FIG 8.

Rab5 and Rab7 are involved in PHEV intracellular trafficking. (A) Neuro-2a cells pretransfected with 50 nM siCtrl, 25 nM siRab5, or 50 nM siRab7 for 48 h were infected with PHEV and incubated for 24 h to allow virus propagation. The knockdown efficiency and viral expression were determined by Western blotting using anti-Rab5, anti-Rab7, or anti-PHEV antibody. Additionally, the infected cells were lysed to quantitate viral RNA copy numbers by qRT-PCR, and the results are presented as means ± SD for data from three independent experiments. (B) Cells transfected with the indicated siRNAs were infected with PHEV. The cells were fixed, stained with anti-PHEV antibody and DAPI, and then visualized by confocal microscopy. Bars, 20 μm. (C) Neuro-2a cells pretransfected with different EGFP-tagged endosomal markers or preprobed with LysoTracker were then exposed to DiD-labeled PHEV (red) on ice for 10 min. After being shifted to 37°C for the indicated times, the cells were washed twice with HS buffer and imaged alive at the indicated time points. Representative micrographs show that DiD-PHEV moved together with Rab5- or Rab7-positive endosomes before 45 min postinternalization, and colocalization with endolysosomes was widely seen by 90 min postinternalization, indicating that PHEV infection requires trafficking through EEs and LEs and that the virus indeed reached the endolysosomes. (D) Percentages of DiD-PHEV (red) colocalized with the endosomal markers EGFP-Rab5wt and EGFP-Rab7wt at different times after warming. The means ± SD of data from each time point for 5 to 10 cells from three different experiments are shown.