FIG 3.

Establishment of a timeline for HSV-1 latency and reactivation by in situ detection of HSV-1 LATs and ICP4 RNA transcripts. (A) Experimental timeline of neuronal differentiation, infection, latency, and reactivation. (B) LUHMES neuronal cultures were plated and differentiated on coverslips for 5 days and then infected with 17syn⁺ at an MOI of 3 in the presence of 50 μM acyclovir (ACV). Forty-eight hours later, the medium was changed to medium without ACV and infection allowed to proceed, with harvesting of coverslips for RNAScope analysis on the indicated days. RNAScope analysis was performed per the manufacturer’s recommendation, and probes for LAT and ICP4 transcripts were designed by ACD. HSV-1 LATs were detected from the C2 channel (red) and the HSV-1 lytic transcripts (ICP4) were detected from the C1 channel (green). Reactivation was induced by incubation of day 11 (p.i.) cultures (labeled as 13d P.I. + 48h WM) for 48 h with the PI3K pathway inhibitor wortmannin (1 μM final concentration).