Spike residue 403 affects binding of coronavirus spikes to human ACE2

SARS-CoV-2, responsible for the COVID-19 pandemic, is a sarbecovirus primarily found in bats. RaTG13, a bat virus closely related to SARS-CoV-2 (96% genome identity), is considered a likely ancestor. However, RaTG13's S protein doesn't interact efficiently with human ACE2, the receptor for SARS-CoV-2 entry. The ability of viral S proteins to bind human ACE2 is a critical factor for zoonotic transmission. While SARS-CoV-2's high infectivity is linked to specific RBD alterations and a furin-cleavage site, RaTG13's inability to effectively bind human ACE2 suggests a significant difference. This study investigates the role of residue 403 in the S protein's interaction with human ACE2 and its implications for zoonotic potential.

Previous studies highlighted the importance of residue 403 in SARS-CoV-2 S protein's interaction with human ACE2. R403 is highly conserved in SARS-CoV-2, and computational analyses suggested its role in molecular interactions and ACE2 binding strength. The presence of a positively charged residue (R or K) at position 403 differentiates SARS-CoV-2 and SARS-CoV-1 from RaTG13 (T403). Molecular modeling predicted that the T403R mutation in RaTG13 S would significantly enhance its ACE2 binding.

The researchers used VSV pseudotyped particles (VSVpp) with parental and mutant S proteins to assess the functional impact of residue 403. They employed various cell lines (Caco-2, Calu-3, A549) and intestinal organoids to evaluate the infection efficiency. Cell-to-cell fusion assays were used to observe syncytia formation. Replication-competent SARS-CoV-2 systems were utilized to investigate viral replication. An in vitro S-ACE2 interaction assay was established to quantify the binding affinity. Molecular dynamics simulations were performed to study the interaction between the S protein and ACE2. The sensitivity of T403R RaTG13 S to inhibitors (e.g., E64d, Camostat) and sera from vaccinated individuals was also examined.

The T403R mutation in RaTG13 S significantly enhanced (~40-fold) its ability to infect human cells, including lung cells and intestinal organoids. Conversely, the R403T mutation in SARS-CoV-2 S decreased infection. The enhancing effect of T403R in RaTG13 S depended on E37 in ACE2; mutating E37A abolished this effect. An in vitro assay confirmed that T403R increased RaTG13 S binding to ACE2. Molecular dynamics simulations supported the experimental findings, showing stronger interactions between R403 and ACE2. The T403R RaTG13 S was sensitive to the fusion inhibitor EK1 and sera from vaccinated individuals but not to Casirivimab. The study also showed that while SARS-CoV-2 S can utilize both human and bat ACE2, RaTG13 S primarily uses human ACE2 after the T403R mutation, suggesting species-specific receptor usage. The RaTG13 T403R mutant's replication was significantly less efficient than WT SARS-CoV-2. Proteolytic processing of the S protein was also examined, showing that ACE2 expression enhanced the processing of SARS-CoV-2 S but not RaTG13 S.

This study demonstrates that a single amino acid change (T403R) drastically alters the ability of RaTG13 S to interact with human ACE2. The dependence on E37 in ACE2 highlights the importance of specific interactions for efficient viral entry. The results underscore the significance of residue 403 in determining the zoonotic potential of bat coronaviruses. The neutralization of T403R RaTG13 S by COVID-19 vaccine sera suggests that current vaccines might offer some protection against future spillover events. The reduced replication efficiency of the T403R RaTG13 S compared to WT SARS-CoV-2 indicates that other factors beyond ACE2 binding contribute to SARS-CoV-2's high infectivity. The species-specific receptor usage of RaTG13 S further emphasizes the complexity of cross-species transmission.

The study reveals that a single amino acid substitution (T403R) in the RaTG13 S protein is sufficient to confer efficient binding to human ACE2, highlighting the critical role of residue 403 in zoonotic potential. The findings have implications for predicting the pandemic risk posed by animal coronaviruses and suggest that current COVID-19 vaccines may offer some protection against related zoonotic viruses. Further research could focus on identifying other key residues and factors influencing cross-species transmission.

The study primarily used VSVpp systems, which may not fully replicate the complexities of natural viral infection. The focus was on a single amino acid change, while other factors could contribute to the zoonotic potential of RaTG13. The sample size of vaccinated individuals' sera tested was limited.