HCV NS4B protein increases the transcriptional activity of SREBP-2. A, Huh7 cells were cotransfected with SRE-Luc reporter plasmid together with the indicated expression plasmids. At 36 h after transfection, cells were harvested, and luciferase activities were determined. The amount of DNA in each transfection was kept constant by adding an appropriate amount of pEF6-Myc empty vector. Data represent the mean of two independent experiments. B, HCV NS4B increases the transcriptional activity of SREBP-2 in a dose-dependent manner. Huh7 cells were cotransfected with SRE-Luc reporter plasmid together with increasing amounts of Myc-tagged NS4B expression plasmid. At 36 h after transfection, cells were harvested, and then luciferase activities were determined (upper panel). Equal amounts of cell lysates were subjected to immunoblotting with anti-Myc monoclonal antibody (lower panel). C, both vector and NS4B-Myc stable cells were transfected with SRE-Luc reporter plasmid. At 36 h after transfection, cells were harvested and then luciferase activities were determined. D, Huh7 cells were transfected with either vector control or NS4B-Myc expression plasmid. At 36 h after transfection, total cell lysates were immunoblotted with the indicated antibodies. E, total cell lysates harvested from both vector stable and NS4B-Myc stable cells were immunoblotted with the indicated antibodies. Protein expression of β-actin was used as a loading control for the same amount of cell lysates.

HCV NS4B protein up-regulates RNA levels of both SREBP-1 and its target genes. A and B, total RNAs were isolated from vector stable, NS4B-Myc stable, IFN-cured, and HCV subgenomic replicon cells. The cDNAs were synthesized by avian myeloblastosis virus reverse transcriptase from 1 μg of total RNAs. To estimate transcriptional levels of SREBP-1 and its target genes (FAS, SCD, and ACC), cDNAs were subjected to PCR amplification using gene-specific primers, respectively. The amplified DNA was analyzed by agarose gel electrophoresis.

AKT is required for NS4B-mediated SREBP activation. A, total cell lysates harvested from both vector stable and NS4B-Myc stable cells were immunoblotted with the indicated antibodies (upper panel). Triplicate experimental data of p-AKT levels were quantified (lower panel). B, Huh7 cells were transiently transfected with either vector or NS4B-Myc expression plasmid. At 24 h after transfection, cells were harvested and immunoblotted with the indicated antibodies (upper panel). Data from triplicate experiments were quantified and each bar represents the average intensity of p-AKT level (lower panel). C, both vector stable and NS4B-Myc stable cells were transfected with either FAS-Luc (upper panel) or SRE-Luc (lower panel) reporter plasmid. At 24 h after transfection, cells were either left untreated (Me₂SO) or treated with LY294002 (5 μm) for an additional 12 h. Cells were harvested and then luciferase activities were determined. Data represent the mean of two independent experiments. D, total cellular extracts used in C were immunoblotted with the indicated antibodies. Quantification of the band intensity was determined by using a calibrated GS-800 densitometer.

HCV NS4B protein increases the transcriptional activity of SREBP-1. A, Huh7 cells were cotransfected with FAS-Luc reporter plasmid together with the indicated expression plasmids. At 36 h after transfection, cells were harvested and then luciferase activities were determined. The amount of DNA in each transfection was kept constant by adding an appropriate amount of pEF6-Myc empty vector. Data represent the mean of two independent experiments. B, HCV NS4B increases the transcriptional activity of SREBP-1 in a dose-dependent manner. Huh7 cells were cotransfected with FAS-Luc reporter plasmid together with increasing amounts of Myc-tagged NS4B expression plasmid. At 36 h after transfection, cells were harvested, and luciferase activities were determined (upper panel). Equal amounts of cell lysates were subjected to immunoblotting with anti-Myc monoclonal antibody (lower panel). C, both vector and NS4B-Myc stable cells were transfected with FAS-Luc reporter plasmid. At 36 h after transfection, cells were harvested and then luciferase activities were determined. D, Huh7 cells were transfected with either vector control or NS4B-Myc expression plasmid. At 36 h after transfection, total cell lysates were immunoblotted with the indicated antibodies. E, both vector stable and NS4B-Myc stable cell (#3) lysates harvested from each cell line were immunoblotted with the indicated antibodies. Protein expression of β-actin was used as a loading control for the same amount of cell lysates. F, Huh7 cells were cotransfected with 550-Luc reporter plasmid together with NS4B-Myc expression plasmid. At 36 h after transfection, cells were harvested and then luciferase activities were determined. Data represent the mean of two independent experiments.

