Novel Permissive Cell Lines for Complete Propagation of Hepatitis C Virus

ABSTRACT Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. Although the HCV life cycle has been clarified by studying laboratory strains of HCV derived from the genotype 2a JFH-1 strain (cell culture-adapted HCV [HCVcc]), the mechanisms of particle formation have not been elucidated. Recently, we showed that exogenous expression of a liver-specific microRNA, miR-122, in nonhepatic cell lines facilitates efficient replication but not particle production of HCVcc, suggesting that liver-specific host factors are required for infectious particle formation. In this study, we screened human cancer cell lines for expression of the liver-specific α-fetoprotein by using a cDNA array database and identified liver-derived JHH-4 cells and stomach-derived FU97 cells, which express liver-specific host factors comparable to Huh7 cells. These cell lines permit not only replication of HCV RNA but also particle formation upon infection with HCVcc, suggesting that hepatic differentiation participates in the expression of liver-specific host factors required for HCV propagation. HCV inhibitors targeting host and viral factors exhibited different antiviral efficacies between Huh7 and FU97 cells. Furthermore, FU97 cells exhibited higher susceptibility for propagation of HCVcc derived from the JFH-2 strain than Huh7 cells. These results suggest that hepatic differentiation participates in the expression of liver-specific host factors required for complete propagation of HCV. IMPORTANCE Previous studies have shown that liver-specific host factors are required for efficient replication of HCV RNA and formation of infectious particles. In this study, we screened human cancer cell lines for expression of the liver-specific α-fetoprotein by using a cDNA array database and identified novel permissive cell lines for complete propagation of HCVcc without any artificial manipulation. In particular, gastric cancer-derived FU97 cells exhibited a much higher susceptibility to HCVcc/JFH-2 infection than observed in Huh7 cells, suggesting that FU97 cells would be useful for further investigation of the HCV life cycle, as well as the development of therapeutic agents for chronic hepatitis C.

M ore than 170 million individuals worldwide are infected with hepatitis C virus (HCV), and the cirrhosis and hepatocellular carcinoma induced by HCV infection are life-threatening diseases (1). Current standard therapy combining pegylated-interferon (peg-IFN) and ribavirin (RBV) has achieved a sustained virological response (SVR) in 50% of individuals infected with HCV genotype 1 (2). Recently, directly acting antiviral (DAA) agents have been applied in a clinical setting (3). An SVR rate of over 80% has been realized by combination therapy with peg-IFN, RBV, and NS3/4A inhibitors in genotype 1 patients (4,5). In addition, several DAAs, including inhibitors for NS3/4A protease, NS5A, and NS5B polymerase, are currently in clinical trials. Several reports have shown that in vitro replication of HCV RNA is significantly inhibited by treatment with daclatasvir (NS5A inhibitor) and asunaprevir (NS3 protease inhibitor), and these two DAAs are also effective for patients infected with genotype 1 HCV who showed no response to previous therapy with peg-IFN-␣ and RBV (6)(7)(8). On the other hand, it has been shown that drug-resistant breakthrough viruses emerge during treatment with DAAs (9)(10)(11)(12). Therefore, identification of host factors crucial for the propagation of HCV is an important task for the development of novel therapeutics for chronic hepatitis C with a low frequency of emergence of drug-resistant viruses.
The establishment of an in vivo infection model has been hampered by the narrow host range and tissue tropism of HCV. Al-though chimpanzees are the only experimental animals susceptible to HCV infection, it is difficult to use a chimpanzee model of experimental infection due to ethical concerns (13,14). In addition, in vitro infection models have also been restricted to the combination of cell culture-adapted clones based on the genotype 2a JFH-1 strain (HCVcc) and human hepatoma cell lines, including Huh7 (15). Recently, several reports have shown that the exogenous expression of microRNA-122 (miR-122) facilitates the efficient propagation of HCVcc in HepG2 and Hep3B cells, which are nonpermissive for propagation of HCVcc (16,17). Furthermore, we reported that nonhepatic cell lines, including Hec1B cells derived from uterine endometrial adenocarcinoma, also permit replication of HCV RNA by exogenous expression of miR-122 (18). These reports indicate that miR-122 is one of the most important determinants for liver tropism of HCV infection. Interestingly, formation of infectious particles was not observed in spite of efficient replication of HCV RNA in nonhepatic cells, suggesting that liver-specific factors other than miR-122 are involved in HCV assembly. Previous reports suggested that verylow-density lipoprotein (VLDL)-associated proteins, including apolipoprotein B (ApoB), apolipoprotein E (ApoE), and microsomal triglyceride transfer protein (MTTP), play important roles in infectious particle production of HCV (19)(20)(21)(22)(23). In addition, Miyanari et al. indicated that lipid droplets (LDs) are crucial organelles for HCV particle assembly (24). These reports suggest that liver-specific lipid metabolism and liver-specific host factors closely participate in assembly of HCV.
Cancer cells are classified into well-differentiated and intermediately and poorly differentiated stages, and these stages have been shown to be strongly related to cancer behaviors, with an immature tumor generally being more aggressive than its more differentiated counterpart. Thus, it is believed that well-differentiated cancer cells maintain the tissue-specific cellular functions and exhibit morphology similar to that of normal cells (25). Permissive cell lines for HCV propagation, including Huh7, HepG2, and Hep3B cells, are derived from well-differentiated hepatocellular carcinoma (HCC) (26,27). In addition, recent reports indicated that hepatocyte-like cells derived from induced pluripotent stem cells (iPS cells/iPSCs) express high levels of miR-122 and VLDLassociated proteins and support propagation of HCVcc (28)(29)(30). These results suggest that the hepatic differentiation required for hepatic functions is involved in HCV propagation.
