Publication date: Jul 28, 2025

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in viral entry and pathogenesis, making it an important target for therapeutic strategies. In this study, we explore the interaction between the S protein and O-GlcNAc transferase (OGT), revealing a physical association between these proteins using immunoprecipitation and glutathione-S-transferase (GST)-pulldown assays. Our results demonstrate that Serine 659 (S659) is the primary site of O-GlcNAcylation on the S protein, as confirmed by ion mobility mass spectrometry and mutagenesis studies. Notably, the S659A mutation significantly reduces O-GlcNAcylation of the S protein, leading to increased ubiquitination and subsequent degradation of the S protein. Cycloheximide pulse-chase assays further corroborate that the wild-type S protein exhibits greater stability compared with the S659A mutant of S. Despite these stability changes, the S659A mutation does not impair the binding affinity of the S protein to its receptor ACE2. However, we find that the S659A mutation significantly decreases the packaging efficiency of SARS-CoV-2 pseudoparticles. Collectively, our findings highlight the critical role of O-GlcNAcylation at S659 in regulating S protein stability and viral particle assembly, underscoring its potential as a target for therapeutic intervention against SARS-CoV-2. This study highlights the critical role of the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the viral infection process and its potential as a target for therapies and vaccines. By identifying Serine 659 (S659) as a key site for O-GlcNAcylation, the research reveals how modifications at this residue can influence the protein’s interactions with host factors, thereby affecting viral replication and pathogenicity. Furthermore, the S659A mutation was shown to lead to a significant increase in ubiquitination and degradation of the S protein, indicating that O-GlcNAcylation is crucial for modulating the protein’s stability and, consequently, its efficiency in facilitating viral entry. Understanding these mechanisms is vital for the development of effective interventions against coronavirus disease 2019 (COVID-19). Overall, this research enhances our understanding of how post-translational modifications impact viral behavior, opening avenues for innovative strategies to combat SARS-CoV-2 and future viral threats.

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