Optimal stability of a highly conserved RNA G4 in PDCoV nsp8 supports viral proliferation

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, primarily causes diarrhea in piglets and has the potential for cross-species transmission to humans. The recent detection of PDCoV in Haitian children underscores the urgent need for developing antiviral therapeutic strategies. G-quadruplexes (G4s) are implicated in the modulation of viral infection; however, their identification and roles in the PDCoV life cycle remain unclear. Here, we identified a highly conserved G4 structure, designated PDCoV-G4, located within the coding region of PDCoV nonstructural protein 8 (nsp8). PDS and TMPyP4, two well-known G4-binding ligands, were found to target PDCoV-G4 and exhibit anti-PDCoV activity. Interestingly, PDS stabilizes the structure of PDCoV-G4, while TMPyP4 disrupts it. The recombinant PDCoV with G4-disruptive mutations (rPDCoV-nsp8mut) displays resistance to both PDS and TMPyP4. Utilizing an embryonated chicken egg (ECE) infection model, we observed that TMPyP4 provides superior protective effects for rPDCoV-wt-infected ECEs compared to PDS. However, both PDS and TMPyP4 exhibited diminished protective effects on chicken embryos infected with rPDCoV-nsp8mut, relative to rPDCoV-wt, further confirming their in vivo antiviral activity through targeting PDCoV-G4. These findings demonstrate that the PDCoV-G4 plays a crucial regulatory role in the PDCoV life cycle and pathogenicity, representing a potential target for antiviral therapy.