Identification of critical residues in RaTG13 and SARS-CoV-2 S protein for virus entry.

(A) Alignment of RaTG13 RBD and SARS-CoV-2 RBD. The RBM of RaTG13 RBD and SARS-CoV-2 RBD are highlighted in yellow background. The mutations chosen are indicated in red. (B) Detection of mutant S proteins of RaTG13 (B) and SARS-CoV-2 (C) in cell lysates and pseudovirions by western blot using anti-FLAG M2 antibody. Top panel, cell lysate; bottom panel, pseudovirions; β-actin and HIV p24 were used as loading controls. Entry mediated by mutant RaTG13 (D) and SARS-CoV-2 (E) S pseudovirions into HEK-293 cells expressing RaACE2 variants. HEK-293 cells transiently expressing RaACE2 variants were transduced with indicated S pseudovirions as well as mutants. Experiments were performed three times, and one representative is shown as the heat map.