Nuclear import of HPV16 vDNA is blocked in interphase cells.

<p>(A) Aphidicolin-treated or untreated HeLa cells were infected with HPV16-GFP. At 16 h p.i., cells were fixed and immunostained with the L1-7 antibody detecting endosomal, conformationally altered virions. Depicted are confocal sections of untreated (left) or aphidicolin-treated (right) cells. (B) As in (A) with quantification of the three-dimensional distance of L1-7 signals to the nuclear border (in µm) ± SD. (C) As in (A) with quantification of the number of discernible L1-7 spots/cell. (D) As in (A) with quantification of the fluorescent intensity (in arbitrary units, AU) of individual L1-7 spots. (E) HeLa H2B-mCherry cells were infected with BrdU-HPV16. Cells were immunostained for BrdU to detect the vDNA after fixation at 24 h p.i.. Depicted are confocal sections of aphidicolin-treated (interphase, right) or untreated (left) cells. Mostly, the vDNA localized intranuclearly as discrete spots in untreated cells (arrowheads) or exclusively perinuclearly in aphidicolin-treated cells. (F) As in (E) with quantification of intranuclear BrdU spots/cell. The number of intranuclear spots is given relative to the total number of cellular spots. (G) As in (E) with quantification of perinuclear BrdU spot/cell as in (F). (H) As in (E) with quantification of the signal intensities of individual perinuclear BrdU spots relative to untreated infected cells. (I) HeLa cells were treated with or without aphidicolin for 16 h prior to infection. 20 h after infection with EdU-HPV16 (green), cells were fixed and immunostained for TGN46 (red) and LAMP1 (light blue). The nucleus was stained by Hoechst (dark blue). Depicted are single confocal sections. The cytosolic EdU-HPV16 signal is depicted after substraction of the EdU-HPV16 signals colocalizing with LAMP1, TGN46 and the nucleus. (J) As (I) with quantification of signal colocalization of EdU-HPV16 with LAMP1 or TGN46. Cytosolic EdU-HPV16 amounts were defined as signals that did not colocalize with LAMP1, TGN46, or the nucleus (see below). (K) As in (I) with quantification of signal colocalization of EdU-HPV16 with the nuclear stain (Hoechst). Statistical significance was determined by a two-tailed, independent t-test; P-values: * <0.05; *** <0.001. All scale bars: 10 µm.