Fig 1.
PML protein dynamics is used to approximate time during and after mitosis.
HaCaT cells were grown on glass coverslips, then fixed, permeabilized, incubated with rabbit anti-PML protein antibody (cyan), and mounted with DAPI (white). Confocal images show cells in all stages of mitosis (A) and end of mitosis and stages of interphase (B).
Fig 2.
PML protein encompasses incoming viral genomes only after nuclear delivery.
HaCaT cells were infected with EdU-labeled PsVs for 24 h, fixed, permeabilized, and treated with Click-iT reaction buffer with AF555 dye to stain EdU-labeled pseudogenomes (red). Next, cells were incubated with mouse anti-PML protein antibody (green) and mounted in DAPI (white). (A) Images show 3D reconstruction of high resolution z-stacks and close-up images were rotated at a 45° angle on the x, y, z axes in the 3D-rendered images. (B) Distance (μm) between center of EdU and center of PML protein puncta was measured in NIS Elements. (C) PML and EdU mean intensity were measured in NIS Elements, normalized to ROI area, and the ratio was calculated. (B and C) Nucleoli were also counted: 7+ nucleoli corresponds to early interphase, 1–6 to late interphase. Results are shown as average of 2 independent experiments and SEM, with 30–50 cells of each condition (early and late interphase cells) collected in z-stacks spanning the whole nucleus. (B) 7+ nucleoli: 0.275 μm ± 0.021 μm; 1–6 nucleoli: 0.113 μm ± 0.006 μm. (C) 7+ nucleoli: 1.146 ± 0.025; 1–6 nucleoli: 1.888 ± 0.069. p value was determined using Student’s t-test comparing Early to Late Interphase. ****: p < 0.0001.
Fig 3.
Viral genome associates with PML protein while still inaccessible to small molecular dyes.
(A and B) HaCaT cells were infected with EdU-labeled PsVs for 24 h, fixed, permeabilized with 0.625 μg/mL digitonin (A) or 0.5% TX-100 (B), and treated with AF555 (green) in Click-iT reaction buffer. Next, the cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, cells were incubated with rabbit anti-PML protein antibody (cyan) and mounted with DAPI (white). (C and D) Percent accessibility of viral genome (C) was determined by manually counting the number of red only (inaccessible [In]) or red/green (accessible [Ac]) EdU puncta over the total number of EdU puncta associated with condensed chromosomes in mitotic cells or nuclear-localized in interphase cells in digitonin- or TX-100-permeabilized cells. Nucleoli were also counted: 7+ nucleoli corresponds to early interphase, 1–6 to late interphase. Co-localization of PML protein and EdU (D) was quantified by manually counting the number of EdU puncta that co-localized with PML protein signal over the total number of accessible or inaccessible EdU puncta associated with condensed chromosomes in mitotic cells or nuclear-localized in interphase in digitonin-permeabilized 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. (C) Mitosis: Dig: %Ac = 4.90% ± 2.55%; TX: %Ac = 91.37% ± 0.77%. 7+ nucleoli: Dig: %Ac = 45.05% ± 3.80%; TX: %Ac = 91.62% ± 3.38%. 1–6 nucleoli: Dig: %Ac = 79.55% ± 4.38%; TX: %Ac = 90.35% ± 2.63%. (D) Mitosis: %In at PML = 2.73% ± 2.73%; %Ac at PML = 0% ± 0%. 7+ nucleoli: %In at PML = 70.09% ± 5.43%; %Ac at PML = 78.85% ± 9.05%. 1–6 nucleoli: %In at PML = 62.22% ± 6.19%; %Ac at PML = 76.43% ± 7.57%. p value was determined using Student’s t-test comparing Dig to TX and Dig to Dig (C) or Interphase to Mitosis (D). ***: p < 0.001. **: p < 0.01. *: p < 0.05. ns: p > 0.05.
Fig 4.
Viral genome associates with SUMO-1 while still inaccessible to small molecular dyes.
