The SARS-CoV-2 fusion domain preferentially initiates fusion in the late endosome.

Abstract

SARS-CoV-2 can enter target cells via both the plasma and endosomal membranes. This ability to utilize either pathway increases the likelihood by which viral entry will succeed in delivering the viral genetic information, a vital component of the viral lifecycle. Most other viruses only utilize one or the other fusion pathway, partly due to vastly different environmental factors such as pH and membrane lipid composition. The coronavirus spike glycoprotein is responsible for facilitating viral entry by first binding to the receptor followed by membrane fusion, the latter of which is initiated by the fusion domain (FD). Improving our understanding of how the FD initiates fusion in both pathways, and whether or not one is preferred over the other, will help to elucidate the molecular mechanisms involved in the membrane fusion process of SARS-CoV-2. Through a FRET-based lipid mixing assay, the SARS-CoV-2 FD (S816-F855) displays a biphasic relationship between pH and fusion, with a pH similar to that of the late endosome (∼pH5.5-4.5) demonstrating the highest fusogenic activity. Intriguingly, when assessing the structure of the FD at pH7.4 and pH5.0 in dodecylphosphocholine, only minor conformational changes occur. This change in conformation results in the elongation of an α-helix within the fusion peptide, which alongside the fusion loop, are the two structural components of the FD that are found to be critical for full functionality of the peptide. Furthermore, the FD also favors the lipid environment of the late endosome to elicit fusion, with a specificity for the endosomal lipid BMP observed. In conclusion, the SARS-CoV-2 FD displays an α-helical extension at low pH when compared to neutral pH, that allows it to preferentially initiate fusion in a lipid and pH environment resembling that of the late endosome.