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Influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic Acid receptors.

ABSTRACT: Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, which supports HA-independent receptor binding. While the wild-type NA efficiently cleaves sialic acid from both ?2-6- and ?2-3-linked glycans, the mutant exhibits much reduced enzymatic activity toward both types of sialosides. Conversely, while wild-type NA shows no detectable binding to sialosides, the D151G NA exhibits avid binding with broad specificity toward ?2-3 sialosides. D151G NA binds the 3' sialyllactosamine (3'-SLN) and 6'-SLN sialosides with equilibrium dissociation constant (K(D)) values of 30.0 ?M and 645 ?M, respectively, which correspond to much higher affinities than the corresponding affinities (low mM) of HA to these glycans. Crystal structures of wild-type and mutant NAs reveal the structural basis for glycan binding in the active site by exclusively impairing the glycosidic bond hydrolysis step. The general significance of D151 among influenza virus NAs was further explored by introducing the D151G mutation into three N1 NAs and one N2 NA, which all exhibited reduced enzymatic activity and preferential binding to ?2-3 sialosides. Since the enzymatic and binding activities of NAs are not routinely assessed, the potential for NA receptor binding to contribute to influenza virus biology may be underappreciated.

SUBMITTER: Zhu X

PROVIDER: S-EPMC3503043 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic Acid receptors.

Zhu Xueyong X McBride Ryan R Nycholat Corwin M CM Yu Wenli W Paulson James C JC Wilson Ian A IA

Journal of virology 20120926 24

Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, whi ...[more]

PMID: 23015718

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Project description: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 ?2,3- over ?2,6-linked SIAs even when their corresponding HA proteins displayed a strict preference for ?2,6-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 in vitro Our results indicate an important role for the N1 second SIA-binding site in binding to and cleavage of multivalent substrates.IMPORTANCE Avian and human influenza A viruses (IAVs) preferentially bind ?2,3- and ?2,6-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.

Project description: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.IMPORTANCE 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.

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Influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic Acid receptors.

Publications

Influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic Acid receptors.

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