Mutation of the Second Sialic Acid-Binding Site, Resulting in Reduced Neuraminidase Activity, Preceded the Emergence of H7N9 Influenza A Virus

Abstract

The emergence of the novel influenza A virus (IAV) H7N9 since 2013 has caused concerns about the ability of the virus to spread between humans. Analysis of the receptor-binding properties of the H7 protein of a human isolate revealed modestly increased binding to α2,6 sialosides and reduced, but still dominant, binding to α2,3-linked sialic acids (SIAs) compared to a closely related avian H7N9 virus from 2008. Here, we show that the corresponding N9 neuraminidases (NAs) display equal enzymatic activities on a soluble monovalent substrate and similar substrate specificities on a glycan array. In contrast, solid-phase activity and binding assays demonstrated reduced specific activity and decreased binding of the novel N9 protein. Mutational analysis showed that these differences resulted from substitution T401A in the 2nd SIA-binding site, indicating that substrate binding via this site enhances NA catalytic activity. Substitution T401A in the novel N9 protein appears to functionally mimic the substitutions that are found in the 2nd SIA-binding site of NA proteins of avian-derived IAVs that became human pandemic viruses. Our phylogenetic analyses show that substitution T401A occurred prior to substitutions in hemagglutinin (HA), causing the altered receptor-binding properties mentioned above. Hence, in contrast to the widespread assumption that such changes in NA are obtained only after acquisition of functional changes in HA, our data indicate that mutations in the 2nd SIA-binding site may have enabled and even driven the acquisition of altered HA receptor-binding properties and may have contributed to the spread of the novel H7N9 viruses. Novel H7N9 IAVs continue to cause human infections and pose an ongoing public health threat. Here, we show that their N9 proteins display reduced binding to and lower enzymatic activity against multivalent substrates, resulting from mutation of the 2nd sialic acid-binding site. This mutation preceded and may have driven the selection of substitutions in H7 that modify H7 receptor-binding properties. Of note, all animal IAVs that managed to cross the host species barrier and became human viruses carry mutated 2nd sialic acid-binding sites. Screening of animal IAVs to monitor their potential to cross the host species barrier should therefore focus not only on the HA protein, but also on the functional properties of NA.

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

“…Multivalent sialylated glycoproteins better mimic the substrates that NA proteins encounter in vivo than the monovalent MUNANA. Furthermore, the contribution of SIA binding via the 2nd SIA-binding site to NA catalytic activity cannot be observed with MUNANA but only with multivalent substrates (9,10). Fetuin contains mono-, bi-, and triantennary glycans with ␣2,3and ␣2,6-linked SIAs in a 2:1 ratio (23), while transferrin contains two biantennary N-linked glycan chains with only ␣2,6-linked SIAs (24,25).…”

“…In addition, the NA proteins of some IAVs have been shown to bind sialosides via a 2nd SIA-binding site that consists of three loops and is located next to the catalytic site (5,7,8). Binding of substrates via the 2nd SIA-binding site enhances the catalytic activity of NA against these substrates (9,10) although this has so far been studied only for N2 and N9 proteins. In general, most residues that contact SIA in the 2nd SIA-binding site are highly conserved in avian IAVs but much less so in human IAVs (9,11).…”

“…Alternatively, the NA enzymatic activity may be decreased by shortening the stalk domain, thereby reducing the substrate accessibility of the NA proteins in the context of virus particles (reviewed in reference 19). More recently, it was shown for N2 and N9 that the enzymatic activity for multivalent substrates may also be adapted by manipulating the binding of these substrates to NA via the 2nd SIA-binding site (9,10,20). N2 proteins of human pandemic viruses were shown to display decreased SIA binding and cleavage due to mutation of an SIA contact residue in the 370 loop of the 2nd SIA-binding site (9).…”

“…N2 proteins of human pandemic viruses were shown to display decreased SIA binding and cleavage due to mutation of an SIA contact residue in the 370 loop of the 2nd SIA-binding site (9). For the N9 protein, which preferentially binds ␣2,3-linked sialosides, it was shown that substitution of a residue lacking SIA contact (here, a noncontact residue) in the 400 loop could also affect binding and cleavage of substrates (10). To what extent other substitutions in the 2nd SIA-binding site and the 2nd SIA-binding site in other NA subtypes affect/contribute to binding and cleavage of multivalent substrates is not known.…”

Spotlights

“…The group of Xander de Haan has developed novel methods to perform in depth analysis of the interaction of HA and NA proteins of human and animal viruses with their sialic acid glycan receptors [15,16].…”