In this study, we identified novel cell lines supporting complete propagation of HCVcc by screening cancer cell lines expressing ␣-fetoprotein (AFP), which is highly expressed in well-differentiated hepatocellular carcinoma and evaluated in most cancer cell lines (31). These cells exhibit high levels of expression of liverspecific host factors and permit complete propagation of HCVcc without any exogenous expression of liver-specific factors, including receptor molecules, miR-122, and apolipoproteins. Our current study suggests that hepatic differentiation participates in the expression of liver-specific host factors required for complete propagation of HCV.

MATERIALS AND METHODS
NextBio Body Atlas. The NextBio Body Atlas application allows the aggregated analysis of gene expression across various normal tissues, normal cell types, and cancer cell lines. It enables us to investigate the expression of individual genes as well as gene sets. Samples for Body Atlas data are obtained from publicly available studies that are internally curated, annotated, and processed (32). Body Atlas measurements are generated from all available RNA expression studies that used Affymetrix U133 Plus or U133A Genechip arrays for human studies. The results for 128 human tissue samples from 1,067 arrays, those for 157 human cell types from 1,474 arrays, and those for 359 human cancer cell lines from 376 arrays are incorporated. In this study, we screened cell lines expressing a high level of AFP. The details of the analysis protocol developed by NextBio were described previously (32). The raw data used in this application are derived from the GSK Cancer Cell Line data deposited at the National Cancer Institute website (https://array.nci.nih.gov/caarray/project/woost-00041).
Plasmids. The cDNA clones of pri-miR-122 and Aequorea coerulescens green fluorescent protein (AcGFP) were inserted between the XhoI and XbaI sites of lentiviral vector pCSII-EF-RfA, which was provided by M. Hijikata, and the resulting plasmids were designated pCSII-EF-miR-122 and pCSII-EF-AcGFP, respectively. Plasmids pHH-JFH1-E2p7NS2mt, encoding a cDNA of a full-length RNA of the JFH-1 strain, and pJFH2/ AS/mtT4, encoding a cDNA of a full-length RNA of the JFH-2 strain, were described previously (33,34). pSGR-Con1, which encodes a subgenomic replicon (SGR) of the Con1 strain, was provided by R. Bartenschlager. pIFN-␤-Luc and pISRE-Luc carrying a firefly luciferase (Luc) gene under  the control of the IFN-␤-and IFN-sensitive response element (ISRE) promoters, respectively, were provided by S. Akira. The internal control plasmid encoding a Renilla luciferase (pRL-SV40) was purchased from Promega (Madison, WI). The plasmids used in this study were confirmed by sequencing with an ABI Prism 3130 genetic analyzer (Applied Biosystems, Tokyo, Japan).
Preparation of viruses. HCVs derived from the genotype 2a JFH-1 strain (HCVcc) were prepared after serial passages of the culture supernatants of Huh7.5.1 cells transfected with pHH-JFH1-E2p7NS2mt into Huh7.5.1 cells (33). HCVs derived from the genotype 2a JFH-2 strain (HCVcc/JFH-2) were prepared by several passages of the culture supernatants of Huh7.5.1 cells electroporated with JFH-2 RNA transcribed in vitro. Infectious titers were determined by a focus-forming assay and expressed as focus-forming units (FFU) (15). The vesicular stomatitis virus (VSV) variant NCP12.1 derived from the Indiana strain was provided by M. Whitt. Pseudotype VSVs bearing HCV E1 and E2 glycoproteins (HCVpv), were prepared as described previously (36), and infectivity was assessed by a luciferase assay system (Promega) according to a protocol provided by the manufacturer and expressed in relative light units (RLU).
Transfection and lentiviral gene transduction. Cells were transfected with the plasmids by using TransIT LT-1 transfection reagents (Mirus, Madison, WI) according to the manufacturer's protocol. LNAs were introduced into cells by Lipofectamine RNAi MAX (Life Technologies). The lentiviral vectors and ViraPower lentiviral packaging mix (Life Technologies) were cotransfected into 293T cells, and the supernatants were recovered at 48 h posttransfection. The lentivirus titer was determined by a lenti-XTM quantitative reverse transcription-PCR (qRT-PCR) titration kit (Clontech, Mountain View, CA), and the expression levels of miR-122 and AcGFP were determined at 48 h postinoculation.
Quantitative RT-PCR. HCV RNA levels were determined by a method described previously (38). Total RNA was extracted from cells by using an RNeasy minikit (Qiagen, Valencia, CA), and the first-strand cDNA synthesis and qRT-PCR were performed with TaqMan EZ RT-PCR core reagents and a ViiA7 system (Life Technologies), respectively, according to the manufacturer's protocol. The primers for TaqMan PCR targeted to the noncoding region of HCV RNA were synthesized as previously reported (39). To determine the expression of miR-122, total miRNAs were prepared by using an miReasy minikit (Qiagen), and miR-122 was determined by using the fully processed miR-122-specific RT and PCR primers provided in the TaqMan microRNA assays (Life Technologies) according to the manufacturer's protocol. U6 small nuclear RNA was used as an internal control. Fluorescent signals were analyzed with the ViiA7 system.
