No abstract

Coronavirus infects the cell by cytoplasmic or endosomal membrane fusion driven by the spike (S) protein, which must be primed by proteolytic cleavage at the S1/S2 furin cleavage site (FCS) and S2′ site by cellular proteases. Exogenous trypsin as a medium additive facilitates isolation and propagation of several coronaviruses in vitro. Here, we showed that trypsin mediated the enhancement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in cultured cells and that SARS-CoV-2 entered the cells via either a non-endosomal or an endosomal fusion pathway depending on the presence of trypsin. Interestingly, trypsin enabled viral entry at the cell surface and led to more efficient infection efficiency than trypsin-independent endosomal entry, suggesting that trypsin production in the target organs may trigger high replication of SARS-CoV-2 and cause severe tissue injury. Extensive syncytia formation and enhanced growth kinetics were observed only in the presence of exogenous trypsin for the cell-adapted SARS-CoV-2 strains. During 50 serial passages without trypsin addition, a specific R685S mutation occurred in the S1/S2 FCS (681PRRAR685) that was completely conserved but with several mutations in the S2 fusion subunit in the presence of trypsin. These findings demonstrate that S1/S2 FCS is essential for S protein proteolytic priming and fusion activity for SARS-CoV-2 entry but not for SARS-CoV-2 replication. Our data can contribute to the improvement of SARS-CoV-2 production to develop vaccines or antivirals and motivate further investigations into the explicit functions of cell adaptation-related genetic drift in SARS-CoV-2 pathogenesis.