Fig 2: Endolysosomal acidification via V-ATPase is essential for poxvirus replication. (A) Schematic diagram of CPXV regulating the acidic environment of endolysosomes. (B) Representative images of LysoTracker Red staining in Vero cells (left), LysoTracker Red staining in Vero cells infected with CPXV (MOI = 0.05) for 24 h (middle), and corresponding GFP fluorescence images of CPXV-infected cells at 24 h (right). (C) Western blotting analysis of V-ATPase subunits (ATP6V1A, ATP6V0A2, and ATP6V0C) in CPXV-infected Vero cells (MOI = 0.05) at different time points post-infection. The mean value was calculated by one‑way ANOVA with Tukey. (mean ± SD, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. (D) Quantification of Cathepsin B and D activity and gene expression in Vero cells. Bar plots show enzymatic activity of Cathepsin B and D measured using fluorogenic substrates. Violin plots represent transcriptomic expression levels of CTSB and CTSD genes in Vero cells following infection with CPXV, ECTV, or VACV for 0 h (blue), 2 h (red) and 16 h (green). UI: uninfected; CPXV: cowpox virus infected. Numerical values above the plots indicate two-tailed t-test P values. (E) Plaque assay and GFP fluorescence imaging results of CPXV (MOI = 0.05) infection in Vero cells after siRNA-mediated knockdown of ATP6V1A (middle panels) and ATP6V0A2 (right panels), compared with control siRNA-treated cells (left panels). (F) Plaque assay and GFP fluorescence imaging results of CPXV-infected Vero cells treated with Diphyllin, Bafilomycin A1 (BafA1), or KM91104 for 48 h under neutral (pH 7.4) and acidic (pH 4.0) conditions