Spike residue 403 affects binding of coronavirus spikes to human ACE2

SARS-CoV-2, a sarbecovirus likely originating from horseshoe bats, has caused a global pandemic. The Spike (S) glycoproteins of SARS-CoV-1 and SARS-CoV-2 use the human ACE2 receptor for entry, a key determinant of zoonotic transmission. RaTG13, a bat sarbecovirus closely related to SARS-CoV-2 (~96% genome identity), likely cannot infect humans directly because its S interacts poorly with human ACE2. Prior work implicated receptor-binding domain features and the polybasic furin-cleavage insertion (PRRA) in efficient ACE2 usage and infectivity. This study investigates whether residue 403 in Spike governs ACE2 binding and whether introducing a positively charged amino acid at this position enables RaTG13 S to utilize human ACE2, thereby informing zoonotic potential.

Previous studies suggested R403 contributes to destabilizing the SARS-CoV-2 S trimer interface and strengthens RBD interaction with human ACE2. The polybasic furin cleavage site in SARS-CoV-2 S enhances infectivity, whereas RaTG13 lacks this site. Multiple residues in the SARS-CoV-2 RBD mediate ACE2 binding; residue 501 has been implicated in ACE2 usage changes, though effects were weaker than those reported here for position 403. The presence of an RGD motif in SARS-CoV-2 S led to hypotheses about integrin usage as attachment factors, though its role remains debated. Entry protease usage differs among cells: TMPRSS2 can activate SARS-CoV-2 S at the plasma membrane, while cathepsins can activate S in endosomes.

• Computational modeling: Reactive molecular dynamics (ReaxFF) simulations based on ACE2-bound SARS-CoV-2 S (PDB 7N8P/7DNH) to assess proximity and electrostatic interactions between Spike residue 403 and ACE2 residue E37; predicted that introducing R at 403 in RaTG13 S strengthens ACE2 binding.
• Mutagenesis: Generated Spike mutants SARS-CoV-2 R403T, RaTG13 T403R, and RaTG13 T403A by site-directed mutagenesis.
• Pseudovirus entry assays: Produced VSVΔG-GFP pseudotyped with parental or mutant S proteins. Infections performed on human cell lines Caco-2, Calu-3, A549 (with ACE2 overexpression), AGS, HEK293T (with ACE2 variants), and human intestinal organoids. Quantified infection by GFP-positive cell counting/automated fluorescence.
• Cell–cell fusion: Assessed syncytia formation in cells co-expressing S proteins and ACE2; quantified effects of protease inhibitors.
• Replication/propagation assays: Complementation and recombinant SARS-CoV-2 systems with S variants to assess cytopathic effects and replication kinetics (qPCR) in Caco-2 at low MOI; Gaussia luciferase reporter readouts.
• ACE2 dependence mapping: Overexpressed ACE2 variants (WT, E37A, D383 mutants) in HEK293T; measured pseudovirus entry and S–ACE2 binding.
• Biochemical analyses: Western blots of whole-cell lysates/supernatants to assess S expression and proteolytic processing (S1/S2 and S2′), with and without ACE2 and TMPRSS2.
• In vitro S–ACE2 binding assay: Quantified S binding to ACE2 using immobilized ACE2 and detection of retained S; compared effects of R403T and T403R.
• Protease/inhibitor studies: Tested TMPRSS2 co-expression and inhibitors Camostat (TMPRSS2) and E64-d (cathepsins) on entry and cell–cell fusion.
• Species specificity: Assessed entry via human, bat (Rhinolophus affinis), and murine ACE2 by expressing orthologs in HEK293T and bat-derived lung epithelial cells (Tadarida brasiliensis cell line).
• Neutralization/sensitivity: Tested fusion inhibitor EK1 (EK1 peptide), monoclonal antibody Casirivimab, and sera from vaccinated individuals (BNT162b2; AZ/BNT162b2) against SARS-CoV-2 and RaTG13 T403R pseudoviruses.
• Cell culture and construct generation: Detailed protocols for HEK293T maintenance, pseudotype production, cloning of expression constructs and recombinant SARS-CoV-2 systems, biosafety compliance, and statistical analysis (GraphPad Prism; Student’s t-test with Welch’s correction).

