SARS-CoV-2 Omicron spike H655Y mutation is responsible for enhancement of the endosomal entry pathway and reduction of cell surface entry pathway

The SARS-CoV-2 Omicron variant reportedly displays decreased usage of the cell surface entry pathway mediated by the host transmembrane protease, serine 2 (TMPRSS2) and increased usage of the endosomal entry pathway mediated by cathepsin B/L. These differences result in different cell tropisms and low fusogenicity from other SARS-CoV-2 variants. Recent studies have revealed that host metalloproteases are also involved in cell surface entry and fusogenic activity of SARS-CoV-2, independent of TMPRSS2. However, the involvement of metalloproteinase-mediated cell entry and fusogenicity in Omicron infections has not  been investigated.  Here, we report that Omicron infection is less sensitive to the metalloproteinase inhibitor marimastat ,  like the TMPRSS2 inhibitor nafamostat, and is more sensitive to the cathepsin B/L inhibitor E-64d than infections with wild-type SARS-CoV-2 and other variants. The findings indicate that Omicron preferentially utilizes the endosomal pathway rather than cell surface pathways for entry. Moreover, the Omicron variant also displays poor syncytia formation mediated by metalloproteinases ,  even when the S cleavage status mediated by fusion-like proteases is unchanged. Intriguingly, the pseudovirus assay showed that a single mutation, H655Y, of  the Omicron spike (S)  is responsible for the preferential entry pathway usage without affecting the S cleavage status. These findings suggest that the Omicron variant has altered entry properties and fusogenicity , probably through the H655Y mutation in its S protein , leading to modulations of tissue and cell tropism, and reduced pathogenicity. ### Competing Interest Statement The authors have declared no competing interest.