Figure 6. Inhibition of glycolytic response attenuates ZIKV replication by restoring AMPK activity.

(A) HRvEC cells were treated with a glycolytic inhibitor, 2-deoxyglucose (2-DG), for 1 h followed by infection with ZIKV at MOI 1 or mock infection for 48 h. The cells were fixed and immunostained for ZIKV envelope antigen (red) and nuclei were counterstained using DAPI (magnification 200X). (B) The percentage of ZIKV antigen-positive cells were determined from at least four fields (mean ± SD). (C) HRvEC cells were treated with 2-DG and infected with ZIKV at MOI 1 and the culture supernatant was collected at 48 hpi for virus titration. (D) HRvEC cell lysates from mock-infected, 2-DG treated, ZIKV infected and ZIKV + 2-DG treated cells were immunoblotted for p-AMPK and total AMPK, ZIKV NS3, and β-actin. (E) Glucose uptake was measured in HRvEC cells infected with ZIKV for 48 h in the presence or absence of 1 mM AICAR. 2DG was used as a positive control for inhibition of glucose uptake. The values were plotted as Relative Luminescence units (RLU). (F) ATP levels were measured in HRvEC cells infected with ZIKV in the presence or absence of AICAR (1 mM). The values represent mean ±SD from three independent experiments. The statistical analysis was performed using one-way ANOVA- Bonferroni’s multiple comparison tests. ** P <0.001, *** P <0.0001.