The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2

Bat coronavirus(CoV)RaTG13 shares the highest genome sequence identity with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)among all known coronaviruses,and also uses human angiotensin converting enzyme 2(hACE2)for virus entry.Thus,SARS-CoV-2 is thought to have originated from bat.However,whether SARS-CoV-2 emerged from bats directly or through an intermediate host remains elusive.Here,we found that Rhinolophus affinis bat ACE2(Ra ACE2)is an entry receptor for both SARSCoV-2 and Ra TG13,although the binding of Ra ACE2 to the receptor-binding domain(RBD)of SARSCoV-2 is markedly weaker than that of h ACE2.We further evaluated the receptor activities of ACE2 s from additional 16 diverse animal species for Ra TG13,SARS-CoV,and SARS-CoV-2 in terms of S protein binding,membrane fusion,and pseudovirus entry.We found that the Ra TG13 spike(S)protein is significantly less fusogenic than SARS-CoV and SARS-CoV-2,and seven out of sixteen different ACE2 s function as entry receptors for all three viruses,indicating that all three viruses might have broad host rages.Of note,Ra TG13 S pseudovirions can use mouse,but not pangolin ACE2,for virus entry,whereas SARS-CoV-2 S pseudovirions can use pangolin,but not mouse,ACE2 enter cells efficiently.Mutagenesis analysis revealed that residues 484 and 498 in Ra TG13 and SARS-CoV-2 S proteins play critical roles in recognition of mouse and human ACE2 s.Finally,two polymorphous Rhinolophous sinicus bat ACE2 s showed different susceptibilities to virus entry by Ra TG13 and SARS-CoV-2 S pseudovirions,suggesting possible coevolution.Our results offer better understanding of the mechanism of coronavirus entry,host range,and virushost coevolution.