FIGURE 1.

Zika virus (ZIKV) infection requires acidic endosomal pH. (A,D) Cells treated with increasing concentrations of NH₄Cl or chloroquine were assessed by the MTT assay to test cell viability. (B,E) Treatment with NH₄Cl or chloroquine inhibited ZIKV infection. T98G cells treated with NH₄Cl (B) or chloroquine (E) were incubated with ZIKV for 1 h at 37°C in the presence of drugs. At 24 h post-infection, the infected cells were lysed to determine viral RNA copy numbers by RT-qPCR. (C,F) Treatment with NH₄Cl or chloroquine inhibited ZIKV infection. T98G cells treated with NH₄Cl (C) or chloroquine (F) were incubated with ZIKV for 1 h at 37°C. At 48 h post-infection, the titers of supernatant viruses were determined by plaque assay. (G) To verify V-ATPase knockdown, protein samples from cells expressing each siRNA construct were analyzed by Western blotting. (H,I) T98G cells were transfected with siRNA targeting V-ATPase or control siRNA for 48 h and then infected with ZIKV at an MOI of 0.5. At 24 h post-infection, the cells were lysed to determine viral RNA copy numbers (H). At 48 h post-infection, the titers of supernatant viruses were determined by plaque assay (I). (J) At the same time, the cells were fixed with 4% paraformaldehyde, incubated with an anti-ZIKV E antibody (green), and visualized by an Olympus microscope. Nuclei were counterstained with DAPI (blue). Scale bars in all panels represent 50 μm. One representative experiment out of three is shown (G,J). The data shown are the mean ± SD of three independent experiments (A–F,H,I). *P < 0.05; **P < 0.01; ***P < 0.001.