum-derived HCV.
HCV infects more than 170 million people worldwide, and is a major cause of chronic liver disease, including hepatic steatosis, cirrhosis, and hepatocellular carcinoma (1). Combination therapy with pegylated IFN and ribavirin has achieved a sustained virological response in 50% of individuals infected with HCV genotype 1 (2). The establishment of cell culture systems using an HCV genotype 2a JFH-1 strain isolated from a patient with fulminant hepatitis C (3–5) and genotype 1a H77 and H77-S strains (6, 7) marked an epoch in the history of HCV study. However, reliable and robust cell culture systems capable of propagating serum-derived HCV from hepatitis C patients have not been established, hampering the development of effective anti-viral measures for HCV.
HCV is an enveloped virus possessing a single-stranded positive-sense RNA genome, and belongs to the genus Hepacivirus in the family Flaviviridae. The genome encodes a large precursor polyprotein composed of about 3000 amino acids. The viral polyprotein is processed by cellular and viral proteases, resulting in structural proteins (core, E1, E2), a putative viropore protein (p7), and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). HCV proteins recruit various host cellular proteins for efficient viral propagation (8–10) and disturb host immune systems to establish persistent and chronic infection (11, 12). Viral serine protease NS3 participates in the processing of HCV nonstructural proteins through a non-covalent interaction with cofactor NS4A (13, 14) and plays a crucial role in HCV replication. In addition, HCV NS3/4A protease plays an important role in the inhibition of both host innate immune response (11, 12) and apoptosis induced by HCV infection (15) through cleavage of IPS-1 (also called MAVS, Cardif or VISA), a mitochondria-resident adaptor molecule of retinoic acid-inducible gene-I.
The infectious titer and amounts of viral RNA and core protein of HCV have so far been determined by time- and cost-consuming methods such as focus-forming assay, qRT-PCR, and enzyme-linked immunosorbent assay, respectively. Although replicon RNA or infectious viruses incorporating various reporter genes in their viral genomes have been developed for high-throughput screening of anti-HCV compounds (16–18), strategies for manipulating viral genomes are not useful for establishing a cell culture system for serum-derived HCV from patients. To identify and purify HCV-infected cells without any genetic manipulation of the HCV genome, several groups have reported HCV NS3/4A-mediated trans-activation of reporter gene systems based on chimeric transcription factors containing the cleavage sequence of the NS3/4A protease derived from the HCV polyprotein (19–21).
In this study, we established a novel indicator cell culture system for HCV based on activation of the chimeric transcription factors containing the HCV NS3/4A cleavage sequence of IPS-1, but not of the HCV polyprotein, as reported in previous studies (19–21). Our system is sensitive and quantitative enough to detect JFH-1 virus propagation with a log-scale dynamic range. The chimeric transcription factors were efficiently cleaved by HCV protease and harnessed for detection of HCV replication and for recovery of HCV-infected cells. The strategy employed herein for establishing an indicator cell culture system may provide clues toward the establishment of cell culture systems for serum-derived HCV from hepatitis C patients.
MATERIALS AND METHODS
The cDNA encoding the N-terminally truncated form of the TBP (94 to 337 aa) and the C-terminal region of IPS-1 (503 to 540 aa; Fig. 1a) were amplified from a human fetal brain library (Clontech, Palo Alto, CA, USA) by PCR. The cDNA encoding the FLAG epitope tag was amplified from pCAG/FLAG-JEC-HA (22). These PCR fragments were introduced into pBIND vector (Promega, Madison, WI, USA) downstream of the cDNA encoding the Gal4 DB domain, and the resulting plasmid (which encodes a fusion protein consisting of a Gal4 DB domain, a truncated TBP, a FLAG epitope and the C-terminal region of IPS-1 [Gal4-TBP/IPS-1] in that order), was designated pBIND-Gal4-TBP/IPS-1. The cDNA encoding the C-terminally truncated form of the Tat protein (1 to 86 aa) of HIV-1 and myc epitope tag were amplified from pQBI-TatGFP (Qbiogene, Montreal, Canada) and pcDNA3.1/myc-His (Invitrogen, Carlsbad, CA, USA), respectively. The cDNA encoding the fusion protein that consists of Tat, a myc epitope tag and the C-terminal region of IPS-1 (Tat/IPS-1) was amplified and connected in frame by the method of ‘splicing by overlap extension’ (23) using the above PCR products as templates, and introduced into pCAGPM vector (24). The resulting plasmid was designated pCAG-Tat/IPS-1. To localize the chimeric transcription factors in the ER membrane, the C-terminal Arg-Arg-Arg sequences in the luminal region of the IPS-1 were replaced with Gly-Gly-Ser (IPS-1ER), and the resulting plasmids encoding the mutant transcription factors were designated pBIND-Gal4-TBP/IPS-1ER and pCAG-Tat/IPS-1ER. To express the chimeric transcriptional factors from a dicistronic mRNA, cDNA clones of Tat/IPS-1ER and Gal4-TBP/IPS-1ER were introduced into the NheI/EcoRI and SalI/NotI sites of pIRES vector (Clontech), respectively, and the resulting plasmid was designated pIRES-TG. Schematic representations of the transcription factors are shown in Figure 1a.
