MERS-related coronavirus circulating in pangolins exhibits strong fusogenicity in human cells and high sensitivity to fusion inhibitors.

Unlike preceding MERS-related coronaviruses, the recently identified MjHKU4r-CoV-1 strain can directly infect human cells. Nonetheless, its potential pathogenic attributes and underlying molecular mechanisms remain unclear. We find that MjHKU4r-CoV-1 induces significant inflammation, including interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α), and exhibits pronounced fusogenicity mediated by its spike (S) protein, leading to extensive syncytium formation. This suggests the possibility that MjHKU4r-CoV-1 possesses strong pathogenic potential in humans. Further, we successfully reveal the molecular mechanism of MjHKU4r-S-driven membrane fusion by crystallizing the six-helix bundle (6-HB) structure, a fusion apparatus composed of HR1 and HR2 domains. Concurrently, we develop a series of peptide-based fusion inhibitors that target the viral HR1 domain to impede the formation of viral 6-HB. Among these fusion inhibitors, a stapled peptide, MjHKU4r-HR2P10, shows the most potent inhibitory activity against MjHKU4r-CoV-1, MERS-CoV, SARS-CoV-2, and HCoV-OC43 infections at nanomolar level and thus holds considerable promise for further development as effective antiviral agents in clinic.