Immunoblotting. Cells were lysed on ice in lysis buffer (20 mM Tris-HCl [pH 7.4], 135 mM NaCl, 1% Triton X-100, 10% glycerol) supplemented with a protease inhibitor mix (Nacalai Tesque). Culture supernatants of cells incubated for 3 days were used for detection of ApoB. The samples were boiled in loading buffer and subjected to a 5% to 20% gradient SDS-PAGE or 3 to 8% Tris-acetate gel (Thermo Scientific, Waltham, MA). The proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) and reacted with the appropriate antibodies. The immune complexes were visualized with SuperSignal West Femto substrate (Pierce, Rockford, IL) and detected with an LAS-3000 image analyzer system (Fujifilm, Tokyo, Japan).
Immunofluorescence assay. Cells cultured on glass slides were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at room temperature for 30 min, permeabilized for 20 min at room temperature with PBS containing 0.2% Triton X-100, washed three times with PBS, and blocked with PBS containing 2% FCS for 1 h at room temperature. Then cells were incubated with PBS containing appropriate primary antibodies at room temperature for 1 h, washed three times with PBS, and incubated with PBS containing AF488-or AF594-conjugated secondary antibodies at room temperature for 45 min. For lipid droplet staining, cells were incubated in medium containing 20 g/ml BODIPY for 20 min at 37°C, washed with prewarmed fresh medium, and incubated for 20 min at 37°C. The stained cells were covered with Vectashield Mounting Medium containing DAPI (Vector Laboratories Inc., Burlingame, CA) and observed with a FluoView FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan).
In vitro transcription, RNA transfection, and colony formation. The plasmid pSGR-Con1 was linearized with ScaI and transcribed in vitro by using a MEGAscript T7 kit (Life Technologies) according to the manufacturer's protocol. The in vitro-transcribed RNA (10 g) was electroporated into FU97 cells at 10 7 cells/0.4 ml under conditions of 210 V and 960 F using a Gene Pulser apparatus (Bio-Rad, Hercules, CA) and plated on DMEM containing 10% FCS. The medium of FU97 cells was replaced with fresh DMEM containing 10% FCS and 400 g/ml G418 at 24 h postelectroporation. The remaining colonies were cloned by using a cloning ring (Asahi Glass, Tokyo, Japan) or fixed with 4% PFA in PBS and stained with crystal violet at 5 weeks postelectroporation.
Flow cytometry. Cultured cells were detached with 0.25% trypsin-EDTA, incubated with PE-conjugated anti-hCD81 antibody or antimouse IgG antibody for 1 h at 4°C, washed twice with PBS containing 1% bovine serum albumin (BSA), and analyzed by using a flow cytometry system (FACSCalibur; BD Biosciences).
Gene silencing. A commercially available small interfering RNA (siRNA) pool targeting ApoB and ApoE (siGENOME SMARTpool human ApoB and ApoE) and a control nontargeting siRNA were purchased from Dharmacon (Buckinghamshire, United Kingdom) and transfected into JHH-4 and FU97 cells using Lipofectamine RNAi MAX (Life Technologies) according to the manufacturer's protocol.
Luciferase assay. Cells seeded onto 24-well plates at a concentration of 5 ϫ 10 4 cells/well were transfected with 250 ng of each of the plasmids, stimulated with the appropriate ligands for 24 h at 24 h posttransfection, and lysed in 100 l of passive lysis buffer (Promega). Luciferase activity was measured in 20-l aliquots of the cell lysates using a dual-luciferase reporter assay system (Promega). Firefly luciferase activity was standardized with that of Renilla luciferase cotransfected with the internal control plasmid pRL-SV40 and was expressed in RLU.
Buoyant density gradient analysis. Culture supernatants of Huh7.5.1 and FU97 cells infected with HCVcc at 72 h postinfection were passed through 0.45-m-pore-size filters and concentrated by a Spin-X Concentrator (100,000-molecular-weight cutoff column; Corning, Lowell, MA). One milliliter of concentrated sample was layered onto the top of a linear gradient formed from 10% to 40% of OptiPrep (Axis-Shield PoC, Oslo, Norway) in PBS and spun at 32,000 rpm for 16 h at 4°C by using an SW41-Ti rotor (Beckman Coulter, Fullerton, CA). Each fraction collected from the top was analyzed by qRT-PCR, focus-forming assay, and immunoblotting.
Statistical analysis. The data for statistical analyses are the averages of three independent experiments. Results were expressed as the means Ϯ standard deviations. The significance of differences in the means was determined by Student's t test.

JHH-4 and FU97 cells express high levels of the liver-specific host factors required for HCV propagation.
