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

MERS-CoV is a lethal pathogen with pandemic potential. Clade A and B MERS-CoV viruses have caused outbreaks in the Middle East since 2012 when they initially spilled over from camels to humans. Clade C viruses, however, are only found in camels across Africa and the spillover potential of these viruses seems to be lower than for clade A/B strains but remains to be fully understood. Here, we report that clade C spikes are less well-cleaved at the S1/S2 boundary than clade A or B viral spikes and that most clade C spikes induce reduced syncytium formation. Additionally, we demonstrate that several East African clade C strains are less able to utilize the TMPRSS2-mediated pathway for viral entry in both cell lines and primary nasal epithelial cultures. We map the molecular basis of this reduced TMPRSS2 usage to the N-terminal domain (NTD) and subdomain 2 (SD2) of East African clade C MERS-CoV. We suggest that reduced usage of the TMPRSS2-mediated entry pathway may underlie the reduced replication of East African clade C strains in humans, while the reduced replication of West African strains remains to be further investigated. Overall, we suggest that altered protease usage may contribute to differential tropism of East African clade C strains and indicate geographically distinct selection pressures on spike between MERS-CoV strains circulating in camels. Significance StatementClade A/B MERS-CoV outbreaks have caused 957 deaths since the first spillover in 2012; meanwhile, Clade C MERS-CoV strains have been found in camels across Africa but have not been reported to cause outbreaks. Investigating why these viruses do not successfully transmit to humans will be key to understanding the pandemic potential of the African MERS-CoV camel reservoir. Our study indicates that clade C viruses exhibit less spike cleavage and that East African clade C isolates are less able to utilize the TMPRSS2-mediated pathway during viral entry of both human cell lines and primary nasal cells. Differences in viral entry pathways could alter cellular and organ tropism and inform our understanding of the pandemic potential of these viruses.