ACE2 binding is an ancestral and evolvable trait of sarbecoviruses

Abstract

Two different sarbecoviruses have caused major human outbreaks in the past two decades1,2. Both of these sarbecoviruses, SARS-CoV-1 and SARS-CoV-2, engage ACE2 through the spike receptor-binding domain2–6. However, binding to ACE2 orthologues of humans, bats and other species has been observed only sporadically among the broader diversity of bat sarbecoviruses7–11. Here we use high-throughput assays12 to trace the evolutionary history of ACE2 binding across a diverse range of sarbecoviruses and ACE2 orthologues. We find that ACE2 binding is an ancestral trait of sarbecovirus receptor-binding domains that has subsequently been lost in some clades. Furthermore, we reveal that bat sarbecoviruses from outside Asia can bind to ACE2. Moreover, ACE2 binding is highly evolvable—for many sarbecovirus receptor-binding domains, there are single amino-acid mutations that enable binding to new ACE2 orthologues. However, the effects of individual mutations can differ considerably between viruses, as shown by the N501Y mutation, which enhances the human ACE2-binding affinity of several SARS-CoV-2 variants of concern12 but substantially decreases it for SARS-CoV-1. Our results point to the deep ancestral origin and evolutionary plasticity of ACE2 binding, broadening the range of sarbecoviruses that should be considered to have spillover potential.

Sequence determinants of human-cell entry identified in ACE2-independent bat sarbecoviruses: A combined laboratory and computational network science approach

“…We and others have previously shown that clade 2 viruses are not capable of using ACE2 from human, mouse, civet, pangolin, or from the natural hosts, Rhinolophus - affinis or - sinicus bats. 7 , 28 , 29 To further assess the ACE2-independence of clade 2 viruses, we tested our panel with an Artibeus jamaicensis cell line (AJ i ) that is selectively permissive to clade 2 entry following trypsin treatment, but not to the ACE2-dependent clade 1 viruses. 7 We included the SARS-CoV-2 RBD in these assays, which has been recently suggested to use alternative, ACE2-independent pathways 30 ( Figure 2 c).…”

“…2 , and 3 ), human liver (Huh7.5), African green monkey kidney (VeroE6), and the AJ i kidney cell line (AJ i ), 7 while ACE2 from these species clearly does not support clade 2 infection when provided in ACE2-defficient cells like BHKs ( Figure 2 c,d). 7 Studies by other groups have further demonstrated that clade 2 RBDs are not capable of binding ACE2 from humans, pangolins, civets, and the Rhinolophus bat species that carry these viruses 29 , 34 [preprint]. Taken together with our previous evidence showing that individual point mutations cannot impart ACE2 binding to clade 2 viruses, it is apparent that at least some clade 2 sarbecoviruses are capable of employing an ACE2-independent cell entry route that is conserved across a range of mammalian species.…”

Shifting mutational constraints in the SARS-CoV-2 receptor-binding domain during viral evolution

“…However, as proteins evolve, the impacts of individual amino acid mutations can shift due to epistasis ( 4 ). For example, the same N501Y mutation that enhances SARS-CoV-2 binding to ACE2 severely impairs ACE2 binding by SARS-CoV-1 and other divergent sarbecoviruses ( 5 ). Furthermore, N501Y epistatically enabled other affinity-enhancing mutations that emerged in the Omicron variant of SARS-CoV-2 ( 6 – 8 ).…”

Selection Analysis Identifies Clusters of Unusual Mutational Changes in Omicron Lineage BA.1 That Likely Impact Spike Function

“…For example, among the bat-infecting sarbecoviruses, the BA.1 S/G339D substitution (in cluster region 1) has primarily to date been found among the bat-infecting viruses within a clade that does not use ACE2 as a cell entry receptor ( fig. 6 ; Starr, Zepeda, et al 2022 ). The change in receptor-binding function in these viruses is, however, most likely due to two receptor-binding motif deletions that are also specific to this clade.…”

“…The exception that proves the rule that sites in this region might not be free to change in response to immune pressures is S/493R. Given that S/493R has a strong antigenic effect, if it was not under selective constraints to sustain optimal degrees of ACE2 interaction ( Starr, Zepeda, et al 2022 ), it should (but does not) display at least intermittently detectable signs of positive selection.…”