Fig. 1. Suppression of HCV RNA by IFN-γ and H₂O₂. SgPC2 cells were treated with various concentrations of H₂O₂ or IFN-γ, as indicated. After 24 h, total RNA was analyzed for HCV RNA and GAPDH mRNA by Northern blots, and the images were analyzed with a phosphoimager (Cyclone; Perkin–Elmer). The intensities of the HCV RNA bands were normalized against that of GAPDH mRNA and expressed as a percentage of the respective control. A representative Northern blot is also shown. Note that these and all other experiments in the subsequent figures were repeated two to eight times, and data are presented as means ± EM of several independent experiments.

Fig. 2. Buffering [Ca²⁺]_i prevented the suppression of subgenomic and genomic HCV RC by ROS. SgPC2 cells were pretreated with 2 μM BAPTA-AM (Molecular Probes, Inc.) or the vehicle control alone (0.02% DMSO) for 0.5 to 1 h. (A) [Ca²⁺]_i was monitored after loading cells with Indo-1 AM. H₂O₂ (100 μM) was added at 60 s. AUC were 935.9 ± 58.9 Ca²⁺/min (without BAPTA) and 128.4 ± 82.3 Ca²⁺/min (with BAPTA), and the difference was significant (p < 0.05). (B) SgPC2 cells were labeled with [³H]uridine while treating them with H₂O₂ for 6 h in the presence of actinomycin D. Then, the RNA was isolated and analyzed, as described under Materials and methods. The gel was stained with ethidium bromide to compare the amount of rRNA present in each lane (bottom), which served as the loading control. (C) SgPC2 cells were treated with H₂O₂ for 30 min or 3 h, and the cytoplasmic extracts were subjected to in vitro replication assay. The RNA products were isolated and analyzed on a 1% RNA gel. The gel was stained with ethidium bromide to compare the amount of rRNA in each lane (bottom) as a loading control. (D) SgPC2 cells were treated with H₂O₂ for 24 h. Then, total RNA was analyzed for HCV RNA and GAPDH mRNA by Northern blots, and the images were analyzed with a phosphoimager. The intensities of the HCV RNA bands were normalized against that of GAPDH mRNA and expressed as a percentage of the respective control: H₂O₂, 36.8 ± 16.6% of untreated control; H₂O₂ + BAPTA, 139.8 ± 20.2% of BAPTA-treated control. (E) BAPTA removed the suppression of genomic HCV RNA replication by H₂O₂. Huh7 cells were transiently transfected with the genomic HCV RNA and then treated with H₂O₂ for 24 h, with 1 h pretreatment with 2 μM BAPTA-AM or control DMSO. Total RNA was analyzed for HCV RNA by Northern blot. GAPDH mRNA was also analyzed as the control. HCV RNA with H₂O₂ was 34.0 ± 4.0% of untreated control; H₂O₂ + BAPTA was 111.5 ± 4.5% of BAPTA-treated control.

Fig. 3. Continuous exposure to H₂O₂ suppressed HCV RNA replication. SgPC2 cells were treated with GO for 24 h, with and without cotreatment with 130–200 U/ml catalase or with 1 h pretreatment with 2 μM BAPTA-AM or DMSO. (A and B) HCV RNA and GAPDH mRNA levels were determined by Northern blot analysis and the images were analyzed with a phosphoimager. The intensities of the HCV RNA bands were normalized against that of GAPDH mRNA and expressed as a percentage of the control. Data represent means ± SEM. (C) HCV NS5A protein level was analyzed by Western blot, as described under Materials and methods.

Fig. 4. GSH and HCV RNA replication. SgPC2 cells were incubated with 20–40 μM BSO with and without H₂O₂ for 24 h and analyzed for (A) GSH or (B) HCV RNA and GAPDH mRNA levels by Northern blots. Total GSH was expressed as nmol/mg total protein. The RNA bands were analyzed with a phosphoimager. GAPDH mRNA served as the control. Data represent means ± SEM. *Statistically significant difference by Student's t test (p ≤ 0.05).

Fig. 5. H₂O₂ induced calcium release from the ER. SgPC2 cells were loaded with Indo-1 AM, and [Ca²⁺]_i was monitored in the absence of extracellular calcium, unless specified otherwise. (A) H₂O₂ or water was added at 60 s and calcium was monitored in the presence and absence of 1.3 mM extracellular calcium. AUC of peroxide-induced calcium elevations were 1278.5 ± 109.5 Ca²⁺/min, with extracellular calcium, and 1105.1 ± 238.1 Ca²⁺/min, without extracellular calcium, which were not different (p > 0.05). (B and C) Cells were pretreated with (B) H₂O₂ for 13 min or (C) 400 nM TG or control DMSO for 10 min before calcium measurement. Arrows indicate the addition of peroxide (A and C) or TG (B). H₂O₂ decreased TG-induced calcium elevation to 9.2 ± 2.6% of control (AUC of TG-induced calcium response in control cells, 772.3 ± 189.3 Ca²⁺/min; p < 0.05) (B). Likewise, TG decreased the peroxide-induced calcium elevation to 58.3 ± 8.4% of the control (p < 0.05) (C).

Fig. 6. The suppression of HCV replication by H₂O₂ required an internal calcium store(s). SgPC2 cells were incubated in normal medium (10% FBS) or medium with 0% FBS, serum-starved overnight with 0.5% FBS and then incubated in 0% FBS for 1 h, incubated in KRPH, or pretreated with DMSO control or 5 μM ionomycin in calcium-free KRPH buffer for 15 min. Then, the cells were treated with H₂O₂ for 30 min. The cytoplasmic lysates were prepared and analyzed for in vitro HCV replication. The rRNA gel shows RNA loading. HCV RNA bands were analyzed with a phosphoimager, normalized against the rRNA bands, and expressed as a percentage of the respective control. Data represent means ± SEM.

Fig. 7. Elevated calcium might be sufficient to suppress HCV replication. (A) SgPC2 cells were treated with 400 nM and 2 μM TG, DMSO control, H₂O₂, or 10 μM ionomycin for 15 min. Then, cytoplasmic lysates were prepared and analyzed for in vitro HCV replication. Data represent means ± SEM. (B) CaCl₂ was added to untreated cytoplasmic lysates at concentrations shown before in vitro replication assay. The rRNA gel shows RNA loading. The RNA bands were analyzed with a phosphoimager, normalized against the rRNA bands, and expressed as a percentage of the respective control. Data represent means ± SEM. (C) CaCl₂ was added to purified full-length NS5B at concentrations shown before in vitro RdRp assay. ZnCl₂ (0.5 mM) was used as a positive control.