ATRX promotes transcription initiation of HSV-1 immediate early genes during early lytic infection

Abstract Herpes simplex virus 1 (HSV-1) transcribes its genome in a highly coordinated temporal cascade, utilizing the host RNA polymerase II (Pol II). Repression of transcription precedes the cascade's progression in a process requiring viral immediate early (IE) genes, in a phenomenon termed Transient Immediate Early gene Mediated Repression (TIEMR). Given that components of promyelocytic leukaemia nuclear bodies (PML-NBs) are known to rapidly engage incoming HSV-1 genomes, we investigated potential roles in TIEMR regulation. Using siRNA knockdown of PML-NB constituents (PML, DAXX, and ATRX), we observed that ATRX depletion significantly reduced nascent viral transcription on viral IE promoters at 1.5 hours post-infection (hpi) while DAXX knockdown increased viral transcription. ChIP-Seq analysis indicated ATRX associates with both highly transcriptionally active viral IE genes and with transcriptionally restricted non-IE viral genes, suggesting diverse functions. We show that ATRX's association with active transcription on IE genes correlates with the presence of G-quadruplexes (G4s). Pharmacologic stabilization of G4s mimicked the effects of ATRX knockdown, significantly reducing transcription initiation on IE genes. These findings suggest that ATRX promotes transcriptional initiation of HSV-1 IE genes by preventing or ablating G4 formation. Our results reveal a previously unrecognized pro-transcriptional role for ATRX early in HSV-1 infection.