Novel Permissive Cell Lines for Complete Propagation of Hepatitis C Virus
- T. S. Dermody, Editor
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FIG 1JHH-4 and FU97 cells express high levels of the liver-specific host factors required for HCV propagation. (A) Expression of AFP, ALB, ApoB, and ApoE in cancer cell lines screened by the NextBio Body Atlas application. The expression levels were standardized by the median expression across all cell lines. (B) Expression of AFP, ALB, ApoB, ApoE, MTTP, and miR-122 in AFP-expressing cell lines including HepG2, Hep3B, FU97, and OV-90 cells identified by NextBio Body Atlas and Huh7, JHH-4, and 293T cells was determined by qPCR. The relative expression of AFP, ApoB, ApoE, MTTP, and ALB mRNA was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, and that of miR-122 was normalized to that of U6 snRNA. (C) Secretion of ApoB in the culture supernatants of Huh7, JHH-4, FU97, OV-90, and 293T cells was determined by immunoblotting by using anti-ApoB antibody. The molecular mass of ApoB100 secreted from hepatocyte is about 500 kDa. (D) Expression of CLDN1, SR-BI, and OCLN in these cell lines was determined by immunoblotting. (E) Expression of hCD81 in the cell lines was determined by flow cytometry. (F) HCVpv-bearing HCV envelope proteins and control virus (Ctrlpv) were inoculated into the cell lines, and luciferase activities were determined at 24 h postinfection. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) versus the results for control virus.
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FIG 2JHH-4 and FU97 cells permit HCV propagation. (A) Intracellular HCV RNA levels in Huh7, JHH-4, and FU97 cells inoculated with HCVcc at an MOI of 1, treated with 100 IU/ml of IFN-α or untreated (mock), were determined by qRT-PCR at 12, 24, 48, and 72 h postinfection (hpi). Infectious titers in the culture supernatants of Huh7, JHH-4, FU97, and 293T-CLDN/miR-122 cells infected with HCVcc at an MOI of 1 were determined by a focus-forming assay at 72 h postinfection (bar graph). (B) Exogenous expression of miR-122 in Huh7, JHH-4, and FU97 cells by lentiviral vector (bar graph). Total cellular miRNA extracted from the cells was subjected to qRT-PCR. U6 was used as an internal control. Intracellular HCV RNA in Huh7, JHH-4, and FU97 cells inoculated with HCVcc at an MOI of 1 was determined by qRT-PCR at 12, 24, 48, and 72 h postinfection. Solid and broken lines indicate HCV RNA abundances in miR-122-expressing and GFP-expressing control cells, respectively. (C) Huh7, JHH-4, and FU97 cells were infected with HCVcc at an MOI of 1, fixed with 4% PFA, and subjected to immunofluorescence analyses by using antibodies against core, NS5A, dsRNA, and calregulin. Lipid droplets and cell nuclei were stained by BODIPY and DAPI, respectively. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) versus the results for control cells.
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FIG 3JHH-4 and FU97 cells permit complete propagation of HCVcc without any exogenous expression of host factors crucial for propagation of HCVcc. (A) Effect of inhibitors on the propagation of HCVcc in Huh7, JHH-4, and FU97 cells. (Left panels) HCVcc was preincubated with anti-E2 antibody and inoculated into cells. Cells were preincubated with anti-hCD81 antibody or isotype control antibody (Ctrl IgG) and then infected with HCVcc. (Right panels) Cells were infected with HCVcc and treated with miR-122-LNA (30 nM) or Ctrl-LNA (30 nM) at 6 h postinfection. (B) Huh7, JHH-4, and FU97 cells infected with HCVcc at an MOI of 1 were treated with dimethyl sulfoxide (DMSO) or MTTP inhibitor, CP-346086 (5 μM)or BMS-200150 (10 μM), at 3 h postinfection. Intracellular HCV RNA in cells at 12, 24, 48, and 72 h postinfection was determined by qRT-PCR (left panels). Infectious titers in the culture supernatants of cells infected with HCVcc at an MOI of 1 and treated with 5 μM CP-346086, 10 μM BMS-200150, or dimethyl sulfoxide alone (DMSO) at 3 h postinfection were determined at 72 h postinfection by a focus-forming assay (right graphs). (C) mRNA and protein expression levels of ApoB and ApoE (left panels) in Huh7, JHH-4, and FU97 cells at 48 h posttransfection with siRNA targeting either ApoB or ApoE or a control siRNA (siApoB, siApoE, or siCtrl, respectively) were determined by qRT-PCR and immunoblotting, respectively. Huh7, JHH-4, and FU97 cells were infected with HCVcc at an MOI of 1 at 6 h posttransfection with siRNA targeting either ApoB or ApoE or a control siRNA (siApoB, siApoE, or siCtrl, respectively) (right panels). Intracellular HCV RNA at 12, 24, 48, and 72 h postinfection and infectious titers in the culture supernatants at 72 h postinfection were determined by qRT-PCR and focus-forming assay, respectively. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) versus the results for control cells.
