Clade C MERS-CoV camel strains vary in protease utilization during viral entry

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a lethal pathogen with pandemic potential. Clades A and B MERS-CoV strains have caused periodic outbreaks in the Middle East since 2012 when they spilled over from camels to humans. Meanwhile, Clade C MERS-CoV strains are found only in camels across Africa and do not appear to cause outbreaks in humans. Here, we sought to understand whether differences in viral entry pathways underlie the reduced spread from camels to humans. We characterized the replication and viral entry of a panel of six clade C strains from West and East Africa and found that both West and East clade C strains were attenuated for replication in human lung cell lines relative to clade A/B viruses. We report that clade C spike proteins are less well-cleaved at the S1/S2 boundary than clade A or B viral spikes during viral infection and that a majority of this clade C panel induce reduced syncytium formation. Additionally, we demonstrate that while West African clade C strains have a similar propensity to use the TMPRSS2-mediated pathway for viral entry, East African clade C strains are less able to utilize this protease for entry in both lung-derived cell lines and primary nasal epithelial cultures. We map the molecular determinants of this reduced TMPRSS2 usage to both the N-terminal domain (NTD) and subdomain 2 (SD2) of spike. We suggest that reduced usage of the TMPRSS2-mediated entry pathway may contribute to the reduced replication of East African clade C strains in humans by altering cellular and organ tropism; moreover this indicates geographically distinct selection pressures on spike among MERS-CoV strains circulating in camels. Deciphering the barriers to spillover of the camel reservoir of MERS-CoV in Africa will facilitate pandemic preparedness and improve our understanding of species-specific factors driving viral evolution.