Identification of i-motifs in Alphaherpesvirus immediate early promoters and their dynamic folding with G4s during infection.

I-motifs (iMs) and G-quadruplexes (G4s) are non-canonical DNA secondary structures formed by C-rich and G-rich sequences, respectively. While G4s have been identified as regulatory elements in both human and viral genomes, iMs remain largely unexplored. This study investigates the presence of iMs in the immediate-early (IE) promoters of human Alphaherpesviruses (αHHVs), namely herpes simplex virus 1 (HSV-1), herpes simplex virus 2 and varicella-zoster virus. αHHVs IE promoters, crucial for initiating an efficient viral cycle, embed G4s, but the potential presence of iMs in these regions is unknown. We identified highly conserved putative iM-forming sequences in IE promoters of all αHHVs. Biophysical characterization by circular dichroism, thermal difference spectroscopy and bromine footprinting, confirmed the ability of these sequences to form iMs in vitro, with varying conformations and distributions among the three viruses. Using a CUT&Tag-qPCR assay, we detected and quantified both iM and G4 structures in the HSV-1 genome during active viral infection. Our results show the dynamic formation of iMs and G4s in IE promoters, with enrichment levels changing over time. These structural changes correlated with variations in IE gene expression, indicating a functional role in HSV-1 biology. This study provides the first evidence of iMs in a viral genome, revealing a novel layer of genome organization through iMs folding alongside G4s in viruses.