The cDNA of the HIV-1 LTR core promoter (−40 to +78) was amplified from pHIV-LTR-nEGFP-bsr (Tatsumi et al., unpublished) and introduced between the NheI and HindIII sites of pGL4.31 vector (Promega). The resulting plasmid pGL4.31LTR-Luc encodes a GAL4UAS, HIV-1 LTR core promoter (TATA box and TAR) and firefly luciferase cDNA (Fig. 1b). The luciferase gene of pGL4.31LTR-Luc was replaced with cDNA of human LNGFR amplified from pMACS-LNGFR (Miltenyi Biotech, Bergisch Gladbach, Germany), and designated as pGL4.31LTR-LNGFR. The cDNA encoding NS3/4A protease of JFH-1 virus amplified from pHH21-JFH1 (25) was introduced into pCAGGS-PM3-NHA vector (8) and designated pCAG-HA-NS3/4A. All PCR products were confirmed by sequencing using an ABI Prism 3130 genetic analyzer (Applied Biosystems, Tokyo, Japan).
Human 293T embryonic kidney cells and human Huh7OK1 hepatoma cells, which exhibit high susceptibility to JFH-1 virus (26), were maintained in DMEM (Sigma, St. Louis, MO) containing 10% FBS and nonessential amino acids. The Huh9–13 cell line, an Huh7-derived cell line harboring a subgenomic HCV replicon (27), was maintained in DMEM containing 1 mg/ml G418 (Nacalai Tesque, Kyoto, Japan). Huh7OK1 cells transfected with pIRES-TG were selected with 1 mg/ml G418 and the drug-resistant clone was designated Huh7OK1/TG. Furthermore, Huh7OK1/TG cells transfected with pGL4.31LTR-Luc or pGL4.31LTR-LNGFR were treated with 50 μg/ml hygromycin B (Wako, Tokyo, Japan) and the drug-resistant clones were designated Huh7OK1/TG-Luc and Huh7OK1/TG-LNGFR, respectively. All cell lines were cultured at 37°C in a humidified atmosphere with 5% CO2.
Viruses and human sera
The HCV JFH-1 strain was prepared by transfection of the plasmid pHH21-JFH1 (25) into Huh7OK1 cells, and the infected cells or supernatants were serially passaged. The infectivity of JFH-1 virus was determined by focus-forming assay and expressed in FFU as described previously (28). Recombinant VSV encoding E1 and E2 envelope proteins of HCV (HCVrv) and JEV were prepared as described previously (22, 29). Sera from healthy donors and hepatitis C patients were collected at the Kyushu University Hospital after obtaining full informed consent.
Rabbit polyclonal antibody to NS5A was prepared as described previously (10). Mouse monoclonal and rabbit polyclonal antibodies to the influenza virus HA tag and mouse monoclonal antibody to the myc epitope tag were purchased from Covance (Richmond, CA, USA). A rabbit antibody to the Gal4 DB domain was purchased from Upstate Biotechnology (Charlottesville, VA, USA). A mouse anti-β-actin monoclonal antibody was purchased from Sigma. A FITC-conjugated anti-LNGFR antibody was purchased from Miltenyi Biotech.
