Effect of polymorphism in Rhinolophus affinis ACE2 on entry of SARS-CoV-2 related bat coronaviruses

Author summary Bat CoV RaTG13 has zoonotic transmission potential, and interactions between S protein and ACE2 play essential roles. RaTG13 uses RaACE2 as an entry receptor, and there are at least seven polymorphic RaACE2 variants. The effect of RaACE2 polymorphism on entry of RaTG13 and other SARS-CoV-2 related bat CoVs (SC2r-CoVs) has not been determined. In this study, we find that RaTG13 S pseudovirions only highly efficiently transduce cells expressing RaACE2 among available 18 bat species commonly habituating in Southwestern China and Southeastern Asia, suggesting that there might be limited bat species including R. affinis susceptible to infection of RaTG13 virus. The RaACE2 polymorphism has a marked effect on the entry of RaTG13, whereas it shows a minimal effect on the entry of SARS-CoV-2 and SC2r-CoVs like BANAL-20-52, BANAL-20-236, and pangolin-CoV GD. Further sequence and mutagenesis analyses identify residues 34, 38, and 83 in RaACE2 and residues 501 and 505 in the S proteins critical for S protein and receptor interactions, indicating that RaTG13 might have not been well adapted to R. affinis bats. We also find that T372A substitution in RaTG13 S significantly enhances entry on all RaACE2 variants and several bat ACE2s, and immune evasion might contribute to the natural selection of T372 over A372 in bat S proteins. Altogether, our work provides detailed analyses of the effect of RaACE2 polymorphism on the entry of RaTG13 and other SC2r-CoVs and a better understanding of the entry and evolution of CoVs.