Stabilization of RNA G-quadruplexes in the SARS-CoV-2 genome inhibits viral infection via translational suppression.

The G-quadruplex (G4) formed in single-stranded DNAs or RNAs plays a key role in diverse biological processes and is considered as a potential antiviral target. In the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 25 putative G4-forming sequences are predicted; however, the effects of G4-binding ligands on SARS-CoV-2 replication have not been studied in the context of viral infection. In this study, we investigated whether G4-ligands suppressed SARS-CoV-2 replication and whether their antiviral activity involved stabilization of viral RNA G4s and suppression of viral gene expression. We found that pyridostatin (PDS) suppressed viral gene expression and genome replication as effectively as the RNA polymerase inhibitor remdesivir. Biophysical analyses revealed that the 25 predicted G4s in the SARS-CoV-2 genome formed a parallel G4 structure. In particular, G4-644 and G4-3467 located in the 5' region of ORF1a, formed a G4 structure that could be effectively stabilized by PDS. We also showed that PDS significantly suppressed translation of the reporter genes containing these G4s. Taken together, our results demonstrate that stabilization of RNA G4s by PDS in the SARS-CoV-2 genome inhibits viral infection via translational suppression, highlighting the therapeutic potential of G4-ligands in SARS-CoV-2 infection.