Cells transfected with plasmids and/or infected with JFH-1 virus were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min. After washing twice with PBS, cells were permeabilized for 15 min at room temperature with PBS containing 0.2% Triton X-100 and then treated with PBS-FBS for 30 min at room temperature. The cells were incubated with PBS-FBS or Can Get Signal immunostain (Toyobo, Osaka, Japan) containing the appropriate primary antibodies for 60 min at room temperature and washed three times with PBS. The immuno-complexes were detected with AlexaFlour 488- or 594-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA). Nuclei were labeled with TO-PRO-3 iodide (Molecular Probes). For staining of mitochondria, cells were pre-incubated with culture medium containing 200 nM Mitotracker Deep-Red (Molecular Probes) for 20 min at 37°C. Cells were observed using a FluoView FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan).
Transfection and immunoblotting
Huh7OK1 cells were transfected with plasmids by using FuGene6 (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer's protocol. Cells were lysed on ice in Triton lysis buffer (20 mM Tris-HCl [pH7.4], 135 mM NaCl, 1% Trition-X 100, 10% glycerol) supplemented with a protease inhibitor mix (Nacalai Tesque) at 24- or 48-hr post-transfection, and subjected to SDS-PAGE using Tris-glycine buffer and immunoblotting using appropriate antibodies. The stained protein bands were visualized using a SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA) and an LAS3000 imaging system (Fujifilm, Tokyo, Japan).
Transfected and/or infected cells were lysed with passive lysis buffer (Promega). Luciferase activity was measured in 10 μl aliquots of cell lysates using the Dual-Luciferase reporter assay system (Promega) or Bright-Glo luciferase assay system (Promega) according to the manufacturer's instructions. The activity of firefly luciferase was standardized to that of Renilla luciferase and expressed as the increase in RLU.
Magnetic cell sorting and flow cytometry
Immunomagnetic cell sorting was performed according to the manufacturer's instructions (Miltenyi Biotech). Cells were harvested with PBE buffer. Cells transfected with 4 μg of pMACS-LNGFR were used as a positive control. Cells (2 × 106) suspended in 600 μl of PBE buffer were mixed with 8 μl of anti-LNGFR magnetic beads (Miltenyi Biotech) at 4°C for 15 min, washed once with PBE buffer, and applied to the MS column of a MiniMACS magnetic separation kit (Miltenyi Biotech). For flow cytometry, cells were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min. After washing once with PBE buffer, cells were permeabilized for 15 min at room temperature with PBS containing 0.2% Triton X-100 and then washed once with PBE buffer. The cells were incubated with PBE containing anti-NS5A rabbit polyclonal antibody for 2 hr, washed twice with PBE buffer and treated with MACSelect control FITC-conjugated antibody against magnetic beads and phycoerythrin-conjugated anti-rabbit IgG antibody for 1 hr. After washing twice with PBE buffer, cells were analyzed by a flow cytometer FACS Calibur (BD Biosciences, Franklin Lakes, NJ, USA).
HCV RNA was determined as described previously (9). Briefly, total RNA was prepared from cells using an RNeasy kit (Qiagen, Tokyo, Japan) and first-strand cDNA was synthesized using an RNA LA PCR kit (Takara Bio, Shiga, Japan) with random primers. Expression of the appropriate gene was estimated by using platinum SYBR green quantitative PCR SuperMix UDG (Invitrogen) and a Prism 7000 system (Applied Biosystems). The 5′ untranslated region of the HCV genome and GAPDH mRNA were amplified using primer pairs as described previously (9). The amount of HCV genomic RNA was normalized with that of GAPDH mRNA.
Screening for HCV inhibitors
Huh7OK1/TG-Luc cells seeded at a density of 5000 cells per well in 96-well plates were incubated with 100 μl of culture medium containing JFH-1 virus at a MOI of 1.0 and various concentrations (up to 20 μM) of chemical compounds obtained from the Chemical Biology Research Initiative at The University of Tokyo. After 4 days of incubation, the supernatant was replaced with 50 μl of DMEM and then 10 μl of Bright-Glo luciferase assay buffer was added. Luciferase activity was determined using a Tropix TR717 Microplate Luminometer (Applied Biosystems). Cell viability was determined by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer's instructions.