AFP is known as a marker for not only well-differentiated HCC (31) but also the early stage of differentiation to hepatocytes in embryonic stem (ES)/iPS cells (29,40,41). Generally, well-differentiated cancer cells show better maintenance of their cell-specific functions than poorly differentiated cancer cells. Therefore, we hypothesized that cancer cell lines with high levels of AFP expression retain sufficient hepatic function for HCV propagation. To examine this hypothesis, we first screened cancer cell lines by using the NextBio Body Atlas application and identified the following cell lines that expressed high levels of AFP: Takigawa and FU97 cells derived from gastric cancer, HepG2 and Hep3B cells from hepatocellular carcinoma, Caco-2 cells from colon cancer, and OV-90 cells from ovarian cancer. To evaluate the correlation of AFP expression with the hepatic functions in these cell lines, we examined the expression of liver-specific host factors, including albumin (ALB), ApoB, and ApoE, by using the Web-based NextBio search engine and found that these cell lines expressed higher levels of the liver-specific genes than the HEK293 cells used as negative controls (Fig. 1A). These results suggested that the expression of AFP was correlated with that of the examined liver-specific host factors in cancer cell lines. Next, to confirm this correlation, the expression levels of ALB, ApoB, ApoE, MTTP, and miR-122 were determined by qPCR in AFP-expressing cell lines, including FU97, OV-90, and HCC-derived Huh7, HepG2, Hep3B, and JHH-4 cells (Fig. 1B). Takigawa cells were difficult to culture, and Caco-2 cells have previously been reported to permit entry and replication of HCV (42,43); therefore, we excluded these cell lines for further analyses. JHH-4 cells were previously shown to permit a partial propagation of HCV in a three-dimensional cultivation by using a radialflow bioreactor system upon inoculation with plasma from an HCV carrier (44). In contrast to 293T cells, these AFP-expressing cell lines express high levels of the examined liver-specific host factors, suggesting that these cell lines maintain their hepatic functions. Because previous studies have shown that Huh7, HepG2, and Hep3B cells are susceptible to HCVcc infection, we selected JHH-4, FU97, and OV-90 cells for further investigation as new cell line candidates for HCV propagation. It is well known that hepatocytes and intestinal cells produce ApoB100 and ApoB48, respectively. ApoB100 was detected in the culture supernatants of Huh7, JHH-4, FU97, and OV-90 cells but not in those of 293T cells by immunoblotting (Fig. 1C). These results suggest that FU97 and OV-90 cells are differentiated into hepatocyte-like cells and possess liver-specific functions. The expression of entry receptor molecules for HCV, including hCD81, CLDN1, OCLN, and SR-BI (45)(46)(47)(48), in these cell lines was confirmed by immunoblotting and fluorescence-activated cell sorting (FACS) analyses ( Fig. 1D and E). To further determine the authenticity of the receptor candidates for HCV entry, HCVpv was inoculated into these cell lines. Although the infectivity of HCVpv to JHH-4 and FU97 cells was comparable to that in Huh7 cells, OV-90 cells did not show any susceptibility to HCVpv infection (Fig. 1F). Cell surface expression of CLDN1 and OCLN was detected in OV-90 cells (data not shown); therefore, lack of susceptibility of OV-90 cells to HCVpv infection might be attributable to lower expression of SR-B1 and hCD81 in OV-90 cells than in other cell lines ( Fig. 1D and E). Thus, we selected JHH-4 and FU97 cells for further investigation of HCV propagation.

JHH-4 and FU97 cells permit HCV propagation.
To examine the susceptibility of JHH-4 and FU97 cells to HCV propagation, HCVcc was inoculated into these cells at a multiplicity of infection (MOI) of 1, and intracellular HCV RNA and infectious titers in the culture supernatants were determined by qRT-PCR and focusforming assay, respectively. FU97 cells exhibited higher levels of HCV gene expression than JHH-4 cells, and these levels increased continuously until 48 h postinfection; treatment with IFN-␣ significantly inhibited HCV gene expression in both JHH-4 and FU97 cells ( Fig. 2A, left panels). In addition, substantial amounts of infectious particles were detected in the culture supernatants of JHH-4 and FU97 cells infected with HCVcc, in contrast to the lack of infectious particles in the culture supernatants of 293T-CLDN/ miR-122 cells infected with HCVcc ( Fig. 2A, bar graph). Recent reports have shown that exogenous expression of miR-122 enhances HCV RNA abundances in several hepatic or nonhepatic cell lines (16)(17)(18). Therefore, we examined the effect of miR-122 overexpression on HCV RNA abundances in both JHH-4 and FU97 cells. miR-122 was introduced in these cells by a lentiviral vector encoding pri-miR-122, an unprocessed miR-122, and miR-122 expression was confirmed by quantitative PCR (qPCR) analysis (Fig. 2B, bar graph). In contrast to the slight increase of HCV RNA in FU97 cells, JHH-4 cells exhibited a significant increase of HCV RNA, suggesting that the expression level of miR-122 is a key determinant for the efficient propagation of HCV (Fig. 2B, right panels). A previous study has shown that NS5A proteins were localized around the endoplasmic reticulum (ER) membrane, and accumulation of core protein around lipid droplets (LDs) facilitates efficient assembly of infectious particles in Huh7 cells (24). Immunofluorescence microscopy observation revealed that core and NS5A proteins in JHH-4 and FU97 cells infected with HCVcc were detected around LDs and in the ER together with doublestranded RNA (dsRNA), respectively (Fig. 2C). These results suggest that expression of liver-specific factors permits complete propagation of HCVcc in JHH-4 and FU97 cells and that hepatic characteristics play crucial roles in HCV propagation.

JHH-4 and FU97 cells permit complete propagation of HCVcc without any exogenous expression of host factors crucial for propagation of HCV.
