FIG. 3.

Effect of host cell determinants on HCV RNA replication. (A) Transient Con1 replication in naive HuH6 cells and those cells that once contained Con1 replicons but later lost them due to a treatment with IFN-α2 (triangles and squares, respectively). Naive and cured cells were transfected with the replication-competent Con1 replicon I₃₄₁PI-luc/NS3-3′/ET (closed symbols) or the inactive Con1 mutant I₃₄₁PI-luc/NS3-3′/GND (open symbols). After 4, 24, 48, and 72 h, cells were lysed and luciferase activities were determined. Values were corrected for different transfection efficiencies by using the 4-h measurements. The result of a single representative experiment is shown. RLU, relative light units. (B) Transient replication of JFH-1 and Con1 replicons in cured HuH6 cells. Cells were transfected with the in vitro-transcribed JFH-1 replicons I₃₈₉Neo/NS3-3′ and I₃₈₉Neo/NS3-3′/ΔGDD (closed and open circles, respectively) or the Con1 replicons I₃₄₁PI-luc/NS3-3′/ET and I₃₄₁PI-luc/NS3-3′/GND (closed and open squares, respectively). The result of a single representative experiment is shown. RLU, relative light units. (C) Cell colony formation after transfection of Con1 replicons into naive and cured HuH6 cells (top and bottom, respectively). Cells were transfected with in vitro-transcribed RNAs of the Con1 replicons I₃₈₉Neo/NS3-3′/ET and I₃₈₉Neo/NS3-3′/ΔGDD or with total RNA of HuH6 Con1 cells containing the replicon I₃₈₉Neo/NS3-3′/ET. In all cases, similar numbers of HCV RNAs were transfected (10⁹ replicon molecules, as quantified by Northern hybridization). For comparison, cells were also transfected with total RNA from naive HuH6 cells. Transfected cells were seeded in 10-cm dishes, cultured for about 4 weeks in the presence of 500-μg/ml G418, fixed, and stained with Coomassie blue.