Depletion of PML significantly impacts the association of H3.3 with latent/quiescent HSV-1 genomes.

(A) Immuno-FISH performed in e-H3.3-expressing BJ cells transduced with a control (shCTRL) or PML (shPML) shRNA-expressing lentivirus and subsequently infected with in1374 for 2 days. E-H3.3 (green), HSV-1 genomes (red), and PML (blue) were detected. Scale bars = 5 μm. (B) Quantification of the immuno-FISH performed in (A). Means from three independent experiments ± SD. The Student’s t-test was applied to assess the significance of the results. ** = pin1374 for 24 h and previously transduced with a lentivirus expressing a control shRNA (shCTRL, blue) or a PML shRNA (shPML, red). Anti-HA antibody was used for the ChIP experiments. The analyzed viral loci were described previously. Means from three independent experiments ± SD. The Student’s t-test was applied to assess the significance of the results. * = ppml+/+ or pml-/- cells. Actin and total histone H3 were detected as loading controls. (F) ChIP for the detection of e-H3.3 associated with HSV-1 genomes, and performed in e-H3.3-expressing pml+/+ (blue) or pml-/- (red) MEF cells infected with in1374 for 24 h. Anti-HA antibody was used for the ChIP experiments. Analyzed viral loci were described previously. Cellular loci acid Sensing Ion Channel Subunit 2 (Asic2) and Heme Oxygenase 1 (Hmox1) are positive and negative controls for deposition of H3.3 in the absence of Pml, respectively (see [44]). Means from three independent experiments ± SD. The Student’s t-test was applied to assess the significance of the results. * = ppml-/-;e-H3.3 or MEF pml-/-;e-H3.3;myc-PML.I cells not treated (-) or treated (+) with doxycycline for 24 h. Anti-human PML was used for the detection of PML. Actin was detected as a loading control. (H) IF for the detection of PML NBs (green) and nuclei (gray/blue) in MEF pml-/-;e-H3.3;myc-PML.I cells not treated (- dox) or treated (+ dox) with doxycycline for 24 h. PML was detected using the anti myc antibody. Scale bars = 5 μm. (I) ChIP for the detection of e-H3.3 associated with HSV-1 genomes and performed in MEF pml-/-;e-H3.3;myc-PML.I cells not treated (- dox) or treated (+ dox) with doxycycline for 24 h then infected with in1374 for 24 h in the presence of dox. Anti-HA antibody was used for the ChIP experiments. Analyzed viral loci were described previously. Means from three independent experiments ± SD. Student’s t-test was applied to assess the significance of the results. * = p< 0.05, ** = p < 0.01.