Structural and functional analysis of the SARS-CoV internal fusion peptide: Biophysical mechanism of membrane fusion

The spike protein of coronaviruses is critical for the infection process, facilitating the entry of viral genetic material into host cells. Among its functional domains, the internal fusion peptide (IFP) plays a pivotal role in the fusion of viral and host membranes, a process essential for viral entry. Understanding the biophysical properties and mechanisms of the IFP-membrane interaction is key to deciphering the viral fusion process and developing potential antiviral strategies. In this study, the SARS-CoV IFP was recombinantly expressed and purified with high purity. Vesicle aggregation and membrane fusion assays revealed that POPC/POPG vesicles were slightly more susceptible to IFP-induced fusion compared to cholesterol-containing vesicles, indicating that cholesterol does not enhance membrane fusion. The peptide binding was found to increase the zeta potential of anionic vesicles, making the membranes less negative and thereby reducing electrostatic repulsion. This effect, however, was independent of peptide concentration, suggesting that hydrophobic interactions, rather than electrostatic forces, are the main drivers of membrane fusion. Further analysis using electron paramagnetic resonance demonstrated that the IFP promotes local lipid ordering, increases packing, and reduces membrane fluidity. Circular dichroism spectroscopy confirmed that the IFP predominantly adopts an α-helical structure in micelles and bicelles, with a higher α-helical content in neutral lipid environments compared to anionic ones. Additionally, differential scanning calorimetry (experiments showed that IFP binding hampers the fluid-to-hexagonal phase transition in phosphatidylethanolamine (PE) membranes, promoting positive curvature and stabilizing the membrane fluid phase. In summary, these findings suggest a biophysical mechanism where IFP initially binds to the PE-rich outer layer of the membrane, inducing positive curvature that facilitates close contact between viral and cellular membranes. The peptide’s deep insertion into the membrane’s hydrophobic core and the subsequent lipid ordering further drive the fusion process, reducing the energetic barrier for the complete fusion of the lipid bilayers. These insights into the IFP’s role in membrane fusion highlight its potential as a target for developing antiviral therapies aimed at disrupting the viral entry process.