Fig. 4: Molecular determinants affecting hACE2 recognition by NeoCoV and PDF-2180.
From: Close relatives of MERS-CoV in bats use ACE2 as their functional receptors
a, RBD binding modes and receptor (hACE2 or Bat37ACE2) footprints of the four indicated ACE2-using coronaviruses. b, The overlap of the ACE2 footprints. The heat map indicates the per-residue frequency of participation to the virus–ACE2 interfaces identifying residues 329–330 as a virus-binding hot spot. c, Schematic of the hACE2 swap mutants with Bat37ACE2 counterparts. d,e, Expression levels of hACE2 mutants were analysed using western blotting (d) and immunofluorescence (e). h, human. f,g, The receptor function of hACE2 mutants was evaluated by NeoCoV RBD binding (f) and NeoCoV S pseudotyped virus entry in HEK293T-hACE2 (h-WT) cells (g). The fold changes of pseudotyped virus entry relative to wild-type hACE2 are shown. For e and f, scale bars, 100 μm. h, Mutations in the interaction between NeoCoV RBD and Bat37ACE2 were taken as the input of mCSM-PPI2 to predict the change of free binding energy (ΔΔG, kcal mol−1). Mutations with lower ΔΔG are marked in blue and those with higher ΔΔG are marked in red. i, Computational modelling of the NeoCoV RBD–hACE2 complex obtained by superposing the hACE2 structure (PDB: 6M0J, blue) on the Bat37ACE2-bound NeoCoV RBD (red) structure described here. Magnified view of the T510F NeoCoV RBD mutation. j–l, The effect of NeoCoV and PDF-2180 RBM mutations on hACE2 recognition assessed by RBD–hFc binding (j), spike expression (lysate) and virion incorporation (supernatant) (k), and pseudotyped virus entry efficiency (l) in HEK293T-hACE2 and HEK293T-Bat37ACE2 cells. For j, scale bars, 100 μm. m, hACE2-dependent entry of NeoCoV-T510F S pseudotyped virus in Caco-2 cells in the presence of 50 μg ml−1 anti-ACE2 (H11B11) or anti-VSVG (I1) antibodies. Mock, no antibody. For d–g and j–l, data are representative of two infection assays with independent transfections. For m, data are representative of two infection assays. Data are mean ± s.d. for g (n = 4 biologically independent cells) and l and m (n = 3 biologically independent cells). Statistical analysis was performed using two-tailed unpaired Student’s t-tests.