To further characterize the propagation of HCV in JHH-4 and FU97 cells, we examined the effects of the HCV inhibitors on the replication of HCV RNA. Preincubation with anti-HCV E2 antibody and pretreatment of cells with anti-hCD81 monoclonal antibody significantly inhibited HCVcc infection not only in Huh7 cells but also in JHH-4 and FU97 cells, suggesting that hCD81 is also involved in HCV entry into JHH-4 and FU97 cells (Fig. 3A, left panels). To examine the effect of miR-122 expression on HCV RNA abundances, cells were treated with LNA specific to either miR-122 (miR-122-LNA) or a nonspecific LNA (Ctrl-LNA) at 6 h before infection with HCVcc. Treatment with miR-122-LNA but not with Ctrl-LNA significantly reduced the HCV RNA abundances in these cell lines, suggesting that miR-122 also plays a crucial role in the efficient propagation of HCVcc in JHH-4 and FU97 cells (Fig. 3A, right panels). Previous reports showed that treatment with MTTP inhibitors inhibited the production of infectious particles of HCVcc in Huh7 cells (20,22). Although intracellular HCV RNA levels in Huh7, JHH-4, and FU97 cells were not inhibited by the treatment with MTTP inhibitors, including CP-346086 and BMS-200150 (Fig.  3B, left panels), the production of infectious particles was significantly decreased in these cells (Fig. 3B, right panels). These results suggest that the VLDL secretion pathway also participates in the propagation of HCV in JHH-4 and FU97 cells. Furthermore, it was shown that ApoB and ApoE are involved in the production of HCV particles in Huh7 cells (20)(21)(22). To confirm the role of ApoB and ApoE in HCV propagation in JHH-4 and FU97 cells, the expression of ApoB and ApoE was suppressed by siRNAs (Fig. 3C, left panels). The suppression of ApoB and ApoE expression significantly reduced HCV RNA levels in cells infected with HCVcc at an MOI of 1 (Fig. 3C, middle panels) and significantly reduced the infectious titers in the supernatants (Fig. 3C, right panels) at 72 h postinfection. Collectively, these results suggest that the JHH-4 and FU97 cells permit complete propagation of HCVcc without any exogenous expression of the host factors crucial for propagation of HCV, including receptor molecules, miR-122, and VLDLassociated proteins. FU97 cells exhibited higher susceptibility to HCVcc propagation than JHH-4 cells ( Fig. 2A), and thus we characterized the FU97 cells in greater detail.
Establishment of HCV RNA replicon and cured cells by using FU97 cells. To further examine the characteristics of FU97 cells with respect to HCV RNA replication, in vitro-transcribed subgenomic HCV RNA of the Con1 strain was electroporated into Huh7 and FU97 cells and cultured in medium containing G418 for a month, and then subgenomic replicon (SGR) cells of the Con1 strain were established (Fig. 4A). Replication of HCV RNA in four clones of the FU97 replicon cells was examined by qRT-PCR and immunoblotting. All clones contained a high level of HCV RNA (3 ϫ 10 7 to 7 ϫ 10 7 copies/g total RNA) (Fig. 4B,  upper panel), and the NS5A protein was also detected (Fig. 4B,  lower panel). We examined the localization of NS5A and dsRNA in clone 5 of FU97 SGR cells by immunofluorescence analysis. Colocalization of NS5A with dsRNA was observed in clone 5, suggesting that the replication complex required for viral RNA replication was generated in the FU97 SGR cells (Fig. 4C). It has been shown that the infectivity of HCVcc in the cured cells that were established by elimination of the viral genome by treatment with antivirals from Huh7 replicon cells is significantly higher than that in parental Huh7 cells (49). To establish FU97 cured cells, two clones of FU97 replicon cells (clones 5 and 7) were treated with a combination of either 100 IU/ml of IFN-␣ and 100 nM BILN 2061 (clones 5-1 and 7-1) or 10 pM BMS-790052 and 100 nM BILN 2061 (clones 5-2 and 7-2) to eliminate viral RNA. Viral RNA was gradually decreased and was completely eliminated at 26 days posttreatment in four clones (Fig. 4D), and elimination of NS5A expression in cured cells was confirmed by immunoblot analysis (Fig. 4E). Next, to examine the susceptibility of the cured cells to the propagation of HCVcc, FU97 cured cell clones (clones 5-1 and 7-1) and parental FU97 cells were infected with HCVcc at an MOI of 1. The cured cells are more permissive to HCV infection, resulting in increased HCV RNA (Fig. 4F) and NS5A abundances (Fig.  4G) compared to the parental cells. These results suggest that susceptibility of the cured FU97 cells to the propagation of HCVcc is higher than that of parental cells, as seen in previous studies using hepatic and nonhepatic cells (17,18,49).
Cured FU97 cells exhibit normal innate immune response. It has been shown that one of the reasons for the high susceptibility of the cured cell line, Huh7.5 cells, to HCVcc infection is the impairment of the innate immune responses caused by mutation in RIG-I, a key sensor for viral RNA (50). To examine the involvement of the innate immune response in the enhancement of HCVcc propagation in the cured FU97 cells, the expression levels of IFN-stimulated gene 15 (ISG15) were determined upon stimulation with IFN-␣ or infection with VSV. Expression of ISG15 was significantly increased in both parental and cured FU97 cells by treatment with IFN-␣ or infection with VSV (Fig. 5A). To further confirm the innate immune responses in the cured FU97 cells, reporter plasmids encoding the luciferase gene under the control of either the IFN-␤ (Fig. 5B, left) or ISRE (Fig. 5B, right) promoter were transfected into both parental and cured FU97 cells and treated with IFN-␣ or inoculated with VSV. Activation of these promoters in the cured cells was comparable to that in the parental cells. To further assess the authenticity of viral RNA recognition and ISG induction pathways in the cured cells, nuclear localization of IRF3 and STAT2 upon stimulation was determined by immunofluorescence analysis. IRF3 and STAT2 in both parental and cured FU97 cells were translocated at similar levels into the nucleus upon infection with VSV or treatment with IFN-␣ (Fig.  5C). These results suggest that the efficient propagation of HCVcc in the FU97 cured cells is attributable to reasons other than impairment of innate immunity.
