Structure of the Sialic Acid Binding Site in Influenza A Virus: Hemagglutinin

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

PLoS Pathogens ◽

10.1371/journal.ppat.1008816 ◽

2020 ◽

Vol 16 (8) ◽

pp. e1008816 ◽

Cited By ~ 1

Author(s):

Wenjuan Du ◽

Margreet A. Wolfert ◽

Ben Peeters ◽

Frank J. M. van Kuppeveld ◽

Geert-Jan Boons ◽

...

Keyword(s):

Sialic Acid ◽

Binding Site ◽

Influenza A Virus ◽

Influenza A ◽

Sialic Acid Binding ◽

Compensatory Mutations

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Second sialic acid‐binding site of influenza A virus neuraminidase: binding receptors for efficient release

FEBS Journal ◽

10.1111/febs.15668 ◽

2020 ◽

Author(s):

Wenjuan Du ◽

Erik de Vries ◽

Frank J. M. van Kuppeveld ◽

Mikhail Matrosovich ◽

Cornelis A. M. de Haan

Keyword(s):

Sialic Acid ◽

Binding Site ◽

Influenza A Virus ◽

Influenza A ◽

Sialic Acid Binding

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Potent sialic acid inhibitors that target influenza A virus hemagglutinin

Scientific Reports ◽

10.1038/s41598-021-87845-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yu-Jen Chang ◽

Cheng-Yun Yeh ◽

Ju-Chien Cheng ◽

Yu-Qi Huang ◽

Kai-Cheng Hsu ◽

...

Keyword(s):

Sialic Acid ◽

Antiviral Activity ◽

Binding Site ◽

Influenza A Virus ◽

Receptor Binding ◽

Influenza A ◽

The United States ◽

Ligand Complex ◽

Receptor Binding Site ◽

Computational Strategy

AbstractEradicating influenza A virus (IAV) is difficult, due to its genetic drift and reassortment ability. As the infectious cycle is initiated by the influenza glycoprotein, hemagglutinin (HA), which mediates the binding of virions to terminal sialic acids moieties, HA is a tempting target of anti-influenza inhibitors. However, the complexity of the HA structure has prevented delineation of the structural characterization of the HA protein–ligand complex. Our computational strategy efficiently analyzed > 200,000 records of compounds held in the United States National Cancer Institute (NCI) database and identified potential HA inhibitors, by modeling the sialic acid (SA) receptor binding site (RBS) for the HA structure. Our modeling revealed that compound NSC85561 showed significant antiviral activity against the IAV H1N1 strain with EC50 values ranging from 2.31 to 2.53 µM and negligible cytotoxicity (CC50 > 700 µM). Using the NSC85561 compound as the template to generate 12 derivatives, robust bioassay results revealed the strongest antiviral efficacies with NSC47715 and NSC7223. Virtual screening clearly identified three SA receptor binding site inhibitors that were successfully validated in experimental data. Thus, our computational strategy has identified SA receptor binding site inhibitors against HA that show IAV-associated antiviral activity.

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Role of Neuraminidase in Influenza A(H7N9) Virus Receptor Binding

Journal of Virology ◽

10.1128/jvi.02293-16 ◽

2017 ◽

Vol 91 (11) ◽

Cited By ~ 31

Author(s):

Donald J. Benton ◽

Stephen A. Wharton ◽

Stephen R. Martin ◽

John W. McCauley

Keyword(s):

Sialic Acid ◽

Cell Surface ◽

Binding Site ◽

Influenza A Virus ◽

Receptor Binding ◽

Influenza A ◽

Health Concern ◽

H7n9 Virus ◽

Virus Receptor ◽

Sialic Acid Receptors

ABSTRACT Influenza A(H7N9) viruses have caused a large number of zoonotic infections since their emergence in 2013. They remain a public health concern due to the repeated high levels of infection with these viruses and their perceived pandemic potential. A major factor that determines influenza A virus fitness and therefore transmissibility is the interaction of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) with the cell surface receptor sialic acid. Typically, the HA is responsible for binding to the sialic acid to allow virus internalization and the NA is a sialidase responsible for cleaving sialic acid to aid virus spread and release. N9 NA has previously been shown to have receptor binding properties mediated by a sialic acid binding site, termed the hemadsorption (Hb) site, which is discrete from the enzymatically active sialidase site. This study investigated the N9 NA from a zoonotic H7N9 virus strain in order to determine its possible role in virus receptor binding. We demonstrate that this N9 NA has an active Hb site which binds to sialic acid, which enhances overall virus binding to sialic acid receptor analogues. We also show that the N9 NA can also contribute to receptor binding due to unusual kinetic characteristics of the sialidase site which specifically enhance binding to human-like α2,6-linked sialic acid receptors. IMPORTANCE The interaction of influenza A virus glycoproteins with cell surface receptors is a major determinant of infectivity and therefore transmissibility. Understanding these interactions is important for understanding which factors are necessary to determine pandemic potential. Influenza A viruses generally mediate binding to cell surface sialic acid receptors via the hemagglutinin (HA) glycoprotein, with the neuraminidase (NA) glycoprotein being responsible for cleaving the receptor to allow virus release. Previous studies showed that the NA proteins of the N9 subtype can bind sialic acid via a separate binding site distinct from the sialidase active site. This study demonstrates for purified protein and virus that the NA of the zoonotic H7N9 viruses has a binding capacity via both the secondary binding site and unusual kinetic properties of the sialidase site which promote receptor binding via this site and which enhance binding to human-like receptors. This could have implications for understanding human-to-human transmission of these viruses.

