The Conserved Fusion Peptide Sequence of SARS Coronaviruses Kinetically Promotes the Fusion Pore Opening

Enveloped viruses enter the cell through the fusion of the viral envelope with the host membrane. These viruses either fuse at the plasma membrane or the endosome after entering the cell through endocytosis. Severe acute respiratory syndrome coronaviruses (SARS-CoVs) fuse at the plasma membrane by harnessing the capability of the spike (S) protein to fuse membranes. The binding of the S1 subunit to angiotensin-converting enzyme 2 (ACE2), the cell surface receptor, leads to the proteolytic cleavage at the S2' site, which exposes the N-terminal fusion peptide (FP) and induces fusion. In this work, we have evaluated the effect of the conserved fusion peptide sequence of SARS-CoVs (FP) in polyethylene glycol (PEG)-induced fusion of small unilamellar vesicles (SUVs) in different lipid compositions. The analysis of kinetic data of fusion observables such as lipid mixing (LM), content mixing (CM), and content leakage (CL) in a one-intermediate fusion model provides a detailed insight into the mechanistic role of FP in membrane fusion. We have further shown that the peptide binding does not alter the hydrodynamic diameters, ζ and dipole potentials of membranes with varying lipid composition. To understand the plausible role of FP in inducing membrane fusion, we have examined the effect of peptide binding on the depth-dependent membrane ordering and polarity using steady-state and time-resolved fluorescence spectroscopy. Evaluation of kinetic data reveals that the FP promotes the rate constant of pore formation without affecting its extent, making the fusion reaction kinetically favorable. Overall, our work provides a mechanistic understanding of the role of the conserved fusion peptide sequence of SARS-CoVs in membrane fusion.