PML nuclear body-residing proteins sequentially associate with HPV genome after infectious nuclear delivery
Fig 5
PML protein is recruited to the viral genome prior to the loss of L1 protein in late interphase.
(A) HaCaT cells were infected with EdU-labeled PsVs for 24 h, fixed, permeabilized, and treated with AF555 (red) in Click-iT reaction buffer. Next, the cells were incubated with rabbit anti-PML protein antibody (cyan) and mouse 33L1-7 antibody (green) for specific detection of L1 protein and mounted with DAPI (white). (B) Percent co-localization of EdU and L1 was determined by manually counting the number of EdU puncta and L1 puncta (red/green) over the total number of EdU puncta (red only) associated with condensed chromosomes in mitotic cells or nuclear-localized in interphase cells. Nucleoli were also counted: 7+ nucleoli corresponds to early interphase, 1–6 to late interphase. (C) Percent co-localization of EdU with PML protein was determined by manually counting the number of EdU-L1 puncta (red/green) co-localizing with PML protein signal over the total number of EdU puncta (red only) co-localizing with PML protein associated with condensed chromosomes in mitotic cells or nuclear-localized in interphase cells. Results are shown as average of 3 independent experiments and SEM, with 30–50 cells of each condition (mitotic, early and late interphase cells) collected in z-stacks spanning the whole nucleus. (B) Mitosis: %EdU-L1 = 81.48% ± 3.41%. 7+ nucleoli: %EdU-L1 = 56.19% ± 1.72%. 1–6 nucleoli: %EdU-L1 = 29.01% ± 5.87%. (C) Mitosis: %EdU-L1 at PML = 0% ± 0%. 7+ nucleoli: %EdU-L1 at PML = 52.94% ± 3.86%. 1–6 nucleoli: %EdU-L1 at PML = 16.79% ± 1.74%. p value was determined using Student’s t-test comparing Interphase to Mitosis. ****: p < 0.0001. ns: p > 0.05.