SARS-CoV S1 domain binds rodent ACE2 less efficiently than human ACE2. (A) 293T cells transfected with 10 μg of plasmid encoding N-terminally Myc-tagged ACE2 of the indicated species were stained with the anti-Myc-tag antibody 9E10 (heavy line), with S1-Ig (a fusion of the SARS-CoV S1 domain with the Fc domain of human IgG1 [shaded area]), or with secondary antibody alone (light line), and analyzed by flow cytometry. (B) Same as for panel A, except that 5 μg of plasmid was used for transfection. (C) 293T cells transfected with plasmids encoding C-terminally tagged ACE2 of the indicated species were metabolically labeled with [³⁵S]cysteine and [³⁵S]methionine and lysed as previously described (20). Cell lysates were immunoprecipitated with either the antitag antibody 1D4 or S1-Ig and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Infection with S protein-pseudotyped retrovirus is less efficient in cells expressing rodent ACE2 than in those expressing human ACE2. (A) An aliquot of cells analyzed as shown in Fig. 1B was incubated with S protein-pseudotyped SIV-GFP. After 48 h, the cells were harvested and analyzed by flow cytometry for GFP expression. GFP levels indicating infection (dark gray) are compared with anti-Myc antibody (light gray) and S1-Ig (medium gray) recognition in the same cells. (B) Same experiment as shown in panel A except that cells analyzed as shown in Fig. 1A were infected with SIV-GFP. (C) Typical image of GFP expression of cells transfected and infected as shown in panel B. The figure is representative of two similar experiments with similar results.

Rodent ACE2 mediates less-efficient syncytial formation with S protein-expressing cells than human ACE2. 293T cells transfected with plasmids encoding ACE2 of the indicated species or with vector alone (control) were mixed at a 1:1 ratio with 293T cells transfected with a plasmid encoding a codon-optimized form of a SARS-CoV S protein. Multinucleated syncytia were observed 48 h after the cells were mixed. The experiment shown is representative of three experiments with similar results.

Replication of SARS-CoV in murine cells is enhanced by human receptor. (A) Murine 3T3 cells were transfected with plasmid encoding Myc-tagged variants of human or mouse ACE2 as indicated, or with vector alone (control), and analyzed by flow cytometry using the anti-Myc-tag antibody 9E10 (light gray bars) or S1-Ig (medium gray bars). (B) An aliquot of the cells analyzed as shown in panel A was incubated with SARS-CoV for 4 days. Peak viral titers (medium gray) were determined by measuring the cytopathicity in Vero E6 cells incubated with cell supernatants as previously described (13). Viral RNA levels (dark gray) were determined by semiquantitative reverse transcription-PCR of cell supernatants, as previously described (13). Viral titers and RNA levels of control cells were below 100 50% tissue culture infective doses (TCID₅₀) per ml and 100 copies per ml, the limits of detection in both assays. The figure shows an experiment representative of two experiments with similar results.