Expression of miR-122 is one of the determinants for HCV RNA abundances. We hypothesized that HCV replicon cells are capable of surviving in the presence of G418 by amplification of the viral genome through the enhancement of miR-122 expression, and cured FU97 cells acquired the ability to propagate HCVcc due to the high-level expression of miR-122. Our previous study also suggested that the expression levels of miR-122 in Huh7, Hep3B, and Hec1B cured cells were higher than those in parental cells (17,18). To test this hypothesis, the expression levels of miR-122 in the cured FU97 cells were compared with those in parental cells. Interestingly, the cured FU97 cell clones exhibited a 1.8-fold increase in miR-122 expression (Fig. 6A). These results suggested that the efficient propagation of HCVcc in the cured FU97 cells was attributable to enhanced expression of miR-122 rather than the impairment of the innate immunity. To further confirm the correlation between the expression of miR-122 and HCV RNA abundances, we established FU97 cell lines expressing various concentrations of miR-122 by using a lentiviral vector (Fig. 6B), and HCV RNA abundances in these cell lines upon infection with HCVcc were determined by qRT-PCR (Fig. 6C). HCV RNA abundances increased in accord with the expression of miR-122, suggesting that expression of miR-122 is one of the determinants for HCV RNA abundances in cells infected with HCVcc.
HCV particles produced in FU97 cells exhibit similar characteristics to those in hepatic cells. To examine the characteristics of viral particles produced in FU97 cells, HCV particles recovered from the culture supernatants of Huh7.5.1 and FU97 cells infected with HCVcc were fractionated by buoyant density gradient analysis. Previous reports indicated that viral RNA and infectious particles were broadly distributed, with peaks in fractions from 1.13 to 1.14 g/ml and from 1.09 to 1.10 g/ml, respectively (51,52). In agreement with the previous data, major peaks of HCV RNA and infectious particles in culture supernatants of both Huh7.5.1 and FU97 cells were detected around 1.10 g/ml and 1.09 g/ml, respectively ( Fig. 7A and 7B, upper panels). Furthermore, ApoE was detected around the peak fractions of infectivity in both Huh7.5.1 and FU97 cells ( Fig. 7A and B, lower panels). These results suggest that HCV particles produced in FU97 cells exhibit characteristics similar to those in hepatic cells. NS5B, and NS3/4A, respectively, were treated with various concentrations at 3 h postinfection with HCVcc, and the intracellular HCV RNA level was determined by qRT-PCR at 48 h postinfection. Treatment with these DAAs inhibited the HCV RNA level in a dose-dependent manner in both Huh7 and FU97 cells (Fig. 8A, bar graphs) and exhibited no cell toxicity at all even at the highest dose (Fig. 8A, line graphs). The inhibitory effects of BMS-790052 (Fig. 8A, top graphs) on the propagation of HCVcc in FU97 cells were higher than those in Huh7 cells, and the 50% effective concentration (EC 50 ) values of BMS-790052 against propagation of HCVcc in FU97 and Huh7 cells were 7.2 and 21.8 pM, respectively (P Ͻ 0.05). On the other hand, the antiviral effects of BILN 2061 (Fig. 8A, bottom graphs) on the propagation of HCVcc in FU97 cells were lower than those in Huh7 cells, and EC 50 s of BILN 2061 against propagation of HCVcc in FU97 and Huh7 cells were 65.0 and 38.9 nM, respectively (P Ͻ 0.01). PSI-7977 showed almost equivalent inhibitory effects to HCV propagated in FU97 and Huh7 cells, and the EC 50 s of PSI-7977 against propagation of HCVcc in FU97 and Huh7 cells were 34.6 and 44.1 nM, respectively (Fig. 8A, middle graphs). These results suggest that the antiviral effect of DAAs on the propagation of HCVcc varied between Huh7 and FU97 cells.

Effects of anti-HCV drugs on the propagation of HCV in
Next, we examined the efficacy of IFN-␣, RBV, and cyclosporine, which are inhibitors for HCV targeting host factors involved in the propagation of HCVcc (53)(54)(55), on the propagation of HCVcc in Huh7 and FU97 cells. Cells were treated with various concentrations of the reagents at 3 h postinfection with HCVcc, and the level of intracellular HCV RNA was determined by qRT-PCR at 48 h postinfection. In contrast to the treatment with DAAs, both Huh7 and FU97 cells exhibited cell toxicity by the treatment with RBV and cyclosporine but not with IFN-␣ at higher concentrations (Fig. 6B, line graphs). The inhibitory efficacies of IFN-␣ (Fig. 8B, top graphs) and cyclosporine (Fig. 8B, bottom graphs) on the propagation of HCVcc in FU97 cells were lower than those in Huh7 cells, and the EC 50 s of IFN-␣ against propagation of HCVcc in FU97 and Huh7 cells were 4.3 and 2.5 IU/ml, (P Ͻ 0.05), respectively; those of cyclosporine were 6.9 and 3.2 g/ml (P Ͻ 0.01), respectively. On the other hand, the antiviral effect of RBV on the propagation of HCVcc in FU97 cells was higher than that in Huh7 cells, and the EC 50 s of RBV against propagation of HCVcc in FU97 and Huh7 cells were 99.0 and 198.9 M, respectively (P Ͻ 0.05) (Fig. 8B, middle graphs). These results suggest that the efficacies of anti-HCV drugs targeting host factors involved in the infection of HCV were also different between Huh7 and FU97 cells.
