Fig. 2: RhGB07 can bind and use human ACE2 for cell entry in vitro.

a Entry of different spike pseudoviruses expressing viral glycoproteins into HEK293T cells transfected with (a) human receptors known to allow entry of human coronaviruses or (e) ACE2 homologues from different species. a, e The raw entry values for each pseudoviruses were normalised by their entry into cells transfected with a vector containing no receptor sequence (i.e., “empty”). The raw entry values of representative repeats (n = 3 independent experiments) are also shown for direct comparisons of absolute entry. Representative biolayer interferometry binding curves showing the association and dissociation of SARS-CoV-2 and RhGB07 spike proteins with (b) hACE2 (n = 2 and 3 independent experiments, at three and seven protein concentrations, respectively), or (f) with R. ferrumequinum or M. lucifugus ACE2 (n = 1 independent experiment, at seven protein concentrations). c Entry of pseudoviruses into different “normal” human-cell lines that stably express lower or physiological levels of hACE2. All entry measurements are normalised to those for the “bald” pseudovirus not expressing any spike protein. d Entry of pseudoviruses into Huh7.5 cells transduced with a human TMPRSS2 vector, normalised to “bald”. Data from panels (a, c–e) are compiled from n = 3–8 independent experiments and plotted as mean + s.d. Statistical significance was determined by (a, e) two-way ANOVA or c, d one-way ANOVA on log-transformed data (after determining log normality by the Shapiro–Wilk test and QQ plot) with multiple comparisons against “empty” vector or “bald” pseudovirus, respectively. *0.05 ≥ P > 0.01; **0.01 ≥ P > 0.001; ***0.001 ≥ P > 0.0001; ****P ≤ 0.0001. Exact P values annotated for each graph are as follows (from left to right), (a) <0.0001, <0.0001, <0.0001, <0.0001; (c, Calu-3) < 0.0001, <0.0001, 0.001, <0.0001, <0.0001; (c, Caco-2) < 0.0001, <0.0001, 0.022, <0.0001, <0.0001; (c, HEK293T-ACE2) < 0.0001, <0.0001, <0.0001; d <0.0001, <0.0001, <0.0001. (e, SARS-CoV-2) < 0.0001, 0.0003, 0.12, <0.0001, <0.0001 (e, BANAL-20-52) < 0.0001, <0.0001, <0.0001, <0.0001, <0.0001; (e, RatG13) < 0.0001, <0.0001, <0.0001, <0.0001 (e, RhGB07) < 0.0001, <0.0001; (e, RfGB02) < 0.0001.