FIGURE 2.

Endogenous EGFR is critical for HEV infection. (A) EGFR protein expression in HepG2 cells 48 h after transfection with EGFR-specific siRNAs or nontargeting control siRNA (siCtrl) analyzed by western blot (left) and immunofluorescence (right). (B) HEVcc (p6) infection in HepG2 cells transfected with EGFR-specific siRNAs and control siRNA. Cells were infected with HEVcc (p6) 2 d after transfection and FFU/well determined after fixation 5 d post infection. Left: quantification of FFU of the full well normalized to cells transfected with control siRNA. Right: representative immunofluorescence images stained for ORF2 protein. (C, D) HEVcc p6 nonenveloped and enveloped, as well as 83-2 nonenveloped infection in HepG2 cells under treatment of EGFR modulators erlotinib (33 µM, Erlo), EGF (16.5 nM), and cetuximab (34 nM, Cetu) compared with UTCs, whereas the HEV inhibitor ribavirin (50 µM, Rbv) served as control. (C) Representative immunofluorescence images stained for ORF2 protein in HEVcc (p6) infected HepG2 cells under EGFR modulator treatment. (D) FFUs/well were counted in HEVcc p6 nonenveloped (left), p6 enveloped (middle), or 83-2 (right) infected HepG2 cells under EGFR modulator treatment and normalized to UTC. To test the significance of mean differences, Student t test (B) and one-way ANOVA, followed by Dunnett multiple comparison test (D), were used. p-values <0.05 (*), <0.01 (**), <0.001 (***), and <0.0001 (****). p-values >0.05 were considered to be not significant. All infection experiments were performed in triplicates. Mean and SEM are depicted from at least three independent experiments. Scale bars = 100 µm. Abbreviations: EGFR, EGF receptor; FFU, focus forming units; ns, nonsignificant; ORF2, open reading frame 2; UTC, untreated control cells.