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 α2-6 sialoglycan-binding viruses being inhibited more by NA inhibitor (oseltamivir carboxylate; OsC) than α2-3 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. IMPORTANCE Influenza A viruses (IAVs) contain hemagglutinin (HA) proteins involved in sialoglycan receptor binding and neuraminidase (NA) proteins that cleave sialic acids. While the importance of the NA protein in virion egress is well established, its role in virus entry remains to be fully elucidated. NA activity is needed for the release of virions from mucus decoy receptors, but conflicting results have been reported on the importance of NA activity in virus entry in the absence of decoy receptors. We now show that inhibition of NA activity affects virus entry depending on the receptor-binding properties of HA and the receptor repertoire present on cells. Inhibition of entry by the presence of mucus correlated with the importance of NA activity for virus entry, with the strongest inhibition being observed when mucus and OsC were combined. These results shed light on the importance in virus entry of the NA protein, an important antiviral drug target.