RNA G-Quadruplexes in the Porcine Deltacoronavirus Genome: Structural Regulation and Therapeutic Targeting for Antiviral Strategies.

Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that poses a considerable risk to swine production worldwide. G-quadruplexes (G4s) are noncanonical nucleic acid secondary structures known to regulate critical steps of viral life cycles in multiple viruses; however, the existence and biological role of RNA G4s (RG4s) in PDCoV remain unclear. In this study, we identified putative G4-forming sequences (PQSs) in the nonstructural proteins 2 (Nsp2) and M genes of the PDCoV genome, which are highly conserved across PDCoV strains, based on bioinformatic analysis. Fluorescence turn-on assays and circular dichroism (CD) spectroscopy revealed that these PQSs fold into typical parallel RG4 structures in vitro, and their RG4 formation in living cells was further confirmed using G4-specific probes. Notably, treatment with the G4 ligands TMPyP4 and pyridostatin (PDS) significantly reduced PDCoV N mRNA abundance and corresponding protein expression, concomitant with a marked decrease in viral titers in LLC-PK1 cells. In addition, RG4 structures identified in the Nsp2 and M mRNAs were shown to inhibit translation through a posttranscriptional mechanism. Moreover, G4 ligands, such as PDS and TMPyP4, targeted the RG4 structure in Nsp2 mRNA and significantly inhibited Nsp2-EGFP fusion protein expression without affecting the expression of the M-EGFP fusion protein. In summary, our work supports the presence of sequence-conserved functional RG4 structures in the PDCoV genome and provides new insights into the development of antiviral strategies targeting PDCoV RG4s.