Abstract

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.