α-Herpesviruses exploit host ssDNA gap prevention pathways for GC-rich genome replication and reactivation.

Many α-herpesviruses, such as herpes simplex virus type 1 (HSV-1) and pseudorabies virus, possess GC-rich genomes prone to forming G-quadruplex (G4) structures. However, the impact of these G4 structures on viral genome maintenance is unknown. Here, we demonstrate that G4s impede lagging strand DNA synthesis in the viral genome, generating single-stranded DNA (ssDNA) gaps that activate an ATR-mediated replication checkpoint. To complete genome duplication, these viruses co-opt host ssDNA gap prevention pathways, including PARP-mediated backup Okazaki fragment maturation and post-replicative gap-filling mechanisms, to resolve G4-induced ssDNA gaps. Pharmacological G4 stabilization, combined with inhibition of these pathways, synergistically inhibits viral replication. In terminally differentiated neurons, only gap-filling pathways are essential for virus replication, and disrupting them or stabilizing G4s robustly impairs HSV-1 reactivation in trigeminal ganglion explants. Collectively, our findings identify G4-induced ssDNA gaps as key barriers to α-herpesvirus replication and reactivation and highlight host ssDNA gap prevention pathways as promising therapeutic targets.