<p>Cryo-EM structure of RhGB07 trimeric spike in a 3-RBD down state.</p>

Highlighted residues of RhGB07 show the putative interacting residues that differ in identity to SARS-CoV-2. <b>(C)</b> Schematic depicting mutants generated of RhGB07 and SARS-CoV-2 Spikes with either full RBD swaps, or insertions/deletions, respectively. Lentiviral pseudotypes bearing Spikes of RhGB07 or SARS-CoV-2 mutants (RhGB07 wildtype, RhGB07_{SARS-CoV-2 RBD}, RhGB07437_438insGGNY, SARS-CoV-2 wildtype, SARS-CoV-2_{RhGB07 RBD}, SARS-CoV-2 Δ446−449), were used to infect BHK-21 cells overexpressing human ACE2.

A heatmap illustrating entry is shown, representing the mean log RLU of 3 separate experiments. <b>(D)</b> ELISA data depicting the binding curves and calculated EC₅₀ values (µg/ml) of recombinant RhGB07, SARS-CoV-2, RhGB07 437_438insGGNY or SARS-CoV-2 Δ446−449 RBDs with recombinant human ACE2. <b>(E)</b> Alignment of the Δ4-loop region of RhGB07 with SARS-CoV-2, SARS-CoV-2, RaTG13, Rs4231, and Rc-o319 (RhGB07 numbering), with differences in amino acid residues shown.

RhGB07 was mutated to introduce an insertion (437_438insGGNY, underlined) or additional mutations (mutated residues coloured in pink) in the Δ4-loop. A heatmap illustrating entry data is shown for pseudoviruses bearing these mutant Spikes, used to infect BHK-21 cells overexpressing <i>Rh pusillus</i>, <i>Rh. affinis</i>, <i>Rh. ferrumequinum</i>, <i>Rh. cornutus</i>, <i>Rh. alcyone and M. lucifugus</i> ACE2s. Data represents the mean log RLU of 4 separate experiments.

A vector-only control (pDISPLAY) was included to demonstrate specificity and to set the background signal. The data underlying this Figure can be found in <a href="plosbiology.org/article/info:doi/10.1371/journal.pbio.3003944#pbio.3003944.s017" target="_blank">S1 Data</a>.</p>