Sialic acid functions as a primary attachment factor for avian coronavirus and initiates macropinocytosis-mediated viral entry

Coronavirus cellular invasion relies on initial binding to cell-surface ligands classified as functional receptors, co-receptors, or attachment factors. Sialic acid is widely implicated in viral attachment, yet its mechanistic role during infection by infectious bronchitis virus (IBV)-a globally prevalent avian coronavirus devastating poultry production-remains poorly defined. Here, neuraminidase-mediated sialic acid depletion drastically impaired IBV attachment and internalization, while confocal imaging confirmed direct physical association between IBV particles and cell-surface sialic acids. Additionally, molecular docking revealed multiple stable residues that mediate binding between sialic acid and IBV S1 proteins of distinct lineages. Notably, soluble free sialic acid failed to competitively block IBV entry, whereas lectin competitively inhibited viral adsorption, demonstrating that sialic acid acts as an auxiliary attachment factor rather than a primary functional receptor. IBV infection alters the expression of sialic acid-related genes, and interference with sialic acid transport and synthesis genes also inhibits IBV entry and replication. Mechanistically, IBV enters host cells through dynamin-dependent and macropinocytosis-mediated endocytosis. IBV infection enhanced co-localization between sialic acids and the macropinosome marker sorting nexin 5 (SNX5), whereas depletion of sialic acid synthetic genes diminished IBV-SNX5 co-localization. These data establish sialic acid as an essential upstream trigger for IBV-induced macropinocytic endocytosis. Collectively, this work dissects the molecular cascade linking sialic acid recognition to IBV cellular entry, advancing mechanistic knowledge of avian coronavirus infection and informing sialic acid-targeted antiviral design.