HCV subgenomic replicon increases both SREBP-1 and SREBP-2 activities. Both IFN-cured and HCV subgenomic replicon cells were transfected with either FAS-Luc (A) or SRE-Luc (B) reporter plasmid. At 36 h after transfection, cells were harvested and then luciferase activities were determined. Data represent the mean of two independent experiments. C, total cell lysates harvested from IFN-cured and HCV subgenomic replicon cells were immunoblotted with the indicated antibodies. Protein expression of β-actin was used as a loading control for the same amount of cell lysates. D, both IFN-cured and HCV subgenomic replicon cells were transfected with 550-Luc reporter plasmid. At 36 h after transfection, cells were harvested, and luciferase activities were determined. Data represent the mean of two independent experiments.

HCV NS4B protein induces lipid accumulation through activation of SREBPs. A, both vector and NS4B-Myc stable cells were cotransfected with FAS-Luc reporter and Flag-SREBP-1a (1–490 amino acids) or HA-SREBP-1c (1–447 amino acids) expression plasmids. At 36 h after transfection, cells were harvested and then luciferase activities were determined (top panel). Data represent the mean of two independent experiments. ^***, p < 0.001, vector stable versus NS4B stable cells transfected with Flag-SREBP-1a. ^*, p < 0.05, vector stable versus NS4B stable cells transfected with HA-SREBP-1c. Equal amounts of cell lysates were subjected to immunoblotting with anti-FLAG, anti-HA, anti-Myc, and anti-β-actin monoclonal antibody (bottom panel). B, Huh7 cells were transfected with NS4B-Myc expression plasmid. At 36 h after transfection, cells were fixed and incubated with anti-Myc monoclonal antibody for 2 h. After being washed with PBS, cells were further incubated with TRITC-conjugated goat anti-mouse IgG and BODIPY 493/503 (1 μm, Invitrogen) for 1 h. Samples were analyzed for immunofluorescence staining using the LSM 510 laser confocal microscopy system and BODIPY intensity was quantified. Each bar represents the average intensity of BODIPY staining. ^***, p < 0.001, NS4B-negative cells versus NS4B-positive cells. C, Huh7 cells were either mock-infected or infected with HCV JFH-1. At 3 days after infection, cells were transfected with FAS-Luc reporter plasmid. At 24 h after transfection, cells were harvested and then luciferase activities were determined. D, at 3 days after infection, total cell lysates were immunoblotted with either anti-FAS antibody (top panel) or anti-NS5A antibody (middle panel). Protein expression of β-actin was used as a loading control for the same amount of cell lysates (bottom panel). E, at 3 days after infection, cells were fixed and incubated with anti-NS5A polyclonal antibody for 2 h. After being washed with PBS, cells were further incubated with TRITC-conjugated goat anti-mouse IgG and BODIPY 493/503 (1 μm, Invitrogen) for 1 h. Immunofluorescence staining was performed as described in B. ^***, p < 0.001, mock-infected cells versus HCV-infected cells. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei.

HCV core-mediated transcriptional activation of SREBP-1 was synergistically elevated by NS4B protein. A, Huh7 cells were transfected with vector, NS4B-Myc, FLAG-core, individually or cotransfected with NS4B-Myc and FLAG-core in the presence of FAS-Luc reporter plasmid. At 36 h after transfection, cells were harvested, and luciferase activities were determined. Data represent the mean of two independent experiments. ^*, p < 0.05, core versus HCV NS4B/core. ^**, p < 0.01, vector versus HCV NS4B. ^***, p < 0.001, vector versus HCV core. B, total cellular extracts used in A were immunoblotted with the indicated antibodies. Quantification of the band intensity was determined by using a calibrated GS-800 densitometer.