RaACE2 polymorphism confers different susceptibility to RaTG13.

(A) Phylogenetic tree of RaACE2 variants. The maximum-likelihood tree (left panel) was produced using MEGA X software, based on the alignment of ACE2 amino acid sequences of R. affinis. The branch lengths are scaled according to the number of amino acids substitutions per site, indicated by the scale bar. (B) The table of non-synonymous mutations present in the RaACE2 variants. The three of eight key residues which are involved in interacting with the RaTG13 and SARS-CoV-2 spike are shaded with a yellow background. The corresponding eight residues of human ACE2 are indicated at the bottom. (C) Analysis of RaACE2 proteins on the cell surface by cell surface protein biotinylation assay. HEK293 cells transiently overexpressing different RaACE2 and human ACE2 proteins were labeled with EZ-link Sulfo-NHS-LCLC-biotin on ice, and lysed with RIPA buffer. Biotinylated proteins were enriched with NeutrAvidin beads and detected by western blot using mouse monoclonal anti-FLAG M2 antibody. WCL, whole cell lysate. Entry mediated by RaTG13 S pseudovirions (D) and SARS-CoV-2 S pseudovirions (E) into cells expressing different RaACE2 variants. HEK-293 cells transiently expressing different RaACE2 variants were transduced with RaTG13 S pseudovirions or SARS-CoV-2 S pseudovirions and transduction efficiency were measured 40 hrs post-transduction. Experiments were done in triplicate and repeated at least three times. One representative is shown with error bars indicating SEM. Statistical significance is set as * p<0.05 and ** p<0.01 and calculated by T-test.