G-quadruplexes in the monkeypox virus are potential antiviral targets.

Monkeypox virus (MPXV) is a member of Orthopoxvirus in the Poxviridae family, causing a Public Health Emergency of International Concern. The number of cases and geographic range has increased significantly in 2022. Identification of MPXV-specific therapeutic targets is urgent. G-quadruplex (GQ) secondary structures attract great attention as potential targets for antiviral strategy. Whether GQs are present in the MPXV genome remains inconclusive. In this study, we aim to characterize the GQs encoded by MPXV. Through a series of biophysical experiments, we characterized the formation potential of MPXV-encoded GQs and evaluated the binding and stabilization abilities of GQ ligands including BRACO-19, pyridostatin, and TMPyP4 to GQs encoded by MPXV. Moreover, GQ ligands suppressed the gene transcription of MPXV sequences containing GQ. BRACO-19 and TMPyP4 were able to inhibit vaccinia virus replication. We demonstrated the existence of MPXV GQ and reinforced the idea that GQs could be novel antiviral targets. Targeting these GQ sequences with GQ-binding molecules may represent a new approach for MPXV therapy.