AbstractThe initial stage of host cell infection by influenza A viruses (IAV) is mediated through interaction of the viral haemagglutinin (HA) with cell surface glycans. The binding requirement of IAVs for Galβ(1,4)Glc/ GlcNAc (lactose/lactosamine) glycans with a terminal α(2,6)-linked (human receptors) or α(2,3)-linked (avian receptors) N-acetylneuraminic residue commonly found on N-glycans, is well-established. However the role and significance of sialylated Galβ(1,3)GalNAc (core 1) epitopes that are typical O-glycoforms in influenza virus pathogenesis remains poorly detailed. Here we report a multidisciplinary study using NMR spectroscopy, virus neutralization assays and molecular modelling, into the potential for IAV to engage sialyl-Galβ(1,3)GalNAc O-glycoforms for cell attachment. H5 containing virus like particles (VLPs) derived from an H5N1 avian IAV strain show a significant involvement of the O-glycan-specific GalNAc residue, coordinated by a EQTKLY motif conserved in highly pathogenic avian influenza (HPAI) strains. Notably, human pandemic H1N1 influenza viruses shift the preference from ‘human-like’ α(2,6)-linkages in sialylated Galβ(1,4)Glc/GlcNAc fragments to ‘avian-like’ α(2,3)-linkages in sialylated Galβ(1,3)GalNAc without involvement of the GalNAc residue. Overall, our study suggests that sialylated Galβ(1,3)GalNAc as O-glycan core 1 glycoforms are involved in the influenza A virus life cycle and play a particularly crucial role during infection of HPAI strains.