FIG. 3.

Testing cured cells for RBV resistance. (A) Crystal violet-stained cells after incubation with RBV. The original RBV-resistant cell lines from pool 1 and pool 4 were cured of replicon RNA and then transfected with wild-type replicon RNA to yield cured pool 1 plus replicon and cured pool 4 plus replicon lines. RBV resistance of these lines and the Huh-7 replicon-containing reference line were tested by passage in 300 μM RBV for 7 days. (B) Quantitation of cell survival in the presence of RBV. Surviving cells from two plates for each condition shown in panel A were trypsinized, stained with trypan blue, and counted using a hemocytometer. The experiment was repeated twice, and similar results were obtained. (C) [³H]RBV uptake kinetics. Huh-7, cured pool 1 (squares) or cured pool 4 (triangles) cells wereincubated with medium containing 5 μM ³H-labeled RBV. At indicated times the cells were washed, harvested, and lysed. Cell-associated ³H was quantified by scintillation counting. (D) RBV toxicity in the absence of viral genomes. RBV-sensitive (Huh-7) or RBV-resistant (cured pool 4) cell lines lacking HCV replicons were passaged for 7 days with or without 300 μM RBV and 300 μM guanosine. Surviving cells were stained with crystal violet.