Neuraminidase-dependent entry of influenza A virus is determined by hemagglutinin receptor-binding specificity

Influenza A viruses (IAVs) contain sialoglycan-binding hemagglutinin (HA) and sialoglycan-cleaving neuraminidase (NA) proteins, the concerted action of which is needed for escape from decoy receptors and for virion motility ultimately resulting in infection of epithelial cells of the respiratory tract. The importance of NA in egress of newly assembled virions has been well established, whereas its role in entry has yet to be fully elucidated. In this study, we systematically analyzed the role of NA in viral entry in relation to HA receptor-binding preference, the receptor repertoire displayed on cells and the presence of mucus decoy receptors. Utilizing recombinant viruses that differ only in their HA-NA composition, it was observed that the dependence on NA activity for IAV entry largely depends on HA and not NA, with entry of a2-6 sialoglycan-binding viruses being inhibited more by NA inhibitor (oseltamivir carboxylate; OsC) than a2-3 sialoglycan-preferring viruses. In agreement with this, inhibition of virus entry by OsC could be modified by altering the sialoglycan receptor repertoire of cells. Entry inhibition by OsC correlated with the ability of mucus to inhibit infection, with the combination of the two having the largest effect. Our results indicate that the dependency of IAV on NA activity and, thus, virion motility for entry are determined by the receptor-binding properties of HA in combination with the receptor repertoire present on cells. This dependency is larger when fewer preferred receptors are displayed, which coincides with increased inhibition by mucus decoy receptors.