FU97 cells exhibit higher susceptibility to HCVcc/JFH-2 propagation than Huh7 cells. HCVcc/JFH-2 was cloned from a patient with fulminant hepatitis and exhibited efficient propagation in Huh7 cured cells (34). In vitro-transcribed RNA of pJFH2/ AS/mtT4 encoding a full-length JFH-2 strain was electroporated into Huh7.5.1 cells, and HCVcc/JFH-2 of 1.5 ϫ 10 5 FFU/ml was recovered in the supernatants after serial passages. To examine the susceptibility of FU97 cells to the propagation of HCVcc/JFH-2, cells were infected with HCVcc/JFH-2 at an MOI of 1, and the intracellular HCV RNA level was determined by qRT-PCR. Intracellular HCV RNA in parental and cured FU97 cells increased until 72 h postinfection, while it reached a peak at 48 h postinfection in Huh7 cells, and the highest HCV RNA level was observed in the cured FU97 clones upon infection with HCVcc/JFH-2 (Fig.  9A). Infectious titers in the culture supernatants at 72 h postinfection with HCVcc/JFH-2 were also highest in the cured FU97 7-1 cells (2.5 ϫ 10 4 FFU/ml), followed by parental FU97 (1.2 ϫ 10 4 FFU/ml) and Huh7 (9 ϫ 10 3 FFU/ml) cells (Fig. 9B). Next, we examined the expression and subcellular localization of HCV proteins in cells infected with HCVcc/JFH-2 by immunofluorescence analysis. Expression of NS5A in cells upon infection with HCVcc/ JFH-2 was highest in the cured FU97 7-1 cells, followed by parental FU97 cells, and that in Huh7 cells was low (Fig. 9C, left panels). Core protein was detected around LDs in cells infected with HCVcc/JFH-2, as seen in those infected with the HCVcc/JFH-1 strain (Fig. 9C, right). To further confirm the efficient propagation of HCVcc/JFH-2 in FU97 cells, in vitro-transcribed viral RNAs of the JFH-1 and JFH-2 strains of HCVcc were electroporated into Huh7, FU97, and cured FU97 cells. Although the infectious titers of the JFH-1 strain in FU97 cells were lower than those in Huh7 cells, those of the JFH-2 strain in FU97 and cured FU97 cells were significantly higher than those in Huh7 cells (Fig. 9D). These results suggest that FU97 cells are more susceptible to propagate HCVcc/JFH-2 than Huh7 cells.

DISCUSSION
Several reports have shown that hepatic differentiation is involved in the susceptibility of ES/iPS cells to HCVcc infection (28,30,41). In addition, in hepatic cancer cell lines, including Huh7, HepG2, and Hep3B, cells derived from not poorly but well-differentiated HCC permit complete propagation of HCVcc (15)(16)(17), suggesting that hepatic differentiation is closely related to the susceptibility of cells to HCVcc propagation. In this study, we identified two cell lines susceptible to HCVcc infection by the screening of cancer cell lines expressing AFP as a marker of hepatic differentiation. HCCderived JHH-4 cells and gastric cancer-derived FU97 cells permit complete propagation of HCVcc without any exogenous expression of the host factors required for HCVcc propagation, including HCV receptor candidates, miR-122, and apolipoproteins. In particular, FU97 cells exhibited higher susceptibility to HCVcc/ JFH-2 infection than Huh7 cells, suggesting that FU97 cells would be useful tools for further HCV analyses.
Although HCV has been classified into seven major genotypes and a series of subtypes (56,57), the in vitro infection model had been restricted to the JFH-1 strain based on the genotype 2a until  recently (15). To clarify the pathogenesis of HCV depending on the genotypes, the establishment of cell-culture-adapted clones derived from various genotypes is essential (58). Viable JFH1based intergenotypic recombinants, containing genotype-specific structural proteins, p7 and the complete or partial NS2, were generated for various genotypes of HCV (56,59,60). Although robust propagation systems of full-length HCV infectious clones of the H77 strain (genotype 1a) (61), TN strain (1a) (62), JFH-2 strain (2a) (34), and S310 strain (3a) (63) were established, the construction of infectious clones of other genotypes has not succeeded yet.
Because permissive cell lines for HCVcc infection in vitro had been limited to Huh7 cells due to cell tropism and the narrow host range (13,14), the establishment of a novel cell culture system supporting HCV propagation is needed for further HCV analyses. Previous reports have demonstrated that HepG2, Hep3B, and HEK293 cells permit HCVcc propagation (16,17,64). However, exogenous expression of host factors is necessary for complete propagation of HCVcc in these cell lines. In HepG2 and Hep3B cells, overexpression of miR-122 is essential for efficient replication of HCV RNA (16,17). In HEK293 cells, the exogenous expression of CLDN1, miR-122, and ApoE was required for infectious particle formation upon infection with HCVcc (64). On the other hand, JHH-4 and FU97 cells permit complete propagation of HCVcc without any exogenous expression of the host factors required for propagation of HCVcc. JHH-4 cells grown in a threedimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier and transfection of HCV RNA transcribed from full-length cDNA (44). In addition, JHH-4 cells were suggested to possess some host factors involved in the enhanced translation of HCV RNA (64,65). Furthermore, high susceptibility of FU97 cells to HCVcc/JFH-2 infection compared to Huh7 cells raises the possibility of using FU97 cells for the propagation of HCVcc derived from other genotypes, including the H77, TN, and S310 strains.
