FIGURE 8.

Effects of Rab5 and Rab7 knockdown on Zika virus (ZIKV) infection. (A) The knockdown efficiency of Rab5 or Rab7 was determined by Western blotting. T98G cells were transfected with either siControl, siRab5, or siRab7 for 48 h. Cell lysates were reacted with an anti-Rab5 antibody or an anti-Rab7 antibody specific for the indicated proteins. β-actin was used as an internal loading control. One representative experiment out of three is shown. (B) Rab5 or Rab7 depletion reduced ZIKV propagation. T98G cells transfected with the indicated siRNAs were infected with ZIKV at a multiplicity of infection (MOI) of 0.5 and incubated for 48 h to allow virus propagation. The infected cells were lysed to quantitate viral RNA copy numbers by RT-qPCR. (C) At 48 h post-infection, the titers of supernatant viruses were determined by plaque assay. (D) ZIKV localized in Rab5- and Rab7-postitive endosomes. T98G cells were transfected with EGFP-tagged Rab5 or Rab7 for 24 h, followed by infection with ZIKV at an MOI of 10 at 4°C for 1 h and then shifted to 37°C. At 24 h post-infection, the cells were fixed and stained with an anti-ZIKV envelop antibody (red). Nuclei were stained with DAPI (blue). One representative experiment out of three is shown (A). The immunofluorescence was examined under a confocal microscope. Representative confocal images from three independent experiments are shown. Scale bars in all panels represent 10 μm (D). The data shown are the mean ± SD of three independent experiments (B,C). *P < 0.05; **P < 0.01; ***P < 0.001.