Single-virus fusion measurements yield an opportunistic model for SARS-CoV-2 fusion
SARS-CoV-2 binds to cell-surface receptors and is activated for membrane fusion and cell entry via proteolytic cleavage. Phenomenological data have shown that SARS-CoV-2 can be activated for entry at either the cell surface or in endosomes, but the relative roles in different cell types and mechanisms of entry have been debated. Here we use single-virus fusion experiments and exogenously controlled proteases to probe activation directly. We find that plasma membrane and an appropriate protease are sufficient to support SARS-CoV-2 pseudovirus fusion. Furthermore, fusion kinetics of SARS-CoV-2 pseudoviruses are indistinguishable no matter which of a broad range of proteases was used to activate the virus. This suggests that fusion mechanism is insensitive to protease identity or even whether activation occurs before or after receptor binding. These data support a model for opportunistic fusion by SARS-CoV-2, where subcellular location of entry likely depends on the differential activity of airway, cell-surface, and endosomal proteases, but all support infection. Inhibiting any single host protease may thus reduce infection in some cells but may be less clinically robust. ImportanceSARS-CoV-2 can use multiple pathways to infect cells, as demonstrated recently when new viral variants switched dominant infection pathways. Here, we use single-virus fusion experiments together with biochemical reconstitution to show that these multiple pathways coexist simultaneously and specifically that the virus can be activated by different proteases in different cellular compartments with mechanistically identical effect. The consequences of this are that the virus is evolutionarily plastic and that therapies targeting viral entry should address multiple pathways at once to achieve optimal clinical effects.