FIG 2.

Inhibition of endosomal acidification prevents PHEV entry. (A) The PHEV receptor NCAM translocates with virus particles from the plasma membrane to cytoplasmic compartments. Neuro-2a cells were treated with PHEV for the indicated times at 37°C, and immunostaining was conducted using anti-NCAM and anti-PHEV antibodies. (B) PHEV cargos were internalized into EEA1-positive endosomes. Cells infected with PHEV were fixed and immunostained with anti-EEA1 and anti-PHEV antibodies. As a positive control, cells were probed with Tf-AF594 for 30 min at 37°C and then subjected to IFA. The insets show colocalization of PHEV or Tf with EEA1 inside the cytoplasm. (C) Neuro-2a cells were infected with PHEV in the presence of NH₄Cl or CQ at the indicated doses for 1 h, after which the endocytosed viral loads were estimated by qRT-PCR and Western blotting. (D) Neuro-2a cells grown on coverslips were either left untreated or treated with NH₄Cl or CQ for 1 h and subsequently incubated with PHEV. At 12 hpi, cells were fixed and stained with anti-PHEV-S primary antibodies. Bars, 10 μm (A and B) and 50 μm (D). *, P < 0.05; **, P < 0.01.