Construction of plasmids for cell culture systems for the detection of HCV propagation by reporter gene expression
To establish cell culture systems capable of monitoring the replication of HCV without any artificial manipulation of the viral genome, we employed the concept of the indicator cell line for HIV-1 infection, also known as the MAGI assay (30). This assay depends on the transactivation activity of Tat protein, which is produced de novo upon HIV-1 replication and activates the HIV-1 LTR promoter. Furthermore, we utilized a chimeric transcriptional factor Gal4-TBP which consists of a Gal4 DB domain and truncated TBP, and synergistically enhances transcription under the control of GAL4UAS and HIV-1 LTR tandem promoter with Tat protein (31). To harness these transcription factors so they would be specifically activated in HCV-infected cells, we added the C-terminal region of IPS-1 to the C-terminus of the chimeric transcription factors. IPS-1 localizes on the mitochondrial outer membrane and is efficiently cleaved by HCV NS3/4A protease upstream of the transmembrane region (11). Although these transcription factors reside on the mitochondrial outer membrane and exhibit no activity under normal conditions, upon infection with HCV these transacting domains are translocated into the nucleus after cleavage by HCV NS3/4A protease and activate appropriate reporters. We employed luciferase and human LNGFR genes as reporters and inserted them under the control of the GAL4UAS and the HIV-1 LTR tandem promoter (Fig. 1b).
Subcellular localization of the chimeric transcription factors
To determine the intracellular localization of Tat/IPS-1, Huh7OK1 cells were transfected with pCAG-Tat/IPS-1 and examined by immunofluorescent assay (Fig. 2a). Although Tat/IPS-1 was co-localized with the mitochondrial marker Mitotracker as we expected, expression of Tat/IPS-1 induced severe aggregation of mitochondria and cell death. Accumulation of artificial proteins on the mitochondria membrane may induce mitochondrial dysfunction that leads to cell death. Horie et al. have reported that the C-terminal net positive charge of C-tail-anchored proteins, such as Tom5, is crucial for mitochondrial targeting, and that mutants reducing the net charge are distributed throughout the intracellular membrane (32). Four out of five C-terminal residues of IPS-1 (RRRLH) are positively charged (Fig. 1a). To modify the intracellular localization of Tat/IPS-1, we constructed an expression plasmid pCAG-Tat/IPS-1ER encoding Tat/IPS-1 possessing substitution of three arginine residues in the C-terminal five residues with non-charged amino acid glycine residues (GGGLH) (Fig. 1a) and examined the intracellular localization (Fig. 2b). Tat/IPS-1ER was co-localized with an ER marker Calnexin but not with Mitotracker and neither mitochondrial accumulation nor cell death was observed in the cells expressing Tat/IPS-1ER. These results indicate that Tat/IPS-1ER resides in the ER membrane and, unlike Tat/IPS-1, exhibits no severe cell toxicity. Therefore, we prepared Gal4-TBP/IPS-1ER in a similar way and used these ER-localized transcription factors in the following experiments.
Activation of chimeric transcription factors by HCV NS3/4A protease and establishment of indicator cell lines for HCV
To confirm that the chimeric transcription factors had been cleaved by NS3/4A protease, 293T cells were transfected with pCAG-Tat/IPS-1ER and/or pBIND-Gal4-TBP/IPS-1ER together with a plasmid encoding HCV NS3/4A protease or its defective mutant NS3/4A S139A (Fig. 3a). The cleaved products of Tat/IPS-1ER and Gal4-TBP/IPS-1ER were detected by anti-myc and anti-Gal4 DB antibodies, respectively, in cells co-expressing with NS3/4A but not with a S139A mutant. Immunofluorescent analyses revealed clear nuclear localization of the transcription factors cleaved off from the ER by expression of NS3/4A protease (Fig. 3b). Next, to examine whether or not reporter gene expression under the control of GAL4UAS and the HIV-1 LTR tandem promoter was dependent on the cleavage of transcription factors by HCV NS3/4A protease, Huh7OK1 cells were transfected with the plasmids encoding the transcription factors, pBIND-Gal4-TBP/IPS-1ER and pCAG-Tat/IPS-1ER, together with pGL4.31LTR-Luc, and luciferase activity was determined (Fig. 3c). No significant reporter gene activation was observed in cells co-transfected with pGL4.31LTR-Luc and pCAG-Tat/IPS-1ER irrespective of the expression of NS3/4A protease, and only a two-fold increase in luciferase was observed in cells co-transfected with pGL4.31LTR-Luc and pBIND-Gal4-TBP/IPS-1ER and then expressed with NS3/4A protease but not with NS3/4A S139A. Gal4-TBP and Tat have previously been shown to cooperatively accelerate initiation and elongation of the polymerase reaction (33). Synergistic enhancement (a more than forty-fold increase) of luciferase expression with a low background was achieved by expression of NS3/4A, but not by expression of NS3/4A S139A, in cells co-transfected with pGL4.31LTR-Luc, pCAG-Tat/IPS-1ER and pBIND-Gal4-TBP/IPS-1ER. In addition, specific induction of the reporter gene was detected in Huh9–13 cells harboring the subgenomic replicon of a genotype 1b Con1 strain (Fig. 3d). These results clearly indicate that the chimeric transcriptional factors, Tat/IPS-1ER and Gal4-TBP-1ER, were released from the ER membrane by HCV NS3/4A protease, and synergistically activated reporter gene expression under the control of GAL4UAS and the HIV-1 LTR tandem promoter.
