The furin cleavage site is required for pathogenesis, but not transmission, of SARS-CoV-2

The SARS-CoV-2 spike, key to viral entry, has two features that differentiate it from other sarbecoviruses: the presence of a furin cleavage site (FCS; PRRAR sequence) and an extended S1/S2 loop characterized by an upstream QTQTN amino acid motif. Our prior works show that shortening the S1/S2 loop by deleting either the FCS (ΔPRRA) or an upstream sequence (ΔQTQTN) ablates spike processing, alters host protease usage, and attenuates infection in vitro and in vivo. With the importance of the loop length established, we evaluated the impact of disrupting the FCS while preserving the S1/S2 loop length. Using reverse genetics, we generated a SARS-CoV-2 mutant that disrupts the FCS (PQQAR) but maintains its extended S1/S2 loop. The SARS-CoV-2 PQQAR mutant has reduced replication, decreased spike processing, and attenuated disease in vivo compared to wild-type SARS-CoV-2. These data, similar to those from the FCS deletion mutant, indicate that loss of the furin cleavage site attenuates SARS-CoV-2 pathogenesis. Importantly, we subsequently found that the PQQAR mutant can be transmitted in the direct contact hamster model despite lacking an intact FCS. However, competition transmission showed that the mutant was attenuated compared to WT SARS-CoV-2. Together, the data suggest that the FCS is required for SARS-CoV-2 pathogenesis but is not strictly required for viral transmission.