AFP-producing gastric cancer (AFPGC) cell lines, FU97 and Takigawa cells (66), which were identified by using a cDNA array database, were shown to express high levels of liver-specific factors. AFPGC is a rare case and exhibits a worse prognosis and the characteristics of early hepatic metastasis (67). It is hypothesized that production of AFP, which is suppressed in mature hepatocytes, is induced in HCC by the dedifferentiation of cancer cells or the increase in oval cells in the oncogenic pathway (68). Oval cells are believed to be capable of producing AFP, are candidates for hepatic stem cells, have bipotentiality to differentiate into hepatocytes and bile duct epithelial cells, and play an important role in liver regeneration (69,70). These hypotheses suggest that cancer cells acquired a new function, such as the ability to produce AFP through an alteration in differentiation status. Although the mechanism of AFP production in gastric cancer remains unknown, hepatic dedifferentiation might be induced in gastric cancer. Furthermore, previous reports have proposed the concept of "hepatoid adenocarcinoma" based on the differentiation of AFPGC into hepatocyte-like cells (71,72), suggesting that FU97 and Takigawa cells obtained the hepatocyte-like characteristics required for HCV propagation through dedifferentiation during the oncogenic process. In addition, recent studies demonstrated that hepatocyte-like cells derived from induced pluripotent stem cells (iPS cells/iPSCs) express high levels of miR-122 and VLDLassociated proteins and support propagation of HCVcc and HCV derived from patient serum (28)(29)(30). These results suggest that hepatic differentiation required for hepatic functions plays crucial roles in HCV propagation. In accord with these observations, our data suggest that cancer cell lines differentiated into hepatocyte-   FU97 cells were harvested at 72 h postinfection with HCVcc and analyzed by using iodixanol density gradient centrifugation. HCV RNA and infectious titers of each fraction were determined by qRT-PCR and focus-forming assay, respectively. Buoyant density was plotted for each fraction (upper panels). Expression of ApoE in each fraction was detected by immunoblotting using anti-ApoE antibody (lower panels). like cells to gain hepatic functions could permit complete propagation of HCVcc.
Treatment with DAAs including BMS-790052 (NS5A inhibitor) (73), PSI-7977 (NS5B polymerase inhibitor) (74), and BILN 2061 (NS3/4A protease inhibitor) (75) inhibited propagation of HCV in both Huh7 and FU97 cells infected with HCVcc without any cell toxicity. Antiviral effects of BMS-790052 and BILN 2061 were significantly different between Huh7 and FU97 cells, suggesting that efficacies of DAAs are varied, depending on cell lines. Although anti-HCV drugs targeting host factors including IFN-␣, RBV, and cyclosporine also inhibited propagation of HCVcc in a dose-dependent manner in both Huh7 and FU97 cells, treatment with RBV and cyclosporine produced cell toxicity at higher concentrations than treatment with DAAs. Although the antiviral mechanism of RBV against HCV has not been well elucidated yet (53), inhibitory effects of RBV against HCV infection were significantly higher in Li23 cells than those in Huh7 cells (76,77), and RBV also exhibited a low inhibitory effect upon infection with HCVcc in Huh7 cells compared to that in FU97 cells. Although adenosine kinase (ADK) was shown to be a determinant for the sensitivity of RBV (78), the expression levels of ADK in Huh7 and FU97 cell lines were comparable (data not shown).
The IL28B genotype is associated with the sensitivity of IFN treatment for chronic hepatitis C patients (79)(80)(81), and patients with the minor IL28B genotype exhibit lower susceptibility to the treatment than those with major genotypes. Although FU97 cells showed lower sensitivity to the IFN-␣ treatment than Huh7 cells, FU97 and Huh7 cells possess major and minor IL28B genotypes , and FU97 cured 7-1 cells were infected with HCVcc/JFH-2 at an MOI of 1, and infectious titers in the culture supernatants were determined by focus-forming assay. (C) Huh7, FU97, and FU97 cured 7-1 cells were infected with HCVcc/JFH-2 at an MOI of 1, fixed with 4% PFA at 72 h postinfection, and subjected to immunofluorescence assay using antibodies against NS5A or core. Lipid droplets and cell nuclei were stained with BODIPY and DAPI, respectively. (D) In vitro-transcribed JFH-1 and JFH-2 RNAs were electroporated into Huh7, FU97, and FU97 cured 7-1 cells. The infectious titers of JFH-1 and JFH-2 in the culture supernatants from these cells were determined by focus-forming assay up to 14 days postransduction. Asterisks indicate significant differences (*, P Ͻ 0.05; **, P Ͻ 0.01) versus the results for control cells.
(data not shown), respectively. Furthermore, induction of ISG15 by treatment with IFN-␣ was almost comparable between Huh7 and FU97 cells (Fig. 5A), and expression levels of IFN-␣ receptor in the cell lines were the same (data not shown), suggesting the involvement of other factors in the difference in the IFN responses between FU97 and Huh7 cells.
Cyclophilins possess peptidyl-prolyl cis/trans isomerase (PPIase) activity and are involved in protein folding and assembly. Cyclophilin A (CypA), the most abundant cyclophilin, localizes in the cytoplasm and interacts with the immunosuppressive drug cyclosporine (82). In addition, CypA has been shown to be involved in the propagation of human immunodeficiency virus (83,84), hepatitis B virus (85,86), influenza A virus (87), and HCV (88). Replication of HCV RNA was inhibited by suppression of the PPIase activity of CypA by treatments with cyclosporine, mutation in the active site of CypA, and knockdown of CypA (55,(89)(90)(91). The same level of CypA expression in Huh7 and FU97 cells (data not shown) suggests that the difference in inhibitory effect of cyclosporine in the cell lines may be attributable to other reasons, such as a difference in PPIase activity of CypA in these cell lines. The differences in the efficacy of anti-HCV drugs between Huh7 and FU97 cells were small; however, FU97 cells have the possibility to possess antiviral activity different from that of Huh7 cells.
In summary, we identified novel permissive cell lines for complete propagation of HCVcc without any artificial manipulation. In particular, gastric cancer-derived FU97 cells exhibited a much higher susceptibility to HCVcc/JFH-2 infection than observed in Huh7 cells, suggesting that FU97 cells would be useful for further investigation of the HCV life cycle, as well as the development of therapeutic agents for chronic hepatitis C.