Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2

Author summary The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), like the closely related virus SARS-CoV (SARS-CoV-1), infects cells by interacting with the cellular receptor angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the viral spike (S) protein through an independently folded subdomain, described as its receptor-binding domain (RBD). The susceptibility of a species to SARS-CoV-1 or -2 infection correlates with the binding affinity of their respective RBD for the ACE2 orthologs of that species. We therefore investigated the binding affinity of the RBD regions of multiple SARS-like coronaviruses with a range of ACE2 orthologs. Our results are consistent with the hypothesis that pangolins serve as an intermediate between humans and horseshoe bats. We further observed a high level of variability in the ability of the SARS-CoV-2 RBD to bind horseshoe bat ACE2 orthologs, suggesting ongoing selection pressure on their receptor ACE2 proteins from SARS-like viruses. Indeed, mutations derived from different horseshoe bat orthologs introduced into a soluble form of human ACE2 differentially impacted SARS-CoV-1 and SARS-CoV-2 S-protein-mediated infection. A combination of five residues present in multiple horseshoe bats increased the ability of a soluble form of ACE2 to neutralize SARS-CoV-2 S-protein-mediated infection. Thus horseshoe bats ACE2 orthologs can provide insight useful to improving the potency of ACE2-based therapeutics. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.

Automated summary

The research suggests that pangolins may serve as an intermediate host between humans and bats, and that bat ACE2 orthologs could provide insights useful for enhancing the potency of ACE2-based therapeutics.