PML cages sequester mature and immature VZV nucleocapsids.
(A-C) Representative immunogold-EM images of uninfected (A) or VZV-infected (B and C) HELF cells at 48 hr after infection and processing by high-pressure freezing (HPF) and freeze substitution (FS) to optimally preserve ultra-structural details. PML protein is specifically labeled with (A) 15 nm or (B and C) 10 nm gold particles (arrowheads indicate PML gold particles). Arrows indicate examples of viral NCs. (A) PML-labeled endogenous NBs in uninfected cells; examples are shown in upper and lower panels. (B) Individual VZV NCs (arrows) are associated with varying amounts of PML protein (arrowheads) as shown in the upper and lower panels. (C) Clusters of VZV NCs (arrows) are sequestered in an endogenous fibrous PML cage (arrowheads). The black square in the upper panel demarks the area (i) that is shown at higher magnification on the lower panel. Scale bars are as indicated. (D–F) Quantitative analysis of PML-specific gold particles (N = 1,611) and viral NCs (N = 450) in 30 VZV-infected cell nuclei. (D) Percentage of the total number of PML-specific gold particles associated with VZV capsids (black bar) or found ‘free’ within the nuclear area excluding PML-NBs (grey bar). (E) Density of viral NCs determined as NCs/µm2 (mean ± SD) within PML-NBs (black bar) or within the nuclear area outside PML-NBs (white bar). (F) Correlation between the percentage of NCs sequestered within PML-NBs (y-axis) and the PML labeling density in each nucleus, which is the number of PML-specific gold particles/µm2 of the total nuclear area (x-axis). Since only profiles of infected cell nuclei that contained at least 10 NCs were considered, N = 22 for this analysis. Pearson correlation: r = 0.74, R2 = 0.54, p<0.0002.