The fusion peptide of the spike protein S2 domain may be a mimetic analog of β-coronaviruses and serve as a novel virus-host membrane fusion inhibitor

Coronavirus has garnered more attention recently, particularly in the aftermath of the 2019 pandemic. The β genus of the coronavirus family has demonstrated a significant threat to humanity. Current mitigation strategies involve the development of vaccines and repurposing drugs for symptomatic management of coronavirus infection, specifically SARS-Cov 2. Fusion inhibitors that are available as antiviral drugs for coronavirus have targeted the heptad repeat (HR) 1 and 2 in the S2 domain of the spike protein. The current study identified a fusion peptide (FP) upstream of HR1 as a potential target for developing membrane fusion inhibitors, and mimetic peptides analogous to the FP segment were tested for antiviral activity. Four mimetic fusion peptides (MFPs) (RSA59PP (MFP633), RSA59P (MFP634), RSMHV2P (MFP635), and RSMHV2PP (MFP636)) that are analogous to the FP of murine β coronavirus mouse hepatitis virus (MHV), MHV-A59/RSA59 (PP) and MHV-2/RSMHV2 (P) with central proline mutations, were tested. Results show the ability of MFPs to reduce cell-to-cell fusion and viral replication in vitro. MFP633, which contains a central double proline, exhibited the most potent inhibitory effect in spike protein-mediated membrane fusion assays. Biophysical experiments also demonstrated the strongest interactions between double-proline containing MFPs (MFP633 and MFP636) with biomimetic liposomes. In vivo studies using a liposome-mediated delivery system in mice confirmed the antiviral activity of MFP633. These findings suggest that targeting FPs could develop effective fusion inhibitors against coronaviruses. MFPs act on the host cell membrane by competing with the viral FP during the early stage of host-viral membrane fusion events. MFP633 is a promising peptide drug candidate that warrants future examination to assess whether this and other dual-proline containing peptides may exert similar anti-viral effects in other coronaviruses with conserved FP structures.