1Traditionally, the emergence of coronaviruses (CoVs) has been attributed to a gain in receptor 2 binding in a new host. Our previous work with SARS-like viruses argued that bats already 3 harbor CoVs with the ability to infect humans without adaptation. These results suggested that 4 additional barriers limit the emergence of zoonotic CoV. In this work, we describe overcoming 5 host restriction of two MERS-like bat CoVs using exogenous protease treatment. We found that 6 the spike protein of PDF2180-CoV, a MERS-like virus found in a Ugandan bat, could mediate 7 infection of Vero and human cells in the presence of exogenous trypsin. We subsequently show 8 that the bat virus spike can mediate infection of human gut cells, but is unable to infect human 9 lung cells. Using receptor-blocking antibodies, we show that infection with the PDF2180 spike 10 does not require MERS-CoV receptor DPP4 and antibodies developed against the MERS spike 11 receptor-binding domain and S2 portion are ineffective in neutralizing the PDF2180 chimera.
12Finally, we found that addition of exogenous trypsin also rescues replication of HKU5-CoV, a 13 second MERS-like group 2c CoV. Together, these results indicate that proteolytic cleavage of 14 the spike, not receptor binding, is the primary infection barrier for these two group 2c CoVs.
15Coupled with receptor binding, proteolytic activation offers a new parameter to evaluate 16 emergence potential of CoVs and offer a means to recover previously unrecoverable zoonotic 17 CoV strains.
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Importance
19Overall, our studies demonstrate that proteolytic cleavage is the primary barrier to infection for a 20 subset of zoonotic coronaviruses. Moving forward, the results argue that both receptor binding 21 and proteolytic cleavage of the spike are critical factors that must be considered for evaluating 22 the emergence potential and risk posed by zoonotic coronaviruses. In addition, the findings also 23 offer a novel means to recover previously uncultivable zoonotic coronavirus strains and argue 24 that other tissues, including the digestive tract, could be a site for future coronavirus emergence 25 events in humans.