Tandemly Repeated G-Quadruplex Structures in the Pseudorabies Virus Genome: Implications for Epiberberine-Based...

G-quadruplex (G4) structures have emerged as critical regulatory elements in viral genomes and represent potential targets for antiviral intervention. In this study, we identified and characterized G4 structures in the unique long (UL) region of the Pseudorabies virus (PRV) genome, highlighting their role as novel antiviral targets. Bioinformatic analysis revealed two guanine-rich regions (R1 and R2) that form stable G4 structures, as confirmed by fluorescence assays, circular dichroism (CD) spectroscopy, and immunofluorescence staining. Notably, these G4 structures exhibit a tandem repeat arrangement, a previously unreported feature in the PRV genome. Epiberberine (EPI), a natural G4-stabilizing ligand, bound to and stabilized these structures, leading to the inhibition of Taq polymerase progression. Functional assays demonstrated that EPI effectively suppressed PRV replication in vitro while having no significant impact on viral entry or release. In vivo, EPI treatment significantly improved survival rates and reduced viral loads in multiple organs, including the brain, heart, lungs, and kidneys of infected mice. These findings provide new insights into the role of G4 structures in PRV replication and demonstrate that EPI exhibits potential antiviral activity by targeting G4 structures.