FIG 3.

CHIKV E1-V156A and K211T variants modulate cell entry and increase sensitivity to endosomal pH. (A and B) Fusion-from-without assay. BHK-21 cells were preincubated with NH₄Cl and adsorbed with ZsGreen-expressing viruses (MOI, 1), fusion was triggered for 2 min at the indicated range of pH, and viral spread was blocked by replenishing NH₄Cl-containing media. At 18 h postinfection, cells were fixed and DAPI stained, and the percentage of infected cells was assessed by fluorescence imaging. (C to F) Bafilomycin A1 (C and D) and NH₄Cl (E and F) sensitivity assays. BHK-21 cells were preincubated with bafilomycin A1 or NH₄Cl, incubated with ZsGreen-expressing virus (MOI, 1) for 1 h, washed twice, and incubated in medium containing each treatment for 18 h. Cells were then fixed and DAPI stained, and the percentage of infected cells was assessed by fluorescence imaging. All data are representative of at least three independent experiments performed in duplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P values were determined by two-way ANOVA with Bonferroni’s multiple-comparison test.