In a groundbreaking exploration of coronavirus-host interactions, researchers have unveiled the molecular underpinnings governing how the FCoV-23 virus engages with feline aminopeptidase N (FcAPN), a crucial receptor mediating viral entry. Using state-of-the-art cryo-electron microscopy (cryo-EM), the team resolved the structure of the FCoV-23 receptor-binding domain (RBD) complexed with FcAPN at an impressive 2.5-angstrom resolution, illuminating the intricate details of this viral handshake. This high-resolution map exposes a sophisticated network of interactions critical for viral recognition and attachment, refining our understanding of receptor specificity among alphacoronaviruses.
The structure reveals that each protomer within the FcAPN dimer accommodates one FCoV-23 RBD, establishing a bivalent mode of engagement that echoes binding paradigms observed in related alphacoronaviruses, such as canine coronavirus (CCoV)-HuPn-2018 and porcine respiratory coronavirus (PRCV). Central to this interface are two receptor-binding loops on the viral spike protein, each orchestrating precise interactions with both the protein and glycan components of FcAPN. In receptor-binding loop 1, the aromatic tyrosine residue at position 549 (Y549_FCoV-23) engages in CH–π interactions with the core fucose moiety of the N740 glycan on FcAPN. This interaction is further stabilized by hydrogen bonding to critical amino acids E735 and W741, establishing a glycan-mediated foothold that anchors the viral RBD.
Adjacent to this, the glutamine residue Q551_FCoV-23 forms hydrogen bonds with the side chain of N740_FcAPN and intimately contacts the proximal N-acetyl-glucosamine and core fucose units of the glycan. These glycan-protein partnerships underscore the multifaceted nature of viral recognition, where carbohydrate moieties on host receptors serve as essential determinants of viral binding affinity and specificity. Receptor-binding loop 2 presents a complementary set of interactions: the bulky tryptophan residue W592_FCoV-23 nestles against histidine (H790) and proline (P791) residues on FcAPN, while its imino group forms a stabilizing hydrogen bond with the main-chain carbonyl of N787_FcAPN. Such detailed contacts highlight the convergence of protein-protein and protein-glycan interactions in dictating viral tropism.
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Interestingly, a comparative analysis with the related CCoV-HuPn-2018 strain reveals a critical substitution at position 540 in the spike protein, where an arginine residue (R540_CCoV-HuPn-2018) is replaced by leucine (L546_FCoV-23) in FCoV-23. This mutation abolishes a salt bridge previously formed with glutamic acid (E786) on CfAPN, the canine receptor orthologue, corresponding to glutamine 779 (Q779) in FcAPN. Such subtle yet impactful amino acid changes demonstrate how variations in spike protein composition can modulate receptor engagement and species specificity.
Delving deeper into host receptor tropism, the study highlights the pivotal role of an oligosaccharide at position N739 on human APN, a glycan notably absent in this orthologue but present in CfAPN, FcAPN, SsAPN (swine), and GgAPN (guinea pig). The absence of this glycan appears to underlie the inability of FCoV-23 and similar alphacoronaviruses like CCoV-HuPn-2018 and transmissible gastroenteritis virus (TGEV) to effectively bind human APN. By engineering a human APN mutant that reintroduces this glycan via the R741T substitution, the researchers rendered human cells permissive to viral entry mediated by the FCoV-23 spike protein, underscoring the glycan’s indispensability in receptor recognition.
Conversely, removal of the oligosaccharide from FcAPN—achieved by the T742R substitution—dramatically impaired viral entry by reducing infection efficiency by two orders of magnitude. This functional assay reinforces the structural observations, demonstrating that glycan-mediated interactions at this critical site are not only structurally significant but also biologically consequential, influencing viral entry kinetics and tropism.
In a fascinating twist, the team observed that entry mediated by the spike protein of the human coronavirus 229E (HCoV-229E) was efficient regardless of the presence or absence of glycans at N739 (human APN) or N740 (FcAPN). This phenomenon correlates with earlier structural insights revealing that HCoV-229E targets an APN binding site distinct from those exploited by FCoV-23, CCoV-HuPn-2018, and PRCV, reflecting divergent evolutionary strategies among alphacoronaviruses for host receptor engagement.
Taken together, these findings illuminate the nuanced molecular dialogue between alphacoronaviruses and their cognate aminopeptidase N receptors, emphasizing the combined importance of protein-protein and protein-glycan interactions in determining host range and cross-species transmission potential. The revelation that a single glycan moiety can act as a molecular switch governing viral entry highlights the exquisite specificity embedded within these virus-host interfaces.
Moreover, understanding how mutations within the spike RBD influence the formation or disruption of salt bridges and hydrogen bonds provides critical insights into viral adaptations that may enable spillover events or alter viral infectivity. Such refinements in receptor engagement mechanics may serve as potential targets for antiviral strategies aiming to disrupt viral attachment and entry.
The structural elucidation presented in this study was made possible by advances in cryo-EM technology, enabling researchers to capture intricate interaction details at near-atomic resolution. This level of detail opens avenues for rational drug design and vaccine development, offering templates for molecules that could competitively inhibit viral binding or mask critical receptor epitopes.
Beyond receptor recognition, the implications of these discoveries extend to viral entry kinetics and fusion efficiency. Notably, the loss of domain 0 in the FCoV-23 spike protein was previously shown to enhance fusogenicity, but its exact interplay with receptor binding remained unclear until now. The current research bridges this gap by correlating structural receptor engagement with functional fusion enhancement.
This comprehensive understanding of how specific amino acid substitutions, glycosylation patterns, and receptor orthologue variability impact FCoV-23 infectivity enhances our predictive capabilities regarding coronavirus zoonosis. It also sets the stage for broader comparative studies across alphacoronaviruses, fostering preparedness against emergent viral threats.
As the world continues to grapple with coronavirus pandemics, these mechanistic insights underscore the importance of deciphering viral entry pathways at a molecular level. Such knowledge empowers the scientific community to anticipate viral evolution trajectories, design precision therapeutics, and potentially thwart future outbreaks before they manifest.
Subject of Research: Molecular mechanisms of FCoV-23 spike protein recognition and engagement with feline aminopeptidase N (FcAPN) and implications for coronavirus host specificity and viral entry kinetics.
Article Title: Loss of FCoV-23 spike domain 0 enhances fusogenicity and entry kinetics.
Article References: Tortorici, M.A., Choi, A., Gibson, C.A. et al. Loss of FCoV-23 spike domain 0 enhances fusogenicity and entry kinetics. Nature (2025). https://doi.org/10.1038/s41586-025-09155-z
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Tags: amino acid interactions in receptor bindingbivalent engagement in viral receptorscryo-electron microscopy viral structureFCoV-23 coronavirus receptor interactionsfeline aminopeptidase N bindingglycan-mediated viral attachmenthigh-resolution cryo-EM studiesmolecular interactions in viral recognitionreceptor specificity in coronavirusesreceptor-binding domain complexstructural biology of FCoV-23viral entry mechanisms in alphacoronaviruses