ATRX degradation and aggregation in the presence of HSV protein ICP0.
(A) HSV protein ICP0 promotes ATRX aggregate formation post PML-NB dispersion. HFFs were infected with HSV 7134 or 7134R (7134 rescued for ICP0 expression) at MOI 5. Total protein was harvested at times indicated followed by immunoblot detection of ATRX, ICP4, ICP0, and GAPDH. (B) HFFs were infected with KOS-EdC at MOI 5. Cells were fixed by 2% formaldehyde at times indicated followed by antibody staining for ATRX and PML. Click chemistry was used to biotinylate HSV DNA followed by detection by a fluorophore-conjugated streptavidin probe. Cells were imaged at 100x. (C) Quantification of ATRX-aggregates in imaged cells. A total of 184 infected cells from 2 independent experiments were used to assess the prevalence of ATRX aggregates in fibroblasts infected with KOS-EdC at MOI 5. D) HFFs infected with HSV 7134 were fixed at 6 hpi and stained with antibodies for ATRX and ICP8. Cells were imaged at 100x.