N-linked glycosylation at hemagglutinin position 162 alters the fitness of influenza A (H1N1) pdm09 virus

N-linked glycosylation in hemagglutinin (HA) plays a pivotal role in modulating the biological properties of influenza virus. Sequence analysis of the HA revealed that the influenza A (H1N1) pdm09 virus acquired an N-linked glycosylation site at HA amino acid position 162 since 2012, which coincided with its emergence as a dominant global strain. To investigate the functional implication of this glycosylation on A(H1N1) pdm09 virus, we rescued a wild-type A(H1N1) pdm09 virus possessing the HA162-glycosylation (162N) and two glycosylation-deficient H1N1 mutants (N162S and N162K) using reverse genetics. Comparative characterization revealed that deglycosylation of HA position 162 significantly enhanced viral replication in chicken embryos and increased the hemagglutination affinity for chicken and turkey red blood cells. The N162K mutation altered the viral preference for both human and avian-like receptors and significantly prolonged the de-aggregation time with guinea pig red blood cells. Furthermore, the deglycosylation at HA position 162 altered the antigenicity of A(H1N1) pdm09 virus as measured by both monoclonal antibodies and antisera. In the mouse challenge study, the deglycosylation enabled the virus to acquire the ability to replicate in lungs of the infected mice. These findings demonstrated that N-glycosylation at HA residue 162 is a critical determinant of H1N1 replication efficiency, receptor-binding specificity, and tissue tropism, providing mechanistic insights into the evolutionary selection of this glycosylation motif in circulating A(H1N1) pdm09 strains.