• Conservation of R403: Among ~4.3 million SARS-CoV-2 S sequences, R403 is highly conserved; only 294 showed R403K and ~132 other substitutions or rare deletion, indicating strong selection for a positive residue at 403.
• Modeling predicted charge interaction between Spike 403 and ACE2 E37; RaTG13 T403R was predicted to enhance ACE2 binding.
• Entry assays: • SARS-CoV-2 R403T reduced VSV pseudovirus entry into Caco-2 cells by ~40%. • RaTG13 T403R increased pseudovirus infectiousness for Caco-2 by ~40-fold compared to WT RaTG13; T403A blunted this enhancement. • Similar enhancement patterns observed in Calu-3 and A549(ACE2) cells; TMPRSS2 co-expression did not further boost RaTG13 T403R entry. • In human intestinal organoids, WT RaTG13 S did not mediate entry, whereas T403R enabled significant infection; WT SARS-CoV-2 S infected efficiently.
• Cell–cell fusion: WT and T403A RaTG13 S did not induce syncytia with human ACE2, while T403R did; SARS-CoV-2 S formed large syncytia.
• Replication systems: SARS-CoV-2 R403T mutant showed reduced cytopathic effects and replicated with significantly lower efficiency than WT in Caco-2 at low MOI.
• ACE2 determinant: Mutation E37A in ACE2 abolished the enhancing effect of RaTG13 T403R and reduced SARS-CoV-2 S-mediated infection; a D383 ACE2 mutation did not disrupt enhancement.
• Binding: In vitro S–ACE2 interaction assays showed SARS-CoV-2 R403T moderately reduced binding (with lower S expression), while RaTG13 T403R specifically and strongly enhanced ACE2 binding without affecting S expression levels.
• Proteolysis: RaTG13 S lacks the polybasic S1/S2 furin site; ACE2 expression promoted S processing. Nevertheless, the enhanced entry of RaTG13 T403R is attributed to improved ACE2 interaction rather than altered integrin usage.
• Integrins: An integrin inhibitor did not reduce SARS-CoV-2 or RaTG13 T403R S-mediated infection of Caco-2, arguing against integrin dependence under tested conditions.
• Protease usage: In AGS cells, entry mediated by SARS-CoV-2 and RaTG13 T403R was inhibited by cathepsin inhibitor E64-d but not by TMPRSS2 inhibitor Camostat, indicating endosomal cathepsin dependence in this context.
• Species specificity: SARS-CoV-2 S (WT and, to a lesser extent, R403T) used bat (R. affinis) ACE2 for entry at low efficiency; WT RaTG13 S used human ACE2 poorly and could not use murine ACE2. RaTG13 T403R enabled efficient use of human but not bat ACE2, suggesting species-specific effects.
• Neutralization/sensitivity: EK1 efficiently inhibited SARS-CoV-2 and RaTG13 T403R S-mediated infection. Casirivimab neutralized SARS-CoV-2 but showed little activity against RaTG13 T403R. Sera from vaccinated individuals (BNT162b2 and AZ/BNT162b2) neutralized RaTG13 T403R, suggesting potential cross-protection.

The study demonstrates that a single substitution introducing a positive charge at Spike residue 403 (T403R) enables the bat sarbecovirus RaTG13 S to bind human ACE2 more efficiently and mediate entry into human lung cells and intestinal organoids. This addresses the central question of how specific Spike residues influence cross-species ACE2 usage and zoonotic potential. Functional, biochemical, and computational data converge on a mechanistic model in which R403 in Spike engages E37 in human ACE2 to strengthen receptor interaction. The partial reduction of SARS-CoV-2 infectivity upon R403T and the diminished infection with ACE2 E37A support the relevance of the R403–E37 interaction for optimal SARS-CoV-2 entry. The enhancement is not attributable to integrin engagement and is consistent with endosomal cathepsin-dependent activation in certain cell types. Species-specific assessments indicate that while RaTG13 T403R gains human ACE2 usage, it does not equivalently improve usage of bat ACE2, underscoring host-specific receptor compatibilities beyond a single residue. Neutralization by vaccinee sera of RaTG13 T403R suggests that current immunogens may confer some protection against closely related bat CoVs should they acquire analogous adaptations at position 403.

• Main contributions: Identifies Spike residue 403 as a critical determinant of human ACE2 utilization across sarbecoviruses; experimentally validates that introducing a positively charged residue (R) at 403 enables RaTG13 S to efficiently bind human ACE2 and mediate entry and fusion in human cells and organoids. Establishes the functional importance of the Spike R403–ACE2 E37 interaction. Shows that vaccine-elicited sera neutralize RaTG13 T403R, implying potential cross-protection.
• Implications: Surveillance of animal coronaviruses for residue 403 and ACE2-contacting positions may improve prediction of zoonotic risk. Therapeutics targeting conserved entry mechanisms (e.g., EK1) may retain activity against emergent sarbecoviruses.
• Future directions: Validate findings with authentic RaTG13 or closely related live viruses; map additional Spike–ACE2 contact residues governing cross-species usage; assess the impact of natural ACE2 polymorphisms (e.g., at position 37) on susceptibility; evaluate in vivo relevance and transmission potential; explore combinatorial effects with other Spike features such as the furin site and N501 variants.

The study primarily uses pseudotyped VSV systems, overexpression models, and recombinant SARS-CoV-2 constructs rather than authentic RaTG13 virus, which may affect generalizability to natural infection. Several results depend on in vitro cell lines and organoids, and protease usage can be cell-type dependent. Some molecular insights rely on computational modeling. Species-specific conclusions are based on selected ACE2 orthologs and may not encompass ACE2 diversity across bat species.