FIG 1.

CypI and NS5Ai do not block HCV RNA synthesis of isolated replication complexes (RCs). RCs were isolated from JFH-1-infected Huh7.5.1 cells, as described previously (77, 89). HCV replicase activity was determined in a reaction mixture containing 20 mM Tris-HCl (pH 8), 10 mM MgCl₂, 5 mM dithiothreitol (DTT), 5 mM KCl, 40 μg/ml actinomycin D, 20 μCi of [α-³²P]CTP, 10 μM CTP, 1 mM (each) ATP and UTP, 5 mM GTP, 2.5 mM phosphoenolpyruvate, 1 U of pyruvate kinase, 1 U of RNasin, and 10, 30, or 90 μl of RC fraction in a total volume of 120 μl at 35°C for 60 min, as previously reported (77, 78). Drugs (10 or 1 μM) were added to the reaction mixture prior the addition of the RCs. HCV RNA synthesis was quantified by autoradiography, as illustrated in top panel, in which DMSO, the CypI ALV, the NS5Ai daclatasvir, and the NS5Bi 3′-dCTP were used with 90 μl of RCs. Total HCV RNA was quantified before and after the replicase assay and is represented as the fold increase, as previously reported (78). The error bars represent the standard errors of duplicates. The data are the mean values of five replicates from 3 independent experiments.