Fig. 4.

The peptide inhibits HPV exit from the endosome. HeLa cells were incubated for 1 h with or without 14 μM P16/16, followed by mock-infection or infection with HPV16 PsV at an MOI of 200. At 8 and 16 h.p.i., PLA was performed as described in Fig. 3D with anti-L1 and anti-EEA1 antibody (A) or TGN46 antibody (B). The PLA signal for EEA1-L1 was normalized to that of cells infected with HPV16 PsV in the absence of peptide at 8 h.p.i., and the TGN46-L1 signal was normalized to untreated cells at 16 h.p.i. The graphs show average normalized fluorescence per cell and SD for three independent experiments (n.s., not significant). (C, Left) HeLa cells were mock-infected or infected with HPV16 PsV containing EdU-labeled reporter plasmid DNA at an MOI of 50. Where indicated, cells were pretreated for 1 h with 14 μM P16/16. At 48 h.p.i., cells were fixed and treated with Click-iT chemistry to stain viral DNA (green) and incubated with anti-PML antibody (red). The overlap in EdU and PML staining is pseudocolored yellow. Nuclei are stained blue. (C, Right) Nuclear and nonnuclear EdU staining as in C, Left, was quantified for 60 cells in each condition. Each dot represents data from an individual cell. Horizontal lines indicate mean and SD.