Extended Data Fig. 8: Characterization of NeoCoV and PDF-2180 RBM mutations enhancing human ACE2 binding and pseudovirus entry.
From: Close relatives of MERS-CoV in bats use ACE2 as their functional receptors
a, Analysis of binding of various concentrations of the NeoCoV and PDF-2180 RBD-hFc to HEK293T cells stably expressing hACE2. The SARS-CoV-2 RBD-hFc was used as a positive control. Mock indicates no protein added during protein incubation. Data representative of two live cell binding assays using independent preparations of RBD-hFc proteins. b, Analysis of binding kinetics of the interaction between NeoCoV RBD WT or T510F with hACE2 using BLI. Reported KD values correspond to avidities due to the utilization of dimeric ACE2 constructs. Representative of two independent experiments. Unfitted curves can be found in Supplementary Figure 4b. c, Identification of PDF-2180 S mutations enhancing hACE2 binding. Sequence alignment of the NeoCoV and PDF-2180 RBMs and definition of the PDF-2180 mutants generated. d, Binding of PDF-2180 RBD-hFc mutants to hACE2- or Bat37ACE2-expressing HEK293T cells. Data presented were performed in two independent assays with similar results. e, Western blot analysis of the expression levels of PDF-2180 S protein mutants in HEK293T cells. f, Western blot analysis of the packaging efficiency of PDF-2180 S mutants in VSV pseudotyped viruses. g, Entry efficiency of PDF-2180 S mutant pseudotyped viruses into hACE2- or Bat37ACE2- expressing HEK293T cells. Experiments presented were independently performed twice with similar results for d-g. Representative data of g are presented as mean ± SD (n = 3 biological triplicates). Two-tailed unpaired Student’s t-test; *P < 0.05,**P < 0.01; ***P < 0.005, and ****P < 0.001
