FIG 5.

Dehydrojuncusol inhibits HCV RNA replication of daclatasvir NS5A resistance mutants. (A) Huh-7 cells stably expressing subgenomic replicon SGR-JFH1-NS5AGFP wild type or harboring the L31M or Y93H mutation in NS5A that confer resistance to daclatasvir were incubated for 72 h with 0.5 nM daclatasvir, 1 µM boceprevir, or 10 µg/ml of the methylene chloride partition of J. maritimus (J. mar. MeCl). DMSO (0.0001%) was used as a control. Cells were lysed and lysates subjected to SDS-PAGE followed by Western blot revelation of GFP and tubulin. (B and C) Huh-7 cells stably expressing subgenomic replicon SGR-JFH1-NS5AGFP wild type or harboring the L31M or Y93H mutation in NS5A that confer resistance to daclatasvir were incubated for 72 h with increasing concentrations of dehydrojuncusol (B) or daclatasvir (C). Cells were fixed, and the percentage of GFP-positive cells was quantified as a measure of RNA replication. Replication of subgenomic replicon was expressed as a ratio to carrier control DMSO (0.0001%). Data are means of values obtained in 3 independent experiments performed in triplicate. Error bars represent SEM.