(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, cells were incubated with rabbit anti-SUMO-1 antibody (green) and mouse anti-PML protein antibody (cyan) and mounted in DAPI (white). Images show 3D reconstruction of high resolution z-stacks. Close-up images were rotated at a 45° angle on the x, y, z axes in the 3D-rendered images. (B) HaCaT cells were infected with EdU-labeled PsVs for 24 h, fixed, permeabilized with 0.625 μg/mL digitonin and treated with AF555 (green) in Click-iT reaction buffer. Next, the cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, cells were incubated with rabbit anti-SUMO-1 antibody (cyan) and mounted with DAPI (white). (C and D) Percent accessibility of viral genome (C) was determined by manually counting the number of red only (inaccessible [In]) or red/green (accessible [Ac]) EdU puncta over the total number of EdU puncta associated with condensed chromosomes in mitotic cells or nuclear-localized in interphase cells in digitonin- or TX-100-permeabilized cells. Nucleoli were also counted: 7+ nucleoli corresponds to early interphase, 1–6 to late interphase. Co-localization of SUMO-1 and EdU (D) was quantified by manually counting the number of EdU puncta that co-localized with SUMO-1 signal over the total number of accessible or inaccessible EdU puncta associated with condensed chromosomes in mitotic cells or nuclear-localized in interphase in digitonin-permeabilized 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. (C) Mitosis: Dig: %Ac = 5.85% ± 2.41%; TX: %Ac = 99.09% ± 0.12%. 7+ nucleoli: Dig: %Ac = 36.78% ± 6.21%; TX: %Ac = 97.22% ± 1.10%. 1–6 nucleoli: Dig: %Ac = 74.00% ± 1.73%; TX: %Ac = 98.30% ± 1.18%. (D) Mitosis: %In at SUMO-1 = 1.99% ± 1.48%; %Ac at SUMO-1 = 1.09% ± 0.63%. 7+ nucleoli: %In at SUMO-1 = 50.73% ± 7.14%; %Ac at SUMO-1 = 66.48% ± 0.77%. 1–6 nucleoli: %In at SUMO-1 = 61.45% ± 3.27%; %Ac at SUMO-1 = 76.47% ± 2.79%. p value was determined using Student’s t-test comparing Interphase to Mitosis. ****: p < 0.0001. ***: p < 0.001. **: p < 0.01. *: p < 0.05. ns: p > 0.05.
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.
Fig 6.
L2 protein remains associated with the viral genome 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 33L2-1 antibody (green) for specific detection of L2 protein and mounted with DAPI (white). (B) Percent co-localization of EdU and L2 was determined by manually counting the number of EdU puncta and L2 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-L2 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-L2 = 79.22% ± 4.06%. 7+ nucleoli: %EdU-L2 = 47.11% ± 4.29%. 1–6 nucleoli: %EdU-L2 = 53.59% ± 5.42%. (C) Mitosis: %EdU-L2 at PML = 0.22% ± 0.22%. 7+ nucleoli: %EdU-L2 at PML = 45.20% ± 4.69%. 1–6 nucleoli: %EdU-L2 at PML = 52.11% ± 7.60%. p value was determined using Student’s t-test comparing Interphase to Mitosis. **: p < 0.01. *: p < 0.05.
Fig 7.
Sp100 recruitment to the viral genome is delayed compared to PML protein.
(A and B) HaCaT cells were infected with EdU-labeled PsVs for 24 h, fixed, permeabilized, and treated with Click-iT reaction buffer with AF555 dye to stain EdU-labeled pseudogenomes (red). Next, the cells were incubated with mouse anti-PML protein antibody (cyan) and rabbit anti-Sp100 antibody (green) and mounted in DAPI (white). (A) Images show 3D reconstruction of high resolution z-stacks. Close-up images were rotated at a 45° angle on the x, y, z axes in the 3D-rendered images. (B) Representative confocal images of the following quantification. (C) Percent Sp100-containing PML was determined by manually counting the PML protein puncta co-localizing with Sp100 puncta also co-localizing with EdU puncta (EdU+) or not (EdU-) 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. 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. (C) Mitosis: EdU+ = 0% ± 0%; EdU- = 0.13% ± 0.13%. 7+ nucleoli: EdU+ = 52.56% ± 9.95%; EdU- = 82.48% ± 5.83%. 1–6 nucleoli: EdU+ = 92.10% ± 3.19%; EdU- = 89.38% ± 0.08%. p value was determined using Student’s t-test. ****: p < 0.0001. *: p < 0.05. ns: p > 0.05.
Fig 8.
Sp100 is recruited after the viral genome becomes accessible to small molecular dyes.
