Identification of G-Quadruplexes in Hepatitis E Virus Negative-Sense Genomic RNA as Potent Antiviral Targets

Hepatitis E virus (HEV) causes significant global disease burden with no approved targeted therapies, highlighting the urgent need for innovative treatment strategies. G-quadruplexes (G4s), noncanonical nucleic acid structures formed by guanine-rich sequences, have emerged as important regulators of viral replication. Here, we identified two potential G4 sequences within HEV negative-sense genomic RNA. Circular dichroism spectroscopy confirmed their stable, parallel G4 structures, with structural stability enhanced by the G4-binding ligand pyridostatin (PDS). Using an EGFP reporter system, we demonstrated that these G4s significantly suppressed downstream gene expression, an effect potentiated by PDS treatment. In HEV infection models, PDS substantially inhibited viral RNA synthesis and ORF2 protein expression. This antiviral activity was recapitulated by the structurally distinct G4-binding ligand TMPyP4, but not by the weak-binding control TMPyP2, confirming G4-dependent regulation. Our findings establish G4s as functional regulatory elements in the HEV life cycle and as promising RNA-targeted therapeutic targets against HEV.