To establish indicator cell lines for HCV, Huh7OK1 cells transfected with pIRES-TG encoding the ER-localized transcription factors (Tat/IPS-1ER and Gal4-TBP/IPS-1ER; Fig. 1a) were further transfected with pGL4.31LTR encoding reporter genes under the control of GAL4UAS and the HIV-1 LTR tandem promoter (Fig. 1b), and two indicator cell lines capable of detecting HCV propagation (Huh7OK1/TG-Luc and Huh7OK1/TG-LNGFR) were established (Fig. 1c).
Specific reporter gene activation upon infection with JFH-1 virus
To determine the specificity of reporter gene expression in the indicator cell lines in response to HCV infection, the Huh7OK1/TG-Luc cell line stably expressing Tat/IPS-1ER and Gal4-TBP/IPS-1ER transcription factors and carrying the luciferase gene under the control of the GAL4UAS and HIV-1 LTR promoter was infected with JFH-1 virus at an MOI of 0.0001 to 1.0, and the luciferase activity and intracellular viral RNA levels were determined at 96-hr post-infection. Luciferase activity exhibited a good correlation with HCV RNA in the range of 103 to 105 luciferase units (R2= 0.98704) (Fig. 4a). To confirm the specificity of reporter gene expression of the indicator cell line, Huh7OK1/TG-Luc cells were infected with JFH-1 virus, HCVrv (29), or JEV (a member of the family Flaviviridae) and the luciferase activities determined (Fig. 4b). Dose-dependent induction of luciferase was observed in cells infected with JFH-1 virus, but not in those with infected with HCVrv or JEV, suggesting specific detection of HCV replication by the indicator cell line. To further examine specific induction of the reporter gene upon infection with HCV, we assessed the effects of inhibitors of HCV entry or RNA replication on reporter gene expression (Fig. 4c). Luciferase activity was reduced in a dose-dependent manner by treatment with anti-CD81 antibody (34), but not with the isotype control, consistent with a previous observation (5). Its activity was also suppressed in a dose-dependent manner by treatment with Mβ-CD (35), IFN- α (36) and sera from hepatitis C patients, but not by sera from healthy donors. These results indicate that the Huh7OK1/TG-Luc cell line can be used as a reliable and convenient tool to detect and quantify the propagation of HCV based on luciferase expression.
Establishment of an indicator cell line capable of expressing LNGFR upon infection with HCV
To develop an indicator line capable of selectively collecting HCV-infected cells based on expression of cell surface molecules, we established an Huh7OK1/TG-LNGFR cell line possessing the LNGFR gene under the control of the GAL4UAS and HIV-1 LTR tandem promoter. LNGFR is mainly expressed in the cells of nervous systems, but not in hepatocytes, and immunomagnetic cell sorting systems have been proven to be effective for separating LNGFR-positive cells (37, 38). Expression of LNGFR was induced in Huh7OK1/TG-LNGFR cells upon infection with JFH-1 virus, whereas no expression of LNGFR was detected in uninfected Huh7OK1/TG-LNGFR cells or Huh7OK1/TG-Luc cells infected with JFH-1 virus (Fig. 5a), indicating that Huh7OK1/TG-LNGFR cells can specifically induce expression of LNGFR upon infection with HCV. Next, to demonstrate the ability of the system to isolate HCV-infected cells, Huh7OK1/TG-LNGFR cells were infected with JFH-1 virus at an MOI of 0.1 and sorted by anti-LNGFR antibody-coated magnetic beads at 4 days post-infection. The proportions of cells exhibiting positivity for only NS5A or only LNGFR, or double-positivity for both proteins before sorting were determined to be 14.17%, 5.99% and 3.20% of the total cell population, respectively (Fig. 5b middle panel). The reason for not all HCV-infected cells inducing expression of LNGFR remains unclear at this stage, but it might be because of either a difference in the sensitivity of the antibodies used for detection of NS5A and LNGFR or the presence of an unknown inhibitory mechanism of LNGFR expression on the cell surface. After sorting, the proportion of NS5A and LNGFR double-positive cells was increased to up to 48.05% of the recovered cells inoculated with JFH-1 virus (Fig. 5b right panel). These results suggest that the indicator cell system capable of expressing LNGFR upon infection with HCV is effective for concentrating HCV-infected cells.
