1E). The response to HO-1 induction was found to be less prominent in the replicon cell line, compared with the parental cell line, whereas HO-1 expression in untreated Huh-5-15 cells was elevated compared with untreated Huh-7 cells (Fig. 1E). Induction of HO-1 in both cell lines increased the expression of ferritin (Fig. 1F), indicating bioactivity of HO-1. Similar effects of HO-1 on HCV replication were measured click here in the LucUbiNeo-ET replicon cell line by luciferase assay (Fig. 2A). Reduction of HCV replication by HO-1 induction was also detectable at the protein level. Incubation of Huh-5-15

replicon cells in the presence of 10 μg/mL CoPP resulted in decreased NS5-protein and increased HO-1-protein expression (Fig. 2B). To determine the downstream mediator or mediators of HO-1 responsible for its effects on HCV replication, we first incubated Huh-5-15 or LucUbiNeo-ET replicon cells in the presence of the CO donor MC. Incubation was able to reduce HCV replication, as CP-690550 research buy detected by luciferase reporter assay dose-dependently (Fig. 3A). HO-1 expression in cells incubated in the presence of 100 mM MC for 6 hours was slightly increased, as measured by real-time RT-PCR, but induction was not significant (untreated: 1.011 ± 0.05235, n = 12; MC treated: 1.175 ± 0.1212, n = 10; P = 0.2012). The effect of MC on HCV replication was transient; it was no longer detectable

at 24 hours after the start of incubation (Fig. 3B). Induction of HO-1 results in a release of iron, which in turn induces ferritin (Fig. 1F) and also might contribute to a reduction of HCV replication. We therefore tried to reverse the inhibitory effect of CoPP on HCV replication by co-incubating cells with the iron-trapping agent deferoxamine (Fig. 3C, D). Our results show that, whereas CoPP incubation reduced HCV replication, co-incubation with deferoxamine did not reverse this effect, as measured by real-time RT-PCR for NS5B expression

(Fig. 3C) or luciferase reporter assay (Fig. 3D). We also tried to mimic an iron effect by adding iron 17-DMAG (Alvespimycin) HCl III chloride solution (FeCl3) or iron-saturated lactoferrin to LucUbiNeo-ET replicon cells. Our results show that neither addition of FeCl3 (Fig. 3E) nor addition of lactoferrin (Fig. 3F) reduced HCV replication, as measured by luciferase assay. Incubation of LucUbiNeo-ET replicon cells in the presence of biliverdin dose-dependently interfered with HCV replication, as detected by luciferase assay (Fig. 4A). The same result was obtained in Huh-5-15 cells by real-time RT-PCR (Fig. 4B) and western blot (Fig. 4C) for the HCV nonstructural protein NS5. The effect of biliverdin was not attributable to overall induction of HO-1 (Fig. 4B, C). To exclude unspecific effects of the green biliverdin solution on luciferase activity, we used a reporter construct constitutively expressing casein kinase 2 beta subunit as an unspecific control.