Mutation of the 2(nd) Sialic Acid-Binding Site Resulting in Reduced Neuraminidase Activity Preceded Emergence of H7N9 Influenza A Virus

The emergence of novel influenza A virus (IAV) H7N9 since 2013 has caused concerns about the ability of this 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 display equal enzymatic activity on a soluble monovalent substrate and similar substrate specificity 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 these differences to result from substitution T401A in the 2(nd) 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 2(nd) 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 HA causing the altered receptor-binding properties mentioned above. Hence, in contrast to the widespread assumption that such changes in NA are only obtained after acquisition of functional changes in HA, our data indicate that mutations in the 2(nd) 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.IMPORTANCE Novel H7N9 IAVs continue to cause human infections and pose an ongoing public health threat. Here, we show that their N9 protein displays reduced binding to and lower enzymatic activity against multivalent substrates, resulting from mutation of the 2(nd) 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 2(nd) sialic acid-binding sites. Screening of animal IAVs to monitor their potential to cross the host species barrier should therefore not only focus on the HA protein, but also on the functional properties of NA