Effect of SARS-CoV-2 spike mutations on its activation by TMPRSS2 and TMPRSS13

The continuous emergence of new SARS-CoV-2 variants urges better understanding of the functional motifs in the spike (S) protein and their tolerance towards mutations. We here focus on the S2′ motif which, during virus entry, requires cleavage by a cell surface protease to release the fusion peptide. Though belonging to an immunogenic region, the SARS-CoV-2 S2′ motif (811-KPSKR-815) has shown hardly any variation, with its three basic (K/R) residues being >99.99% conserved thus far. By creating a series of mutant S-pseudotyped viruses, we show that K814, which precedes the scissile R815 residue, is dispensable for SARS-CoV-2 spike activation by TMPRSS2 but not TMPRSS13. The latter protease lost its activity towards SARS-CoV-2 S when the S2′ motif was swapped with that of the low pathogenic 229E coronavirus (685-RVAGR-689) and also the reverse effect was seen. This swap had no impact on TMPRSS2 activation. Also in the MERS-CoV spike, introducing a dibasic scissile motif was fully accepted by TMPRSS13 but less so by TMPRSS2. Our findings are the first to demonstrate which S2′ residues are important for SARS-CoV-2 spike activation by these two airway proteases, with TMPRSS13 exhibiting higher preference for K/R rich motifs than TMPRSS2. This preemptive insight can help to estimate the impact of S2′ motif changes as they may appear in new SARS-CoV-2 variants.