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. 2020 Dec 22;9:e58037. doi: 10.7554/eLife.58037

Figure 4. HSV Reactivation Mediated by Forskolin Requires Neuronal Excitability.

(A) Latently infected cultures were reactivated with forskolin in the presence of the voltage-gated sodium channel blocker tetrodotoxin (TTX; 1 µM) and the number of Us11-GFP-positive neurons quantified at 3 days post-reactivation. (B) Latently infected cultures were reactivated with forskolin in the presence of the voltage-gated potassium channel blocker tetraethylammonium (TEA; 10 mM) and the number of Us11-GFP-positive neurons quantified at 3 days post-reactivation. (C) Forskolin-mediated reactivation in the presence of the HCN channel blockers ZD 7288 (10μM) quantified as the numbers of Us11-GFP-positive neurons at 3 days post-reactivation. (D) The effect of ZD 7288 on the HSV lytic gene transcript ICP27 during Phase I reactivation measured at 20 hr post-forskolin treatment by RT-qPCR. In A-D individual experimental replicates are represented along with the mean and SEM. (E and F) Quantification of the relative nuclear staining for H3K9me3/S10p and γH2AX in SCG neurons at 5 hr post-forskolin treatment and in the presence of ZD 7288 from >800 cells/condition from two independent experiments. Data are plotted around the mean, with the boxes representing the 25th-75th percentiles and the whiskers the 5st-95th percentiles. Statistical comparisons were made using a one-way ANOVA with a Tukey’s multiple comparison (A–D) or two-tailed unpaired t-test (E–F). *p<0.05, **p<0.01, ***p<0.001. In A-D individual experimental replicates are represented.

Figure 4—source data 1. Quantification of GFP-positive neurons, RT-qPCR and nuclear staining intensity for Figure 4.

Figure 4.

Figure 4—figure supplement 1. HSV Reactivation Mediated by Forskolin Requires Neuronal Excitability.

Figure 4—figure supplement 1.

(A and B) Latently infected cultures were reactivated with forskolin in the presence of the HCN channel inhibitors ivabradine (20 µM; A) and CsCl (3 mM; B). Latently infected cultures were reactivated with forskolin in the presence of the HCN inhibitor ZD 7288 (10 µM) and viral lytic transcripts measured at 20 hr post-reactivation (C and D). Individual experimental replicates are represented in addition to the mean and SEM. Statistical comparisons were made using a one-way ANOVA with a Tukey’s multiple comparison. *p<0.05, **p<0.01.
Figure 4—figure supplement 1—source data 1. Quantification of GFP-positive neurons and RT-qPCR for Figure 4—figure supplement 1.