Fig 3. Replicon clearance, replication rebound and resistance analyses.

A. Clearance and replication rebound: Con1 replicon Huh-7 cells were passaged 7 times in the absence or presence of STG-175 or CsA (0.25, 0.5 and 1 μM). Then drugs were removed to allow viral replication rebound to occur. At each passage, samples of cells were collected, RNA extracted and analyzed by RT-PCR for HCV RNA levels. B. Emergence of resistance: Con1 replicon Huh-7 cells were passaged 22 times in the absence or presence of STG-175 or selected DAAs. At each passage, sample of cells were collected, RNA extracted and analyzed by RT-PCR for HCV RNA levels. C. Same as B except that selected CypI were compared–STG-175, ALV and CsA. Data are expressed as number of HCV RNA copies per cell. Data are representative of two independent experiments. D. Degree of resistance of STG-175-resistant colonies: STG-175-resistant colonies, which emerged under drug selection, were analyzed for their resistance to increasing concentrations of STG-175. Data are expressed as mean of relative light units and calculated EC₅₀s are presented. Data are representative of two independent experiments. E. Degree of resistance of D320E and Y321N Con1 variants: Left panel. Wild-type (WT), D320E and D320/Y321N NS5A Con1 RNA were electroporated into Huh7.5.1 cells and replication monitored over 8 days by quantifying the levels of intracellular HCV RNA. Right panel. Same as just above except that increasing concentrations of STG-175 were added at the time of the electroporation. Data were calculated as log of genome equivalents (GE)/μg of total RNA and the RNA amount was normalized to a range of 0–100% in order to calculate the EC₅₀. Results are representative of two independent experiments.