Figure 5.

Differential association of three S proteins with human and palm-civet ACE2. (A) HEK293T cells were transfected with plasmids encoding human or palm-civet ACE2, radiolabeled with [³⁵S]cysteine and [³⁵S]methionine, and lysed. ACE2 proteins were immunoprecipitated with 1D4, which recognizes a tag present at the carboxy-terminus of each receptor, or with S1-Ig variants containing the S1 domains of TOR2, GD, or SZ3 S proteins, and analyzed by SDS–PAGE. TOR2 SARS-CoV was isolated from humans infected during the 2002–2003 outbreak; GD, during the 2003–2004 outbreak; SZ3, from palm civets. (B) Experiment similar to that in (A) except that S-protein residues 318–510, comprising the RBDs of the indicated S proteins fused to the Fc domain of human IgG1 (RBD-Ig), were used to immunoprecipitate human or palm-civet ACE2. The bottom panel shows a Coomassie-stained SDS–PAGE gel of the individual RBDs used in this experiment.