Single-virus fusion measurements yield an opportunistic model for SARS-CoV-2 fusion

Sengar, Anjali; Bondalapati, Sai T.; Kasson, Peter M.

SARS-CoV-2 virus, like many coronaviruses, binds to cell-surface receptors and is then activated for membrane fusion and cell entry via proteolytic cleavage. In some coronaviruses, this occurs primarily at the cell surface, while in others primarily in endosomes. Prior data on SARS-CoV-2 suggest that either can happen, depending on cell type, and there has been debate regarding the physiologically relevant mechanism of entry. Here we use single-virus fusion experiments using isolated plasma membrane and exogenously controlled proteases to probe the question of protease activation directly. We find that a broad range of proteases can activate SARS-CoV-2 pseudoviruses for fusion, that this does not depend on the pseudovirus genetic background, and that the plasma membrane is competent to support fusion regardless of where the protease activation occurs. The resulting data suggest a model of opportunistic fusion by SARS-CoV-2, where the subcellular location of entry depends on airway protease activity, cell-surface protease activity, and endosomal protease activity, but all can support infection. These parallel pathways help explain why targeted host protease inhibition has shown strong efficacy in cell-culture models but weaker efficacy in clinical trials.