Fig. 4: ATRX restricts HSV-1 reactivation and is enhanced by IFNα treatment.

A, B HSV-1 reactivation quantified by Us11-GFP positive neurons at 2 days post-reactivation. ATRX depleted using two independent shRNAs five days pre-infection and treated IFNα for 18 h before infection and 24 h post-infection. Reactivation induced using LY294002 (20 μM). A N = 9 from 3 independent dissections. B N = 12 from 4 independent dissections. Analysis with Ordinary one-way ANOVA with Tukey’s multiple comparison (A: *p = 0.0328; *p = 0.0423; **p = 0.0018. B: ***p = 0.0004; **p = 0.0024). C, D HSV-1 reactivation quantified by Us11-GFP positive neurons at 2 days post-reactivation. SCGs treated with IFNα 18 h pre-infection and 24 h post-infection. ATRX depleted using two independent shRNAs five DPI. Reactivation induced using LY294002. N = 9 from 3 independent dissections. Analysis with Ordinary one-way ANOVA with Tukey’s multiple comparison (C: **p = 0.0016; *p = 0.0135. D: **p = 0.0016; **p = 0.0017; ****p < 0.0001). E SCGs untreated or IFNα-treated and cultured in 5% oxygen. Neurons were infected with EdC/EdA-labeled HSV-1 and depleted of PML at 5 days pre-infection or 5 DPI. Fixation at 10 days post-infection. The viral genome was visualized using click chemistry, and immunofluorescence was done for ATRX. White arrows point to the viral genome. Scale bar, 10 μm. F Signal intensity of ATRX at the viral genome 10-days post-infection. Each data point represents one viral genome. Percentages indicate the proportion of genomes with NucSpotA intensity ratios above the denoted co-localization threshold (dashed line). N > 60 cells from 3 biological replicates. Analysis with Kruskal–Wallis test with Dunn’s multiple comparison (****p < 0.0001). G Signal intensity of ATRX at the viral genome 10 days post-infection in mock-treated neurons. 180 rotated control (CTRL). Each data point represents one viral genome. Percentages indicate the proportion of genomes with NucSpotA intensity ratios above the denoted co-localization threshold (dashed line). N > 60 cells from 3 biological replicates. Analysis with unpaired two-tailed t-test (****p < 0.0001). H HSV-1 reactivation quantified by Us11-GFP positive neurons at 2 days post-reactivation. PML, DAXX, or ATRX were depleted using shRNAs five days post-infection. Reactivation was induced using LY294002. N = 9 from 3 independent dissections. Analysis with Ordinary one-way ANOVA with Tukey’s multiple comparison (**p = 0.0024; *p = 0.0253). Data represent the mean ± SEM.