Molecular basis of convergent evolution of ACE2 receptor utilization among HKU5 coronaviruses

Abstract

DPP4 was considered a canonical receptor for merbecoviruses until the recent discovery of African bat-borne MERS-related coronaviruses using ACE2. The extent and diversity with which merbecoviruses engage ACE2 and their receptor species tropism remain unknown. Here, we reveal that HKU5 enters host cells utilizing Pipistrellus abramus (P.abr) and several non-bat mammalian ACE2s through a binding mode distinct from that of any other known ACE2-using coronaviruses. These results show that several merbecovirus clades independently evolved ACE2 utilization, which appears to be a broadly shared property among these pathogens, through an extraordinary diversity of ACE2 recognition modes. We show that MERS-CoV and HKU5 have markedly distinct antigenicity, due to extensive genetic divergence, and identified several HKU5 inhibitors, including two clinical compounds. Our findings profoundly alter our understanding of coronavirus evolution and pave the way for developing countermeasures against viruses poised for human emergence.

Figure 1.. Identification of several mammalian ACE2s as functional HKU5 entry receptors

(A) Merbecovirus RBD phylogenetic tree based on amino acid sequences defining 6 clades. c/hMERS: camel/human MERS-CoV isolates. Each merbecovirus is listed along with its GenBank ID. The animal symbols represent the hosts in which viruses from a given clade have been detected. (B) Phylogenetic trees of bat (top) or non-bat (bottom) mammalian ACE2 orthologs based on amino acid sequences, with genera and orders indicated for the bat or non-bat mammalian species, respectively. (C-D) Binding of the HKU5–19s RBD-hFc to and entry of HKU5–19s S VSV pseudovirus into HEK293T cells transiently transfected with the indicated bat (C) or non-bat (D) mammalian ACE2 orthologs. Abbr: abbreviations used for species names. (E-F) Propagation of psVSV-HKU5–19s (E) and authentic HKU5–1 (F) in wildtype human Caco-2 cells or Caco-2 cells with stable expression of either human or P.abr ACE2. The propagation of psVSV-HKU5–19s was examined by the expression of the GFP reporter gene at 24 hours post-infection (hpi). The propagation of authentic HKU5–1 was detected by immunofluorescence with an anti-HKU5 N antibody at 24 hpi. The trypsin concentration used is indicated. ACE2 expression was confirmed by immunofluorescence using C-terminal-fused FLAG tags. Scale bars: 200 μm. Mean values are shown in C and D with n=3 biological replicates.

Figure 2.. Molecular basis of HKU5 recognition of the P. abramus ACE2 receptor.

(A) Ribbon diagrams in two orthogonal orientations of the cryoEM structure of the HKU5–19s RBD (light blue) bound to the P. abramus ACE2 peptidase domain (green) at 3.1Å resolution. (B-C) Zoomed-in views of the interface highlighting key interactions between the HKU5–19s RBD and P. abramus ACE2. Selected polar interactions are shown as black dotted lines. (D) Polymorphism of ACE2-interacting residues (RBM) among HKU5 isolates shown as logoplot. (E) RBM amino acid sequence alignment of the HKU5–19s and HKU5–33s isolates. Conserved residues are rendered with a white font over a red background whereas non-conserved residues are rendered with a red font on a white background. The residue numbering corresponds to HKU5–19s. HKU5-g33s insertions are shown as black dots. The blue lines indicate residues outside the RBM shown for visualization purposes around the HKU5–33s insertions. (F-G) Biolayer interferometry analysis of the P.abr ACE2 ectodomain binding to the HKU5–19s and HKU5–33s RBDs immobilized on biolayer interferometry streptavidin (SA) biosensors. Binding avidities were determined by steady state kinetics and are reported as apparent affinities (K_D, app) due to avidity.

Figure 3.. HKU5 molecular determinants of ACE2 host species tropism.

A, Binding of the P.abr ACE2 construct comprising the peptidase and the dimerization domains (residues 20–724) to the wildtype (WT) HKU5–19s and to the listed RBD interface mutants immobilized on biolayer interferometry streptavidin (SA) biosensors. B-C, RBD-hFc binding (B) and pseudovirus entry (C) efficiencies of HKU5–19s mutants in HEK293T cells transiently expressing P.abr ACE2. The entry efficiency of wildtype HKU5–19s S VSV pseudovirus was set as 100%. D-E, HKU5–19s and HKU5–1 RBD-hFc binding to HEK293T cells transiently expressing the indicated P.abr ACE2 or hACE2 mutants assessed by immunofluorescence. F-G, HKU5–19s S VSV pseudovirus entry into HEK293T cells transiently expressing the indicated ACE2 mutants. H, HKU5–19s and HKU5–1 RBD-hFc binding to HEK293T cells transiently expressing wildtype and mutants M.erm ACE2. I, Summary of ACE2 residues governing species tropism for HKU5–19s. Favorable and unfavorable residues in ACE2 orthologs are highlighted in blue and red, respectively, using P.abr ACE2 residue numbering. (+): functional; (−): non-functional. J, Summary of the critical determinants of HKU5–19s receptor recognition. Key residues mentioned in J are highlighted in navy color on the P.abr ACE2 structure rendered as a grey surface. The rest of the HKU5–19s RBD footprint is shown in cyan. Data are shown as the MEAN ± SD of 3 biological replicates for C, F, and G. Statistical analyses used unpaired two-tailed t-tests: *: p < 0.05,**: p < 0.01, ***: p < 0.005, and ****: p < 0.001, NS: not-significant. Data representative of two independent experiments for F and G, and a single experiment for C. Scale bars: 100 μm.

Figure 4.. Identification of countermeasures against HKU5 merbecoviruses.

A, Neutralization of HKU5–19s S VSV pseudovirus mediated by a panel of MERS-CoV infection-elicited human plasma. Bars represent the mean of three biological replicates with SD and data points correspond to the mean of two technical replicates within each biological replicate carried out with distinct batches of pseudoviruses. B-C, Evaluation of inhibition of authentic HKU5–1 propagation in Caco-2 cells stably expressing P.abr ACE2 by the indicated concentration of broadly neutralizing antibodies (B) or small molecule inhibitors (C). HKU5–1 was detected by immunofluorescence using an anti-HKU5 N antibody at 24 hpi. Scale bars: 200 μm.

Figure 5.. Coronaviruses have evolved ACE2 utilization at least five times independently.

(A) RBD footprints of ACE2-using coronaviruses on their cognate receptors. (B) Comparison of the binding modes of the SARS-CoV-2 (PDB 7TN0), NL63 (PDB 3KBH), HKU5 RBDs, NeoCoV (PDB 7WPO) and MOW15–22 (PDB 9C6O) to bat ACE2 (not shown for clarity) or hACE2 (PDB 6M1D, B0AT1 not shown for clarity).