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Anomalous features of human neutrophil activation by influenza A virus are shared by related viruses and sialic acid-binding lectins

Journal of Leukocyte Biology ◽

10.1002/jlb.51.3.230 ◽

1992 ◽

Vol 51 (3) ◽

pp. 230-236 ◽

Cited By ~ 14

Author(s):

Kevan L. Hartshorn ◽

David E. Daigneault ◽

Mitchell R. White ◽

Alfred I. Tauber

Keyword(s):

Sialic Acid ◽

Influenza A Virus ◽

Human Neutrophil ◽

Influenza A ◽

Neutrophil Activation ◽

Sialic Acid Binding

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Breaking the Convention: Sialoglycan Variants, Coreceptors, and Alternative Receptors for Influenza A Virus Entry

Journal of Virology ◽

10.1128/jvi.01357-19 ◽

2019 ◽

Vol 94 (4) ◽

Cited By ~ 4

Author(s):

Umut Karakus ◽

Marie O. Pohl ◽

Silke Stertz

Keyword(s):

Sialic Acid ◽

Influenza A Virus ◽

Mhc Class Ii ◽

Influenza A ◽

Current Knowledge ◽

Cell Receptor ◽

Host Cell Receptor ◽

Histocompatibility Complex ◽

Sialic Acid Binding ◽

Review Current

ABSTRACT The influenza A virus (IAV) envelope protein hemagglutinin binds α2,6- or α2,3-linked sialic acid as a host cell receptor. Bat IAV subtypes H17N10 and H18N11 form an exception to this rule and do not bind sialic acid but enter cells via major histocompatibility complex (MHC) class II. Here, we review current knowledge on IAV receptors with a focus on sialoglycan variants, protein coreceptors, and alternative receptors that impact IAV attachment and internalization beyond the well-described sialic acid binding.

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

Journal of Virology ◽

10.1128/jvi.01243-18 ◽

2018 ◽

Vol 92 (20) ◽

Cited By ~ 9

Author(s):

Wenjuan Du ◽

Meiling Dai ◽

Zeshi Li ◽

Geert-Jan Boons ◽

Ben Peeters ◽

...

Keyword(s):

Sialic Acid ◽

Enzymatic Activity ◽

Binding Site ◽

Influenza A Virus ◽

Influenza A ◽

Substrate Binding ◽

Host Tropism ◽

Contact Residues ◽

Human Viruses

ABSTRACTThe 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 replicationin vitro. Our results indicate an important role for the N1 second SIA-binding site in binding to and cleavage of multivalent substrates.IMPORTANCEAvian 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.

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MD simulation of the interaction between sialoglycans and the second sialic acid binding site of influenza A virus N1 neuraminidase

Biochemical Journal ◽

10.1042/bcj20200670 ◽

2021 ◽

Vol 478 (2) ◽

pp. 423-441

Author(s):

Stefano Elli ◽

Nicola Gambacorta ◽

Timothy R. Rudd ◽

Mikhail Matrosovich ◽

Marco Guerrini

Keyword(s):

Sialic Acid ◽

Binding Site ◽

Influenza A ◽

Md Simulation ◽

Current Knowledge ◽

Influenza Viruses ◽

Molecular Features ◽

Sialic Acid Binding ◽

Glycan Receptors ◽

Glycosidic Conformation

The neuraminidases (NAs) of avian influenza viruses (IAVs) contain a second sialic acid-binding site (2SBS), historically known as the hemadsorption site, which is separated from the sialyl-hydrolase catalytic site and serves to facilitate NA catalytic activity towards multivalent sialyl-capped glycoconjugates. Transmission and adaptation of avian IAVs to humans decreases hemadsorption and catalytic activities of the NA. Here, we report the molecular recognition features of the NA 2SBS of two pandemic H1N1 IAVs, A/Brevig Mission /1/1918 (BM18) and A/California/04/2009 (CA09), differing by their 2SBS activity. Using explicit solvent MD simulation, molecular mechanics, and glycosidic conformation analysis we initially analyzed the interactions of BM18 2SBS with two sialyllacto-N-tetraose pentasaccharides, 3′SLN-LC and 6′SLN-LC, which are models for the glycan receptors of IAVs in birds and humans, respectively. These studies characterize the binding specificity of BM18 2SBS towards human-type and avian-type receptors and identifies the key amino acids that affects binding. We next compared the interactions of the 2SBSs of BM18 and CA09 with 6′SLN-LC, revealing the critical effect of amino acid 372 on binding. Our results expand the current knowledge of the molecular features of NA 2SBSs and its alteration during the adaptation of avian IAVs to humans.

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