Substrate Binding by the Second Sialic Acid-Binding Site of Influenza A Virus N1 Neuraminidase Contributes to Enzymatic Activity

Abstract

The influenza A virus (IAV) neuraminidase (NA) protein plays an essential role in the release of virus particles from cells and decoy receptors. The NA enzymatic activity presumably needs to match the activity of the IAV hemagglutinin (HA) attachment protein and the host sialic acid (SIA) receptor repertoire. We analyzed the enzymatic activities of N1 NA proteins derived from avian (H5N1) and human (H1N1) IAVs and analyzed the role of the second SIA-binding site, located adjacent to the conserved catalytic site, therein. SIA contact residues in the second SIA-binding site of NA are highly conserved in avian, but not human, IAVs. All N1 proteins preferred cleaving α2,3- over α2,6-linked SIAs even when their corresponding HA proteins displayed a strict preference for α2,6-linked SIAs, indicating that the specificity of the NA protein does not need to fully match that of the corresponding HA protein. NA activity was affected by substitutions in the second SIA-binding site that are observed in avian and human IAVs, at least when multivalent rather than monovalent substrates were used. These mutations included both SIA contact residues and residues that do not directly interact with SIA in all three loops of the second SIA-binding site. Substrate binding via the second SIA-binding site enhanced the catalytic activity of N1. Mutation of the second SIA-binding site was also shown to affect virus replication Our results indicate an important role for the N1 second SIA-binding site in binding to and cleavage of multivalent substrates. Avian and human influenza A viruses (IAVs) preferentially bind α2,3- and α2,6-linked sialic acids (SIAs), respectively. A functional balance between the hemagglutinin (HA) attachment and neuraminidase (NA) proteins is thought to be important for host tropism. What this balance entails at the molecular level is, however, not well understood. We now show that N1 proteins of both avian and human viruses prefer cleaving avian- over human-type receptors although human viruses were relatively better in cleavage of the human-type receptors. In addition, we show that substitutions at different positions in the second SIA-binding site found in NA proteins of human IAVs have a profound effect on binding and cleavage of multivalent, but not monovalent, receptors and affect virus replication. Our results indicate that the HA-NA balance can be tuned via modification of substrate binding via this site and suggest an important role of the second SIA-binding site in host tropism.

Antiviral potential of 3′-sialyllactose- and 6′-sialyllactose-conjugated dendritic polymers against human and avian influenza viruses

“…Even though inhibition was minimal, it was interesting to see that the (2020) 10:768 | https://doi.org/10.1038/s41598-020-57608-4 www.nature.com/scientificreports www.nature.com/scientificreports/ (3SL)-conjugated polymers were more potent than the (6SL)-conjugated polymers against both, avian-and human-derived IAV strains. This is in line with published observations for N2 and N1 NA proteins of human viruses (H3N2 and H1N1) that prefer cleaving avian-over human-type receptors 29,30 and indicates that the specificity of the NA protein does not need to fully match that of the corresponding HA protein.…”

Influenza A Virus Hemagglutinin–Neuraminidase–Receptor Balance: Preserving Virus Motility

“…This may be achieved by tuning the receptor (fine-)specificity and affinity of HA [61] and the activity of NA. Of note, all NA proteins preferentially cleave avian-over human-type receptors, although NA of human viruses cleaves human-type receptors relatively more efficiently [62,63]. One way to modulate NA activity is by mutation of the catalytic site which is, however, extremely conserved between avian and human viruses.…”

Mutation of the second sialic acid-binding site of influenza A virus neuraminidase drives compensatory mutations in hemagglutinin

“…We previously generated an H5N1 virus with mutation K432E in the 2SBS of NA (H5N1 432E ) [5] to analyze the importance of the 2SBS for NA catalytic activity and virus replication. This mutation, which reduced catalytic activity of NA on multivalent, but not monovalent substrates [5], resulting from reduced receptor binding via the 2SBS (S1 Fig), rapidly reverted back to wild type NA (432K) [5]. This observation prompted us to examine the effect of mutation K432E in NA on the HA-NA balance of H5N1 virus.…”

“…In our previous work, revertant E432K was readily obtained upon passaging of H5N1 432E [5]. To restore the altered HA-NA balance, compensatory mutations in HA may also suffice to compensate for substitution K432E in NA.…”

“…As yet, little is known about the importance of the NA in host tropism changes. Although the NAs of human IAVs are relatively better at cleaving α2,6 SIA they still, like avian IAVs, display a higher cleavage rate cleavage of α2,3 SIAs [4][5][6][7][8]. It has been suggested that not HA binding per se, but rather the binding properties of HA in relation to the activity of NA are important for optimal virus replication, fitness and host tropism [9][10][11][12].…”