PML-NBs stably entrap vDNA upon nuclear entry.

<p>HFt cells were mock or HSV-1^EdU infected at an MOI of 3 PFU/cell. Cells were fixed and permeabilized at the indicated times (mpi; post-addition of virus). vDNA, or PML-NB host factors and ICP0, were detected by click chemistry and indirect immunofluorescence staining, respectively. (A) Localization of PML (green) and Daxx (cyan) to HSV-1 vDNA (red, white arrows) at 30–90 mpi (as indicated). Insets show magnified regions of interest (dashed boxes) highlighting PML-NB colocalization with vDNA. Cut mask (yellow) highlights regions of colocalization between PML, Daxx, and vDNA (as indicated). Weighted colocalization coefficients are shown. (B) 3D reconstruction of high-resolution confocal Z-series images showing PML (green) entrapment of vDNA (red, white arrow) at 90 mpi. Single channel maximum intensity projections (left-hand panels) and 3D rendered image (right-hand panel). Pearson coefficient for PML-vDNA colocalization is shown. Scale bar 2 μm. (C) Nuclear localization of ICP0 (green) to vDNA (red, white arrow) and PML (cyan) in HSV-1^EdU infected cells at 90 mpi. Cut mask (yellow) highlights regions of colocalization between ICP0, PML, and vDNA (as indicated). Weighted colocalization coefficients are shown. Nuclei stained with DAPI (blue). (D) EdU labelling of viral genomes does not affect the initiation of infection. HFt cells were mock or HSV-1 infected with unlabelled (HSV-1) or EdU labelled (HSV-1^EdU) HSV-1 at an MOI of 3 PFU/cell. Whole cell lysates were collected at the indicated times (hours post-infection; hpi) for western blot analysis to monitor the rate of PML degradation and the accumulation of the viral immediate early (IE) gene products (ICP0, ICP4). Actin is shown as a loading control. Molecular mass markers are shown, < denotes the detection of a non-specific background band.