FIG 6.

Inhibition of the PKA signaling pathway downregulates HCV entry and CLDN1 cell surface localization. (A) Huh-7 cells were treated for 2 h with H89 at different concentrations. An MTS assay was performed at 30 h posttreatment in order to evaluate the cell toxicity of the compound. (B) Huh-7 cells were treated for 3 h with H89 at different concentrations, along with infection by HCVpp JFH1 and RD114pp. At 48 h postinfection, cells were lysed and luciferase activity measured in order to quantify the infection. (C) Huh-7 cells were treated with H89 at different concentrations, in accordance with the schematized kinetics, along with HCVcc JFH1 infection. Infection was quantified at 30 h postinfection by immunofluorescence assay. (D) After 1 h of viral attachment at 4°C, Huh-7 cells were shifted to 37°C and treated with H89 (10 μM), following 15-min kinetics for 2 h. Proteinase K (50 μg/ml) and bafilomycin A (25 nM) were used as controls for early and late entry steps, respectively. Infection was quantified at 30 h postinfection by immunofluorescence assay, and the value for each time point was normalized to that for the corresponding DMSO condition. (E) Huh-7 cells were treated for 2 h with DMSO or H89 (10 μM). Cell surface CLDN1 was analyzed by flow cytometry. Curves from a representative experiment and mean fluorescence intensities relative to that for the DMSO-treated condition are shown. Results are presented as means ± SEM (n = 3 [A to C and E] and n = 2 [D]). Two-tailed Student's t test (E) or one-way (A) or two-way (B and C) ANOVA followed by the Dunnett or Bonferroni posttest was performed for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.001.