Application of Huh7OK1/TG-Luc cells for screening of anti-HCV compounds
To determine the ability of the indicator cell lines to screen anti-HCV compounds, approximately 1300 chemical compounds were obtained from the library of the Chemical Biology Research Initiative at the University of Tokyo and screened by using Huh7OK1/TG-Luc cells. Cells seeded in 96-well plates (5000 cells/well) were incubated with culture medium containing JFH-1 virus at an MOI of 1.0 and various concentrations of each compound, and luciferase activities were determined after 4 days of incubation. Two of the compounds we examined, fiduxosin hydrochloride and flunarizine dihydrochloride, exhibited significant reduction of luciferase expression without severe cell toxicity (Fig. 6a). The antiviral activities of these compounds were confirmed by the reduction of HCV RNA in the cells in a dose-dependent manner (Fig. 6b). Although fiduxosin and flunarizine are known to be antagonists of alpha-1 adrenoceptor (39) and T-type calcium ion channels (40), respectively, the mechanisms by which they inhibit HCV propagation are not known. These results indicate that the Huh7OK1/TG-Luc cell line is applicable for a reliable cell-based assay for high-throughput screening of anti-HCV drugs.
The establishment of indicator cell lines based on MAGI assays (30) has made a great contribution to our understanding of HIV-1 life cycles and the development of anti-HIV-1 reagents (41, 42). In this study, we have established indicator cell lines for HCV infection in the same manner using a MAGI assay. To establish indicator cell lines for HCV, HCV NS3/4A-mediated trans-activation of reporter gene systems based on chimeric transcription factors containing the cleavage sequence of the NS3/4A protease derived from HCV polyprotein have been reported (19–21). Breiman et al. (21) reported construction of a chimeric transcription factor consisting of a fusion protein of the Gal4 DB domain with the VP16 transactivator protein of herpes simplex virus (Gal4VP16), NS3/4A protease-cleavage sequences between HCV NS5A and NS5B proteins and a portion of the ER-resident protein PERK for anchoring at the ER membrane. Upon infection with HCV, the Gal4VP16 was cleaved off by the NS3/4A protease and activated the transcription of reporter genes. NS3/4A protease-cleavage sites from NS4A to NS5B junctions were inserted between green fluorescent protein and SEAP, and HCV infection was quantified by the SEAP activity secreted into culture supernatants (19, 20). However, in the previous studies these chimeric constructs containing NS3/4A protease-cleavage sites derived from HCV proteins exhibited only partial cleavage upon co-expression of NS3/4A protease, suggesting that processing of the HCV sequences in the fusion proteins is not efficiently recognized by HCV protease.
To overcome this obstacle, we utilized the C-terminal portion of the mitochondria-resident IPS-1 as a transmembrane anchor of the chimeric transcriptional factors. It has been reported that cleavages of IPS-1 are observed in cell cultures by expression of NS3/4A derived from several HCV strains of different genotypes (43) and also in the liver tissue of patients with chronic hepatitis C infection (43), suggesting that chimeric constructs fused with the C-terminal region of IPS-1 can be cleaved by NS3/4A protease in a broad range of HCV genotypes. Furthermore, to avoid induction of mitochondrial dysfunction and cell death, we modified the C-terminal residues of IPS-1of the chimeric transcription factors to achieve localization on the ER membrane. HCV NS3/4A is an ER-membrane associated protease, and subcellular localization and distance of the cleavage site of the substrates from the membrane could be crucial for efficient processing. Judging from the processing of the Tat/IPS-1ER and Gal4TBP/IPS-1ER proteins by co-expression of NS3/4A protease, the ER-anchored C-terminal domain of IPS-1 was efficiently cleaved by NS3/4A protease.