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FIG 4Establishment of HCV RNA replicon and cured FU97 cells. (A) Wild-type SGR RNA (Con1-SGR) or replication-defective RNA (Con1-GND) of the HCV Con1 strain was electroporated into Huh7 and FU97 cells and replaced with medium containing 1 mg/ml and 400 μg/ml of G418 at 24 h postelectroporation, respectively. Colonies were stained with crystal violet at 30 days postselection. (B) Four clones derived from FU97 SGR cells (clones 5, 7, 9, and 11) were subjected to qRT-PCR after extraction of total RNA (upper panel) and to immunoblotting using anti-NS5A antibody (lower panel). Huh9-13 cells, which were Huh7-derived Con1-SGR cells, were used as a positive control. (C) Huh9-13 cells, Huh7 parental cells, FU97-derived Con1-SGR cells (FU97 SGR, clone 5), and FU97 parental cells were fixed in 4% PFA and subjected to immunofluorescence assay using anti-NS5A and anti-dsRNA antibodies. Cell nuclei were stained by DAPI. (D) Elimination of HCV RNA from FU97-derived Con1-SGR cells. Two clones derived from FU97 SGR cells (clones 5 and 7) were treated with a combination of either 100 IU/ml of IFN-α and 100 nM BILN 2061 (clones 5-1 and 7-1) or 10 pM of BMS-790052 and 100 nM BILN 2061 (clones 5-2 and 7-2) to eliminate the HCV genome. Clones 5-Ctrl and 7-Ctrl are negative controls, untreated with anti-HCV drugs. Intracellular HCV RNA at 3, 8, 11, 18, 22, and 26 days posttreatment was determined by qRT-PCR. (E) The expression levels of NS5A in FU97 SGR cells (clones 5 and 7) and in FU97 cured cells (clones 5-1 and 7-1) were determined by immunoblot analysis using anti-NS5A antibody. (F) FU97 cured cells (clone 5-1 and clone 7-1) and parental cells were infected with HCVcc at an MOI of 1; the levels of intracellular HCV RNA at 12, 24, 48, and 72 h postinfection were determined by qRT-PCR. (G) The expression of NS5A in Huh7, Huh7.5.1, FU97, and cured FU97 clone 7-1 was determined by immunofluorescence analysis at 72 h postinfection by using anti-NS5A antibody. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) versus the results for control cells.
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FIG 5Innate immune response in cured FU97 cells. (A) Huh7, parental, and cured FU97 cells (clone 7-1) were stimulated with 100 IU/ml of IFN-α or infected with VSV. The expression of mRNA of ISG15 at 4, 8, 12, and 24 h posttreatment (hpt) was determined by qPCR and standardized by that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) Huh7, parental FU97, and cured FU97 (clone 7-1) cells cotransfected with pIFN-β-Luc and pRL-SV40 were infected with VSV at an MOI of 1 at 24 h posttransfection (left). Cells cotransfected with pISRE-Luc and pRL-SV40 were infected with VSV at an MOI of 1 or stimulated with 100 IU/ml of IFN-α at 24 h posttransfection (right). Luciferase activities were determined at 24 h posttreatment. (C) Huh7, parental FU97, and cured FU97 (clone 7-1) cells were infected with VSV at an MOI of 1 or stimulated with 100 IU/ml of IFN-α, fixed with 4% PFA at 18 h posttreatment, and subjected to immunofluorescence assay using anti-IRF3 and -STAT2 antibodies. Cell nuclei were stained by DAPI. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) from the results for control cells.
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FIG 6Expression of miR-122 is one of the determinants for HCV RNA abundances. (A) Total RNA was extracted from Huh7 and parental and cured FU97 (clones 5-1 and 7-1) cells, and the relative expression of miR-122 was determined by qPCR. U6 snRNA was used as an internal control. (B) Establishment of FU97 cell lines stably expressing various concentration of miR-122 by infection with a lentiviral vector. FU97 cells infected with lentiviral vector to express GFP were used as a control. (C) FU97 cell lines expressing various concentrations of miR-122 were infected with HCVcc at an MOI of 1, and HCV RNA abundances were determined at 12, 24, 48, and 72 h postinfection (hpi) by qRT-PCR. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) versus the results for control cells.
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FIG 7HCV particles produced in FU97 cells exhibit similar characteristics to those in hepatic cells. HCV particles in the culture supernatants of Huh7.5.1 and 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).
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FIG 8Effects of anti-HCV drugs on the propagation of HCVcc in FU97 cells. (A) Effect of DAAs on the propagation of HCVcc in Huh7 and FU97 cells. Cells infected with HCVcc at an MOI of 1 were treated with BMS-790052, PSI-7977, and BILN 2061 at 3 h postinfection (identifications in right-hand panels). (B) Effect of HCV inhibitors targeting host factors on the propagation of HCVcc in Huh7 and FU97 cells. Cells infected with HCVcc at an MOI of 1 were treated with IFN-α, RBV (middle), and cyclosporine (CsA) at 3 h postinfection (identifications in right-hand panels). Intracellular HCV RNA levels were determined by qRT-PCR at 48 h postinfection (bar graphs), and cell viability was determined as a percentage of the viability of cells treated with 0.1% dimethyl sulfoxide (DMSO) at 48 h posttreatment (line graphs). From the assay results, the 50% effective concentration (EC50) of each reagent was determined. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) versus the results for control cells.
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FIG 9Propagation of HCVcc/JFH-2 in FU97 cells. (A) Huh7, FU97 parental, FU97 cured 5-1, and FU97 cured 7-1 cells were infected with HCVcc/JFH-2 at an MOI of 1, and the intracellular HCV RNA level was determined by qRT-PCR at 12, 24, 48, and 72 h postinfection. (B) Huh7, FU97, 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 < 0.05; **, P < 0.01) versus the results for control cells.
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