FIG 7.

Rab5 and Rab7 are necessary for PHEV infection. (A) Neuro-2a cells were infected with PHEV, and the lysates were harvested after incubation for the indicated times and then subjected to Western blotting to detect the distribution of intracellular markers and the presence of PHEV particles. (B) Neuro-2a cells were transfected with plasmids expressing EGFP-tagged Rab5 and Rab7 WT, AN, and DN constructs and then incubated with Tf-AF488 for 30 min at 37°C. The uptake of Tf-AF488 was determined by confocal microscopy, and representative micrographs are shown. Bars, 50 μm. (C) Cells pretransfected with the indicated plasmids, pictured as in panel B, were then screened for PHEV infection. At 24 hpi, the cells were fixed, probed with anti-PHEV-S antibody, and visualized by confocal microscopy. Bars, 10 μm. (D) The effects of Rab5 and Rab7 WT, AN, and DN constructs on Tf uptake and PHEV infection were analyzed quantitatively using ImageJ software, and the arithmetic means of data from three independent experiments are shown. *, P < 0.05; **, P < 0.01.