Sarbecovirus RBD indels and specific residues dictating multi-species ACE2 adaptiveness

Figures

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  Fig. 1. Phylogenetic and structural analysis of sarbecoviruses categorized into four indel types. a Flow diagram illustrating the retrieval of 265 non-redundant S glycoprotein sequences from non-human sarbecoviruses and three additional human sarbecoviruses. b The RBD clade information of the 268 sarbecoviruses. c RBM sequence logo plot illustrating the three high variable regions. The SARS-CoV-2 residue numbering is shown. d Phylogenetic tree based on RBD amino acids sequence for the 268 sarbecoviruses (See also Supplementary Fig. 1a, b for the complete tree and selection strategy) and multi-sequence alignment of 35 selected sarbecoviruses displayed with four RBM indel types with relevant sequences were boxed with corresponding colors. The 20 sarbecoviruses for subsequent functional analysis are indicated in red. The two cysteines for disulfide bridging are highlighted in orange. e Summary of the deleted residue numbers in Region 1 and Region 2 compared to the SARS-CoV-2 RBM sequence. The counts of each deletion length among the 268 sarbecoviruses are indicated in parentheses. f The counts of S glycoprotein sequences of the four RBM indel types. g Distribution plot of RBD clades grouped…

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  Fig. 2. Multi-species ACE2 tropism of representative sarbecoviruses and the impact of critical RBM residues. a, b Multi-species ACE2 usage spectra of sarbecoviruses of different indel types. RBD binding of 20 sarbecoviruses (a) and PSV entry of 17 sarbecoviruses (b) were assessed in HEK293T cells stably expressing the 56 ACE2 orthologues from bats and selected mammals. Rhinolophidae as the natural sarbecovirus reservoir is indicated in red. Asterisk: sarbecoviruses not tested in PSV entry experiments. c Efficiencies of selected sarbecovirus RBD-hFc recombinant proteins binding to HEK293T cells stably expressing the indicated ACE2 orthologs from their hosts or preferred species. d BLI analyses of binding kinetics of the soluble dimeric ACE2 ectodomains to immobilized viral RBD-hFc. e, f Structural demonstration (7DRV for RaTG13 and 7DDP for GX_P2V) (e) and S glycoprotein pseudovirus packaging efficiencies (f) of RaTG13 and GX_P2V swap mutants. For f, data are representative blots from two independent experiments. g, h Heat map displaying PSV entry efficiencies of RaTG13 (g) and GX_P2V (h) swap mutants (SARS-CoV-2 residue numbering) in HEK293T cells expressing the indicated ACE2…

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  Fig. 3. The impact of Region 1 and Regon 2 indels on multi-species ACE2 tropism of sarbecoviruses. a Schematic illustration of the Region 1 and Region 2 substitutions in sarbecoviruses of different indel types. Light pink background: insertions corresponding to SARS-CoV-2 counterparts; gray background: deletions corresponding to sequences from type-II or type-III sarbecoviruses. (*) RBM swaps based on the indel boundaries. b Western blot detecting the S glycoprotein packaging efficiency in PSV particles. c, d RBD binding (c) and PSV entry (d) efficiencies of indicated sarbecoviruses and their swap mutants in HEK293T cells stably expressing the 56 ACE2 orthologues. e–h Disulfide bond in Region 2 is critical for multi-species ACE2 usage of sarbecoviruses. Schematic illustration of the Region 2 disulfide-related mutants based on SARS-CoV-2, Rc-o319, and their Region 2 substitution mutants (e), S glycoprotein packaging efficiency (f), RBD binding efficiency (g), and PSV entry efficiency (h) of SARS-CoV-2 and Rc-o319 mutants in HEK293T cells stably expressing the 56 ACE2 orthologues (dots in g and h). Dashed lines: background cut-off of RBD binding and PSV entry assays. For b and f, data are…

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  Fig. 4. Fine mapping of clade 2-specific residues outside the RBM restricting ACE2 recognition. a RBD residue usage (SARS-CoV-2 residue numbering) of 268 sarbecoviruses grouped by ACE2 dependence. Red fonts and arrows: strict clade 2-specific residues. Orange fonts and arrows: limited clade 2 specificity. b RBD amino acid sequences alignment of SARS-CoV-2, SARS-CoV-1, and HKU3. Red: HKU3-specific; Orange: shared by HKU3 and SARS-CoV-1 only. The boundaries of three fragments (Frag.) for subsequent mapping are indicated. c–g Fine mapping of residues restricting ACE2 usage outside the RBM. Mapping strategy for narrowing down the range of determinants restricting ACE2 recognition (c). Orange and green circles: capability of using hACE2 for entry (>1% of SARS-CoV-2 entry) and binding (background subtracted RFU > 0.2), respectively. Gray: unable to use hACE2. Underlines: two critical residues limiting ACE2 binding. PSV entry (d) and RBD binding (e) of the HKU3 mutants carrying SARS-CoV-2 corresponding sequences in HEK293T-hACE2. Yellow highlighted the mutants critical for analyzing ACE2-restricting determinants. Data representative of three independent experiments for d and e. RBD binding (f)…

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  Fig. 5. The proposed coevolution of sarbecovirus RBM indels and their impact on multi-species ACE2 adaptiveness. a The phylogenetic tree based on RBD amino acid sequences using maximum likelihood analysis (See also Supplementary Fig. 1a). The red lines mark the sarbecoviruses tested in this study. b The numbers of supportive ACE2 orthologues (data based on Fig. 2a with RLU > 2 × 105) and hACE2 compatibility of the indicated sarbecoviruses. Coloring is based on RBD clades. c Region 1 sequence logoplot (SARS-CoV-2 numbering) of sarbecoviruses grouped by different indel types in each clade. The conserved D442, F/Y451 for defining the boundary of Region 1 are highlighted in black. The featured glycine (G) and H-bond-associated tyrosine (Y) are highlighted in red and orange, respectively. d The proposed evolutionary pathway of sarbecoviruses RBD clades and RBM indel types. e Details of Region 1 sequence changes along with the emergence of different clades during the evolution of sarbecoviruses in bats, pangolins, and humans. The Region 1 lengths in each group are indicated in blue. The conserved glycine (1 G) and the double G (2 G) in most clade1b sarbecoviruses are highlighted in red. f The…