Characterization of RNA G-quadruplexes in porcine epidemic diarrhea virus genome and the antiviral activity of G-quadruplex ligands

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. However, there are still no anti-PEDV drugs with accurate targets. G-quadruplexes (G4s) are non-canonical secondary structures formed within guanine-rich regions of DNA or RNA, and have attracted great attention as potential targets for antiviral strategy. In this study, we reported two putative G4-forming sequences (PQS) in S and Nsp5 genes of PEDV genome based on bioinformatic analysis, and identified that S-PQS and Nsp5-PQS were enabled to fold into G4 structure by using circular dichroism spectroscopy and fluorescence turn-on assay. Furthermore, we verified that both S-PQS and Nsp5-PQS PQS could form G4 structure in live cells by immunofluorescence microscopy. In addition, G4-specific compounds, such as TMPyP4 and PDS, could signifi-cantly inhibit transcription, translation and proliferation of PEDV in vitro. Importantly, these compounds exert antiviral activity at the post-entry step of PEDV infection cycle, by inhibiting viral genome replication and protein expression. Lastly, we demonstrated that TMPyP4 can inhibit reporter gene expression by targeting G4 structure in Nsp5. Taken together, these findings not only reinforce the presence of viral G-quadruplex sequences in PEDV genome but also provide new insights into developing novel antiviral drugs targeting PEDV RNA G-quadruplexes.