Coronaviruses utilize surface spike glycoproteins for receptor binding and membrane fusion during entry. Understanding the structural dynamics of these spikes, particularly their correlation with infectivity, is critical for vaccine and drug development. Previous studies primarily focused on soluble spike constructs, neglecting the full-length spike's behavior within the viral membrane. This study aims to address this gap by characterizing the native conformations of HCoV-NL63 spikes on intact virions without chemical fixation, employing cryoET to visualize their structures and mass spectrometry to determine their glycan composition and occupancy. The study leverages this structural information in all-atom molecular dynamic simulations to understand the effect of glycosylation on spike flexibility and its implications for infectivity.

Extensive research has characterized the prefusion structures of coronavirus spike crowns using cryo-EM on soluble constructs. These studies revealed the receptor binding domains (RBDs) and their role in determining host tropism and pathogenicity. However, the full-length spike's conformational landscape, particularly the influence of stalk and glycans, remains poorly understood. Previous work on chemically fixed SARS-CoV-2 virions suggested a role for the stalk in bending motions, but the potential artifacts introduced by chemical fixation were unclear. The impact of glycosylation on spike dynamics and infectivity has also been investigated for other coronaviruses, demonstrating the importance of this modification in regulating conformational transitions and immune evasion.

The study used an integrative approach combining cryoET, mass spectrometry (MS), and molecular dynamics (MD) simulations. CryoET was performed on vitrified HCoV-NL63 virions without chemical fixation to determine the structural landscape of full-length spikes. Subtomogram averaging was used to generate high-resolution averages of the spike crown and lower-resolution structures of the stalk. Mass spectrometry was employed to characterize the site-specific glycosylation of the HCoV-NL63 spike protein, identifying the types and occupancies of N-linked glycans at each glycosylation site. This glycan information was incorporated into a fully glycosylated spike model for all-atom MD simulations. MD simulations were performed to elucidate the conformational landscape of the glycosylated spike, examining the role of hinge glycans in modulating spike bending. Infectivity assays were conducted using pseudotyped viruses with various glycan modifications to assess the functional impact of hinge glycans on virus entry.

CryoET analysis revealed significant conformational variability in HCoV-NL63 spikes, with crown tilting up to 80° relative to the stalk. Mass spectrometry identified a high-mannose glycan shield on the HCoV-NL63 spike, more extensive than that of SARS-CoV-2, suggesting higher immune evasion potential. MD simulations, incorporating the experimentally determined glycan structure, demonstrated a crucial role for hinge glycans, especially the glycan at N1242, in modulating spike bending. The simulations showed that glycosylation at N1242 is responsible for the extensive orientational freedom of the spike crown. Infectivity assays confirmed the involvement of N1242-glycan in virus entry, with its removal significantly reducing viral infectivity. Comparison with other coronaviruses revealed that HCoV-NL63 has fewer surface residues accessible to antibody binding, further supporting its enhanced immune evasion capacity. Simulations exploring glycan deletions showed that the removal of hinge glycans, particularly N1242, resulted in significantly reduced spike bending and altered conformational dynamics. Sequence alignment showed that the hinge glycans N1242 and N1247 are highly conserved across different coronavirus genera, suggesting functional importance.

The study's findings provide significant insights into the structure-function relationship of coronavirus spikes. The high-mannose glycan shield contributes significantly to HCoV-NL63's immune evasion. Hinge glycans, particularly at N1242, play a critical role in regulating spike flexibility, affecting virus entry. The high conservation of these hinge glycans across different coronavirus genera underscores their functional significance. The results suggest that targeting hinge glycans could be a promising therapeutic strategy for HCoV-NL63 and potentially other coronaviruses. The integrative approach combining cryoET, MS, and MD simulations effectively elucidated the complex interplay between spike structure, glycosylation, and infectivity.

This study provides a comprehensive understanding of how hinge glycans modulate coronavirus spike tilting and infectivity. The high-mannose glycan shield contributes to immune evasion, while N1242 glycosylation specifically is crucial for spike flexibility and virus entry. Targeting hinge glycans represents a potential therapeutic avenue. Future studies could explore the detailed mechanism of glycan-protein interactions at the hinge region and investigate the potential for developing therapeutics that target this region.

The study focuses primarily on HCoV-NL63. Further studies are needed to determine the generalizability of these findings to other coronaviruses. The transmembrane region of the spike was not included in the computational models, which might affect the interpretation of certain aspects of the results. Although extensive sampling was used in the simulations, longer simulations might capture even larger orientational deviations of the spike crown. The current study does not explore the possibility of other post-translational modifications influencing the spike structure and functionality.