FIG 4 .

AP-1B and AP-4 mediate HCV cell-to-cell spread. (A) Cell-free infectivity measured via luciferase assays 6 h postinoculation of naive cells with supernatants derived from HCV RNA-transfected Huh7.5 cells stably expressing AP shRNA or an NT control. (B) Schematic of the coculture assay. (C) Cell-to-cell spread 48 h after coculturing of HCV RNA-transfected donor cells depleted of AP or NT controls with GFP-expressing target cells measured via FACS analysis following staining of HCV NS5A. Plotted is the percentage of infected target cells (NS5A⁺ GFP⁺) in the target cell population (GFP⁺) pooled from two independent experiments relative to the NT control. (D) Schematic of the experiments displayed in panels E to G. (E, F) Cell-to-cell spread (E and F, top) and expression (see Fig. 3E and F, bottom) in cells concurrently transduced with lentiviruses expressing the shRNAs indicated and transfected with shRNA-resistant GLuc-tagged AP-1A, AP-1B, or AP-4 or an empty control. (G) Cell-to-cell spread in Huh7.5 cells following transfection with the GLuc-tagged APs indicated or an empty control. ns, nonsignificant. Representative experiments of at least two conducted, each with three biological replicates, are shown. Shown are the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (relative to the corresponding NT control; one-way ANOVA with Dunnett’s [A, C, and G] or Tukey’s [E and F] post hoc test).