Sequence determinants of human-cell entry identified in ACE2-independent bat sarbecoviruses: A combined laboratory and computational network science approach

Abstract

Background: The sarbecovirus subgenus of betacoronaviruses is widely distributed throughout bats and other mammals globally and includes human pathogens, SARS-CoV and SARS-CoV-2. The most studied sarbecoviruses use the host protein, ACE2, to infect cells. Curiously, the majority of sarbecoviruses identified to date do not use ACE2 and cannot readily acquire ACE2 binding through point mutations. We previously screened a broad panel of sarbecovirus spikes for cell entry and observed bat-derived viruses that could infect human cells, independent of ACE2. Here we further investigate the sequence determinants of cell entry for ACE2-independent bat sarbecoviruses.

Methods: We employed a network science-based approach to visualize sequence and entry phenotype similarities across the diversity of sarbecovirus spike protein sequences. We then verified these computational results and mapped determinants of viral entry into human cells using recombinant chimeric spike proteins within an established viral pseudotype assay.

Findings: We show ACE2-independent viruses that can infect human and bat cells in culture have a similar putative receptor binding motif, which can impart human cell entry into other bat sarbecovirus spikes that cannot otherwise infect human cells. These sequence determinants of human cell entry map to a surface-exposed protrusion from the predicted bat sarbecovirus spike receptor binding domain structure.

Interpretation: Our findings provide further evidence of a group of bat-derived sarbecoviruses with zoonotic potential and demonstrate the utility in applying network science to phenotypic mapping and prediction.

Funding: This work was supported by Washington State University and the Paul G. Allen School for Global Health.

Keywords: Coronavirus; Entry assay; Mathematical model; Pseudotype; Sequence similarity network; Zoonosis.

Figure. 1

Sequence similarity network (SSN) for sarbecovirus and merbecovirus spike domains. (a) Amino acid SSN of the receptor binding domain (RBD), encompassing spike residues 323–501, from merbeco- (blue) and sarbeco- (red) viruses. Betacoronaviruses that belong to both groups are indicated in fuchsia. (b) Amino acid SSN of the receptor binding motif (RBM), encompassing spike residues 405–481, from merbeco- (blue) and sarbeco-viruses. Sarbecoviruses are further divided into SARS-CoV viruses in green, SARS-CoV-2 in fuchsia, and bat sarbecoviruses in red. Numbering in the spike diagrams corresponds to the SARS-CoV/Urbani amino acid sequence.

Figure. 2

Validation of phenotypes from the receptor binding motif (RBM) sequence similarity network (SSN). (a) Simplified RBM SSN with sarbecovirus receptor binding domain (RBD) clades 1, 2, and 3 indicated in red, gray/purple, and blue, respectively. Sequences from human outbreaks of SARS-CoV and SARS-CoV-2 have each been condensed into single points. Viruses that were functionally tested for cell entry are shown as indicated. (b–d) Luciferase-reporter particles pseudotyped with SARS-CoV chimeric spike protein containing the RBD from the indicated sarbecoviruses were used to infect 293T cells (b), Artibeus jamaicensis kidney cells (c), or BHK cells transfected with Artbieus jamaicensis ACE2 (d), in the presence or absence of trypsin. Shown are experimental triplicates (b) or quadruplicates (c, d) from one experiment representative of at least three biological replicates. Asterisks indicate statistical significance from no spike control (**=p-value < 0.001; *=p-value < 0.05; n.s. = not significant; values are not significant unless otherwise indicated).

Figure. 3

Full-length spikes are functionally similar to chimeric spikes. (a) 293T cells were infected with pseudotypes bearing either the chimeric (hashed bars and semi-filled circles) or full-length spike (solid bars and circles) in the presence or absence of trypsin, and luciferase was measured the next day. Shown are experimental triplicates from one experiment representative of at least three biological replicates. (**=p-value < 0.001; *=p-value < 0.05; bars without asterisks indicate no statistical significance from no spike control). (b) GFP microscopy of cells infected with GFP-luciferase dual-reporter pseudotypes. Scale bar indicated 500um. (C) Expression and incorporation of the spike protein.

Figure. 4

Identification of a putative ACE2-independent sarbecovirus spike receptor binding motif (RBM). (a) RBMs were exchanged between clade 2 RBDs and inserted into the SARS-CoV spike protein backbone. (b) Chimeric sarbecovirus spike expression and pseudotype incorporation by western blot. (c) VSV-luciferase pseudotype reporter particles were used to infect 293T cells in the presence or absence of trypsin. Solid-colored circles indicate a full-length spike RBD in the SARS-CoV spike background, while mixed-color circles indicate a chimeric spike RBD in the SARS-CoV spike background. Shown are experimental triplicates from one experiment representative of at least three biological replicates. (**=p-value < 0.001; *=p-value < 0.05; bars without asterisks indicate no statistical significance from no spike control). (d) Web-logo plot comparison of sarbecovirus RBD clade 2 amino acid sequences, with varying residues highlighted. (e) Amino acid comparison between Rs4081 and Rf1 RBD. (f) SARS-CoV spike RBD (red) complexed with human ACE2 (yellow) (PDB: 2AJF) and a representative clade 2 RBD (grey; predicted, SwissModel). SARS-CoV spike residues that contact ACE2 are indicated in blue, and residues that vary between RBD clade 2 are indicated in khaki.