Concanavalin A targets phylogenetically conserved N-linked glycans on coronavirus spike proteins for broad-spectrum antiviral activity.

The rapid evolution of SARS-CoV-2 variants, driven by antigenic drift in the spike glycoprotein, continues to undermine the efficacy of current vaccines and monoclonal antibody therapies. Targeting conserved features of the spike protein has been a major focus against coronavirus entry and the development of therapeutics. Here, we demonstrate that the plant lectin concanavalin A (ConA) broadly inhibits coronavirus entry through a conserved mechanism. With a combination of cell-cell fusion, pseudoviral entry, and authentic virus infection models, we show that ConA broadly inhibits coronavirus spike-mediated membrane fusion and viral entry. Biochemical analyses reveal that ConA targets two highly conserved N-glycosylation sites outside the receptor binding domain, flanking the S2' cleavage site via its mannose-binding properties. This interaction sterically impedes proteolytic activation of the spike, a molecular step essential for membrane fusion. ConA exhibited nanomolar efficacy against hCoV-NL63 infections in vitro and significantly reduced viral load and mitigated lung pathology in hCoV-NL63-infected mice in vivo. Our findings reveal specific N-linked glycosylation sites as a major vulnerability of the spike and highlight ConA as a prototype for the development of lectin-based therapeutics against emerging coronavirus infections.IMPORTANCEThe rapid evolution of SARS-CoV-2 variants, which evade current vaccines and therapeutics by altering epitopes on the spike protein, highlights a critical need for broad-spectrum antivirals. This study investigates concanavalin A (ConA), a legume lectin that targets highly conserved N-linked glycosylation sites on the spike protein, as a potential pan-coronavirus entry inhibitor. ConA broadly inhibits diverse coronaviruses by blocking spike-mediated membrane fusion. In contrast to previously reported antiviral lectins, ConA binds specifically to high-mannose oligosaccharides by targeting two phylogenetically conserved residues in the S2 subunit outside the receptor-binding domain. Consequently, ConA binding prevents the proteolytic activation of S2' and effectively inhibits membrane fusion and coronavirus infection both in vitro and in vivo. This work identifies conserved N-glycosylation sites on the spike protein as stable, vulnerable targets for antiviral intervention, distinct from the variable epitopes recognized by antibodies. These findings indicate that lectins like ConA may provide a promising approach for developing effective antivirals against emerging coronaviruses.