Effects of viroporin inhibitors on intracellular vesicular pH and infectious virus production.

(A) The ability of inhibitors to affect p7 channel function in vivo was assessed in full-length replicon bearing cells loaded with LysoSensor Yellow/Blue DND-160. Amantadine or rimantadine were added directly to LysoSensor loaded cells and the cells were imaged after 5 min. Fluorescence ratio images are presented as for Fig. 5D and pH is represented by pseudocolor mapping as indicated. Scale bar represents 20 µm. (B) Average pH changes from a minimum of 3 experiments performed as in panel A. pH values were determined from fluorescence ratios by interpolation on the calibration curve as shown in Fig. 5. FL  =  full-length, SG  =  sub-genomic and CFL  =  cured full-length HCV replicons. (C) Comparison of the effect of various concentrations of amantadine on both intravesicular pH (data from panel B) and infectious virus present in the extracellular culture medium. For measurement of virus titers, Huh-7.5 cells were incubated with the indicated concentration of amantadine for 24 hours prior to collection of medium and tittering by the FFU assay as described in Materials and Methods. (D) Intra- and extracellular infectious virus production after amantadine treatment. Huh-7.5 cells were infected with HJ3-5 virus and treated with amantadine as described in Materials and Methods. Medium was collected for determination of infectious virus titer by FFU assay (Extracellular), and cell pellets were washed, disrupted by freeze-thaw and then assayed similarly (Intracellular). Bars represent the proportion of the total infectious virus yield that was present in each compartment.