FIG. 5.

Transient replication of HCV replicons carrying a reporter gene. (A) Structure of a replicon construct harboring a firefly luciferase gene (Ffl-luc). GND indicates the position of the amino acid substitution of the NS5B RdRp. For further details, see the legend to Fig. 1. (B) Flow chart of the experimental approach. Huh-7 cells were transfected by electroporation (Epo), and 1/10 of each was seeded in a culture dish. After 4, 24, 48, 72, and 96 h, cells were lysed and luciferase activities were determined. Cells that were cultured for 72 h were passaged at a dilution of 1:3. One-third of each was harvested at time points (t) corresponding to 127 and 144 h after transfection, whereas the remainder were again passaged at a time point corresponding to 168 h posttransfection. Cells of this second passage were lysed at time points corresponding to 216 and 244 h after transfection. (C) Representative results obtained with the replicons specified at the top. Values for each time point correspond to the mean and the error range of quadruplicate results. Note that, owing to low variations, error bars are in most cases not visible in the graph. Values are corrected for transfection efficiency as determined by measuring the luciferase activity 4 h after transfection. Wt, wild type.