One of the difficulties in development of indicator cells is a low S/N ratio. When we examined the combination of Tat/IPS-1ER and an HIV-1 LTR promoter, a high background of luciferase expression was observed, probably due to spontaneous release of an undetectable population of the transcription factor from the ER and contingent activation of the target promoter (data not shown). A previous report has indicated that Gal4-TBP and Tat proteins accelerate transcription at different steps (33). Gal4-TBP and Tat accelerate recruitment of RNA polymerase II holoenzyme to initiate transcription, and of P-TEFb to enhance elongation of the polymerase reaction, respectively. As we expected, synergistic enhancement of luciferase expression with a high S/N ratio was achieved by the combination of the two chimeric transcription factors and the GAL4UAS and HIV-1 LTR tandem promoter. Furthermore, a close correlation between expression and viral RNA replication upon HCV infection was observed in Huh7OK1/TG-Luc cells. These results indicate that the Huh7OK1/TG-Luc cell line is useful for quantifying HCV replication with a high sensitivity and specificity and applicable for high-throughput screening of anti-HCV compounds. Among the 1300 compounds screened here, we identified fiduxosin hydrochloride and flunarizine dihydrochloride as anti-HCV agents. Fiduxosin is known as an alpha1-adrenoceptor antagonist (39). Previous screening of a whole-genome siRNA library has shown that knockdown of alpha-1 adrenoceptor A, B, or D expression significantly reduces the replication of the genotype 1b subgenomic replicon (44), suggesting that alpha-1 adrenoceptors participate in the replication of HCV. Flunarizine is an antagonist of T-type calcium ion channels (40) and has been shown to induce expression of HO-1 (which is an antioxidant defense and key cytoprotective enzyme) through PI3K/Nrf2 signaling in an auditory cell line (45). It has been reported that overexpression of HO-1 suppresses HCV replication in genotype 1b replicon cells (46). Although further studies are required, it is reasonable to speculate that HO-1induced by treatment with flunarizine participates in the inhibition of HCV replication.
Another advantage of the indicator system is its flexibility in the choice of reporter genes. For easy separation and concentration of HCV-infected cells by immunomagnetic sorting, we established an Huh7OK1/TG-LNGFR cell line and demonstrated that a fifteen-fold concentration of HCV-infected cells could be achieved with a single sorting step. In addition, it would be possible to use this system in research on various cell lines other than Huh7 derivatives and primary hepatocytes in order to isolate novel HCV strains that exhibit no susceptibility to Huh7-derived cell lines. Although a few HCV strains for productive infection in cultured cells have been reported (3, 6, 47), no reliable and robust cell culture system for propagation of serum-derived HCV has thus far been established. We have inoculated sera from the chronic and window periods of patients with hepatitis C, but so far no significant reporter gene expression has been obtained in the indicator cell lines described in this study (data not shown), indicating that more factors are required for efficient propagation of serum-derived, naturally occurring HCV.
While it has been reported that HCV can spread through cell-to-cell pathways even in the presence of neutralizing antibodies (48–51), the precise mechanisms are not known. Although human CD81 and Claudin-1 are known to be major receptor candidates for HCV entry, cell-to-cell transmission between hepatoma cells is dependent on the expression of Claudin-1 but not of human CD81 (49, 50). In addition, peripheral blood B cells, in which JFH-1 virus cannot replicate, are able to transfer JFH-1 virus to hepatoma cells through SR-BI-, DC-SIGN- and L-SIGN-dependent pathways (51). The indicator cell lines established in this study could be used as recipient cells to investigate cell-to-cell transmission.
In conclusion, we have constructed chimeric transcription factors which are specifically and efficiently cleaved by HCV NS3/4A cleavage and established indicator cell lines capable of monitoring infection with JFH-1 virus based on reporter gene activation through cleavage of the transcription factors by HCV protease. By introducing the present system into various cell lines and modifying the reporter gene, it might be possible to establish a cell culture system capable of propagating serum-derived HCV.
We thank H. Murase for her secretarial work. We also thank R. Bartenschlager and T. Wakita for providing cell lines and plasmids. This work was supported in part by grants-in-aid from the Ministry of Health, Labor, and Welfare; the Ministry of Education, Culture, Sports, Science, and Technology; the Osaka University Global Center of Excellence Program; and the Foundation for Biomedical Research and Innovation.