Fig. 5: ATRX is maintained on viral genomes with H3K9me3.

A SCGs untreated or IFNα-treated were infected with EdC/EdA-labeled HSV-1. Fixation at 10DPI. The viral genome visualized using click chemistry, and immunofluorescence for H3K9me3 and ATRX. White arrows indicate viral genome. Scale bar, 10 μm. B Signal intensity of H3K9me3 and ATRX at the viral genome 10 DPI. Percentage of genomes above or below the co-localization threshold (H3K9me3 = 1.1 and ATRX = 1.5) represented as a bar graph. N > 60 cells from 3 biological replicates. C, D Infected SCGs untreated (C) or IFNα-treated (D) were depleted of ATRX using shRNAs at 5 DPI. RNA collected 18 h following LY294002. RT-qPCR used to quantify IE (ICP27), E (ICP8), and L (gC) viral gene expression. Analysis with two-tailed t test or Mann–Whitney test (C: ****p < 0.0001, D: **P = 0.0072; **p = 0.0017; ***p = 0.0003). C N = 9 from 3 independent dissections. D N = 12 from 4 independent dissections. E, F Infected SCGs untreated (E) or IFNα-treated (F) were depleted of ATRX using shRNA at 5 DPI. HSV-1 reactivation quantified by Us11-GFP positive neurons 2 days after LY294002 and DMSO or ML-324 (10 μM; KDM4 inhibitor) treatment. E N = 15 from 5 independent dissections. F N = 9 from 3 independent dissections. Analysis with two-way ANOVA with Šídák’s multiple comparisons test (E; ***0.0003; F: *p = 0.0115). G Model of (E, F) mechanism. ML-324 Inhibits KDM4. Created in BioRender. Whitford, A. (2025) https://BioRender.com/04la4oh. H SCGs untreated or IFNα-treated were infected with EdC/EdA-labeled HSV-1. Fixation at 18 h post-reactivation stimulus. Viral genome visualized using click chemistry, and immunofluorescence done for H3K9me3S10p and ATRX. White arrows indicate viral genome. Scale bar, 10 μm. I Signal intensity of ATRX at the viral genome 10 DPI or 18 h post-reactivation stimulus. Each data point represents one viral genome. Percentages indicate proportion of genomes with NucSpotA intensity ratios above the co-localization threshold (dashed line). N > 60 cells from 3 biological replicates. Analysis with Mann–Whitney. J Signal intensity of H3K9me3S10p and ATRX at the viral genome 18 h post-reactivation stimulus. The percentage of genomes above or below the co-localization threshold (H3K9me3S10p = 1.5 and ATRX = 1.5) are represented as a bar graph. N > 60 cells from 3 biological replicates. Data represent the mean ± SEM.