FIG 3.
HPV16 internalization depends on the tyrosine kinase Abl2. (A to D) HeLa cells were incubated with the small-compound inhibitors saracatinib (Src, Abl) (A), PP2 (Src) (B), nilotinib (Abl) (C), or imatinib (Abl) (D) at the indicated concentrations for 30 min. The cells were then either infected with HPV16 PsVs (black bars) for 48 h, with VSV-GFP for 5 h (white bars), or with reovirus for 16 h (gray bars). In the case of HPV16 infection, the inhibitor was exchanged by NH4Cl at 12 h p.i. to reduce cytotoxicity. Infection was scored by flow cytometry and normalized to solvent-treated control cells. (E) Mouse embryonic fibroblasts with a knockout of Src, Fyn, and Yes (SFY) as well as the littermate control cell line (Src+/+) were infected with HPV16 PsVs (black bars) for 48 h or SFV (white bars) for 5 h. Infection was scored by flow cytometry. (F) HeLa Kyoto cells were lysed 48 h after transfection with indicated siRNAs. Lysates were immunoblotted with Abl1- or Abl2-specific antibodies. (G) HeLa Kyoto cells were transfected with siRNAs against Abl1 and 2. At 48 h posttransfection, cells were infected with HPV16 PsVs for 48 h. Infection was analyzed by microscopy and automated cell scoring, and values were normalized to the control siRNA. (H) HeLa cells were preincubated with 25 μM Iressa, 25 μM imatinib, or 10 μM nilotinib for 30 min and subsequently infected with HPV16 PsVs. Cells were treated with a high-pH buffer at 12 h p.i. Infection was continued in the absence of the inhibitor. Infectious internalization was scored 48 h p.i. by flow cytometry and normalized to solvent-treated controls. All infection values are depicted as the mean of three independent experiments ± SD.