Insights into the mechanism of action of the bipartite fusion module of SARS-CoV-2 spike protein

ABSTRACT SARS-CoV-2 entry into host cells is mediated by the spike glycoprotein, which promotes fusion between viral and host membranes. Despite its importance, the precise location and mode of action of the fusion peptide, a key spike region that inserts into and perturbs the host membrane, remain elusive. Two regions have been proposed as fusion peptides: one located at the N-terminus of the protein (nFP) and the other at an internal position (iFP). Here, we combine computational and experimental approaches to characterize their roles and impact on membrane fusion. Molecular dynamics (MD) simulations indicated that the nFP interacts mostly at the membrane surface. Consistently, experimental biophysical assays revealed that the nFP exhibits low affinity and weak perturbing effects on lipid vesicles. In contrast, the iFP exhibits stronger membrane binding and induces stronger perturbation in vitro . MD simulations show that the iFP inserts deeply into and strongly affects the membrane, inducing lipid tail protrusion and increased water flux through the bilayer. Moreover, spike-pseudotyped lentiviruses carrying mutations in the iFP region showed that residues Y873, F888, and F906 are required for viral entry. Together, our findings suggest that SARS-CoV-2 uses a bipartite fusion module in which the nFP establishes initial contact with the host membrane and primes the bilayer, enabling subsequent deep insertion and further membrane destabilization promoted by the iFP. Given that FPs are conserved across viral families, a similar fusion module may be present in other coronaviruses, making this region a promising target for the development of broad-range antiviral therapeutics. IMPORTANCE We studied how SARS-CoV-2 enters human cells by focusing on a key region of its spike protein, known as the fusion peptide, which interacts with the host membrane. Although this region is essential for infection, its exact location and mechanism of action have remained unclear, with two candidate regions proposed. Using a combination of computational and experimental approaches, we found that these regions act together through a bipartite fusion module. One region initiates contact with the membrane, while the other inserts more deeply and drives the membrane perturbations required for viral entry. Given the relevance of this peptide for viral entry, these insights are expected to guide the development of novel antiviral therapies.