bioRxiv2025
SARS-like betacoronaviruses (sarbecoviruses) endemic in bats pose a significant zoonotic threat to humans. Genetic pathways associated with spillover of bat sarbecoviruses into humans are incompletely understood. We previously showed that the WT spike of the rhinolophid bat coronavirus SHC014-CoV has poor entry activity and uncovered two distinct genetic pathways outside the receptor-binding domain (RBD) that increased spike opening, ACE2 binding, and cell entry. Herein, we show that the widely studied bat sarbecovirus WIV1-CoV is likely a cell culture-adapted variant of Rs3367-CoV, which was sequenced from the same population of rhinolophid bats as SHC014-CoV. We demonstrate that the acquisition of a single amino-acid substitution in the 630 loop of the S1 subunit was the key spike adaptation event during the successful isolation of WIV1-CoV, and that it enhances spike opening, virus-receptor recognition, and cell entry in much the same manner as the substitutions we previously identified in SHC014-CoV using a pseudotype system. The conformational constraints on both the SHC014-CoV and Rs3367-CoV spikes could be alleviated by pre-cleaving them with trypsin, suggesting that the spike-opening substitutions arose to circumvent the lack of S1-S2 cleavage. We propose that the locked-down nature of these spikes and their requirement for S1-S2 cleavage to engage ACE2 represent viral optimizations for a fecal-oral lifestyle and immune evasion in their natural hosts. These adaptations may be a broader property of bat sarbecoviruses than currently recognized. The acquisition of a polybasic furin cleavage site at the S1-S2 boundary is accepted as a key viral adaptation for SARS-CoV-2 emergence that overcame a host protease barrier to viral entry in the mammalian respiratory tract. Our results suggest alternative spillover scenarios in which spike-opening substitutions that promote virus-receptor binding and entry could precede, or even initially replace, substitutions that enhance spike cleavage in the zoonotic host. Author SummaryRecent epidemic-causing coronaviruses, including SARS-CoV-2, originated in bats. Large numbers of such viruses circulate in bats and pose clear and present risks to humans. However, our incomplete understanding of the variables that influence viral spillover into new hosts challenges attempts to stratify viruses by threat level. We showed previously that the entry spike of the bat coronavirus SHC014-CoV, closely related to SARS-CoV-1 and SARS-CoV-2, exists largely in a closed conformation that is incompatible with its binding to the viral receptor, ACE2, and that genetic changes in key control sequences open the spike and unlock its entry activity. Here, we extend these findings to a second bat coronavirus. We demonstrate that WIV1-CoV, a highly studied virus that was previously isolated from the same population of Chinese horseshoe bats, is in fact a variant of the bat coronavirus Rs3367-CoV that acquired a genetic change in the same hotspot region of the spike during its propagation in cell culture. Our findings support the idea that the closed spikes of at least some (and likely, many) bat coronaviruses, while exquisitely adapted to their natural milieu in the bat digestive system, suffer poor functional activity outside this milieu, imposing a barrier to viral spillover that must be overcome through viral adaptation. Further, we identify spike genetic changes that overcome this deficit and may have value as prognostic markers of zoonotic risk.