(A) HaCaT cells were infected with EdU-labeled PsVs for 24 h, fixed, permeabilized with 0.625 μg/mL digitonin and treated with AF555 (green) in Click-iT reaction buffer. Next, the cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, cells were incubated with rabbit anti-Sp100 antibody (cyan) and mounted with DAPI (white). (B and C) Percent accessibility of viral genome (B) was determined by manually counting the number of red only (inaccessible [In]) or red/green (accessible [Ac]) EdU puncta over the total number of EdU puncta associated with condensed chromosomes in mitotic cells or nuclear-localized in interphase cells in digitonin- or TX-100-permeabilized cells. Nucleoli were also counted: 7+ nucleoli corresponds to early interphase, 1–6 to late interphase. Co-localization of Sp100 and EdU (C) was quantified by manually counting the number of EdU puncta that co-localized with Sp100 signal over the total number of accessible or inaccessible EdU puncta associated with condensed chromosomes in mitotic cells or nuclear-localized in interphase in digitonin-permeabilized 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: Dig: %Ac = 5.22% ± 0.69%; TX: %Ac = 93.87% ± 0.38%. 7+ nucleoli: Dig: %Ac = 33.98% ± 3.89%; TX: %Ac = 94.60% ± 2.57%. 1–6 nucleoli: Dig: %Ac = 76.67% ± 2.86%; TX: %Ac = 94.59% ± 3.64%. (C) Mitosis: %In at Sp100 = 0% ± 0%; %Ac at Sp100 = 0% ± 0%. 7+ nucleoli: %In at Sp100 = 26.49% ± 2.46%; %Ac at Sp100 = 48.95% ± 9.61%. 1–6 nucleoli: %In at Sp100 = 55.79% ± 6.74%; %Ac at Sp100 = 72.22% ± 4.66%. p value was determined using Student’s t-test comparing Interphase to Mitosis. ****: p < 0.0001. ***: p < 0.001. **: p < 0.01. *: p < 0.05. ns: p > 0.05.
Fig 9.
Mutations in SUMO motifs on L2 protein render pseudovirions defective for nuclear delivery.
(A and E) HaCaT cells were infected with EdU-labeled WT or mutant PsVs for 24 h, fixed, permeabilized, and treated with AF555 (red) in Click-iT reaction buffer. Next, cells were incubated with rabbit anti-PML protein antibody (cyan) (A) or mouse anti-α-tubulin antibody (white) (E) and mounted with DAPI (white (A), blue (E)). (A) Representative confocal images of EdU and PML localization in interphase cells. (E) Representative confocal images of EdU on microtubules in mitotic cells. (B) Number of EdU in whole cell was determined by manually counting EdU puncta present in z-stacks spanning whole nucleus of interphase cells. WT = 12.99 ± 0.38; K35R = 12.35 ± 0.39; SIM 105-9A = 10.27 ± 0.35; SIM 145-8A = 11.44 ± 0.36; SIM 286 = 9A = 12.83 ± 0.35. (C) Number of EdU in nucleus was determined by manually counting EdU puncta present in z-stacks spanning the whole nucleus of interphase cells. WT = 2.83 ± 0.24; K35R = 0.91 ± 0.10; SIM 105-9A = 0.29 ± 0.43; SIM 145-8A = 0.43 ± 0.06; SIM 286 = 9A = 0.02 ± 0.01. (D) Percent co-localization of EdU and PML protein was determined by manually counting the number of EdU puncta that co-localized with PML protein signal over the total number of EdU puncta present in z-stacks spanning the whole nucleus of interphase cells. WT = 70.93% ± 0.69%; K35R = 68.12% ± 10.58%; SIM 105-9A = 44.48% ± 12.66%; SIM 145-8A = 75.28% ± 1.09%; SIM 286 = 9A = 0% ± 0%. (F) Number of EdU localized on chromosomes was determined by manually counting the number of EdU puncta that co-localized with mitotic chromosomes in z-stacks spanning whole mitotic cells. WT = 11.66 ± 0.92; K35R = 6.73 ± 0.81; SIM 105-9A = 2.92 ± 0.33; SIM 145-8A = 4.47 ± 0.48; SIM 286 = 9A = 0.29 ± 0.10. Each quantification is shown as average of 2 independent experiments and SEM, with 100 cells in each condition and experiment. p value was determined using Student’s t-test comparing mutants to WT. ****: p < 0.0001. **: p < 0.01. ns: p > 0.05.