Dataset Information

An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity.

ABSTRACT: Neuraminidase (NA) is a sialidase expressed on the surface of influenza A viruses that releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. It is a homotetramer, with each monomer consisting of a transmembrane region, a stalk, and a globular head with sialidase activity. We recently characterized two swine viruses of the pandemic H1N1 lineage, A/swine/Virginia/1814-1/2012 (pH1N1low-1) and A/swine/Virginia/1814-2/2012 (pH1N1low-2), with almost undetectable NA enzymatic activity compared to that of the highly homologous A/swine/Pennsylvania/2436/2012 (pH1N1-1) and A/swine/Minnesota/2499/2012 (pH1N1-2) viruses. pH1N1-1 transmitted to aerosol contact ferrets, but pH1N1low-1 did not. The aim of this study was to identify the molecular determinants associated with low NA activity as potential markers of aerosol transmission. We identified the shared unique substitutions M19V, A232V, D248N, and I436V (N1 numbering) in pH1N1low-1 and pH1N1low-2. pH1N1low-1 also had the unique Y66D substitution in the stalk domain, where 66Y was highly conserved in N1 NAs. Restoration of 66Y was critical for the NA activity of pH1N1low-1 NA, although 19M or 248D in conjunction with 66Y was required to recover the level of activity to that of pH1N1 viruses. Studies of NA stability and molecular modeling revealed that 66Y likely stabilized the NA homotetramer. Therefore, 66Y in the stalk domain of N1 NA was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity. IMPORTANCE:Neuraminidase (NA) is a sialidase that is one of the major surface glycoproteins of influenza A viruses and the target for the influenza drugs oseltamivir and zanamivir. NA is important as it releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. Mutations in the globular head domain that decrease enzymatic activity but confer resistance to NA inhibitors have been characterized; however, the importance of specific mutations in the stalk domain is unknown. We identified 66Y (N1 numbering), a highly conserved amino acid that was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

SUBMITTER: Zanin M

PROVIDER: S-EPMC5215329 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity.

Zanin Mark M Duan Susu S Wong Sook-San SS Kumar Gyanendra G Baviskar Pradyumna P Collin Emily E Russell Charles C Barman Subrata S Hause Benjamin B Webby Richard R

Journal of virology 20170103 2

Neuraminidase (NA) is a sialidase expressed on the surface of influenza A viruses that releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. It is a homotetramer, with each monomer consisting of a transmembrane region, a stalk, and a globular head with sialidase activity. We recently characterized two swine viruses of the pandemic H1N1 lineage, A/swine/Virginia/1814-1/2012 (pH1N1<sub>low</sub>-1) and A/swine/Virginia/1814-2/2012 (pH1N1<sub>lo ...[more]

PMID: 27847354

Similar Datasets

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:Oligonucleotides corresponding to neuraminidase (NA) stalk motifs that have been associated with enhanced influenza virulence have been identified using surface-enhanced Raman spectroscopy (SERS). 5'-Thiolated ssDNA oligonucleotides were immobilized onto a hexadecyltrimethylammonium bromide (CTAB) coated Au nanoparticles (AuNP). Three synthetic RNA sequences corresponding to specific amino acid deletions in the influenza NA stalk region were attached to the CTAB-modified AuNPs. Two of these sequences were specific to sequences with amino acid deletions associated with increased virulence, and one was a low virulence sequence with no amino acid deletions. Hybridization of synthetic matched and mismatched DNA-RNA complexes were detected based on the intrinsic SERS spectra. In addition, this platform was used to analyze RNA sequences isolated from laboratory grown influenza viruses having the NA stalk motif associated with enhanced virulence, including A/WSN/33/H1N1, A/Anhui/1/2005/H5N, and A/Vietnam/1203/2004/H5N1 strains. Multivariate feature selection methods were employed to determine the specific wavenumbers in the Raman spectra that contributed the most information for class discrimination. A one-way analysis of variance (ANOVA) test identified 884 and 1196 wavenumbers as being highly significant in the high and low virulence spectra, respectively (p < 0.01). A post-hoc Tukey Honestly Significance Difference (HSD) test identified the wavenumbers that played a major role in differentiating the DNA-RNA hybrid classes. An estimate of the spectral variability, based on the Wilcoxon rank sum test, found the major source of variation to be predominately between the different classes, and not within the classes, thus confirming that the spectra reflected real class differences and not sampling artifacts. The multivariate classification methods partial least squares discriminant analysis (PLS-DA) and support vector machine discriminant analysis (SVM-DA) were able to distinguish between different NA stalk-motifs linked to NA-enhanced influenza virus virulence (NA-EIV) with >95% sensitivity and specificity in both synthetic RNA sequences as well as the isolated viral RNA. This study demonstrates the feasibility of SERS for direct identification of influenza NA stalk mutations associated with virulence without sample amplification or labeling.

Project description:The N1 neuraminidases (NAs) of avian and pandemic human influenza viruses contain tyrosine and asparagine, respectively, at position 347 on the rim of the catalytic site; the biological significance of this difference is not clear. Here, we used molecular dynamics simulation to model the effects of amino acid 347 on N1 NA interactions with sialyllacto-N-tetraoses 6'SLN-LC and 3'SLN-LC, which represent NA substrates in humans and birds, respectively. Our analysis predicted that Y347 plays an important role in the NA preference for the avian-type substrates. The Y347N substitution facilitates hydrolysis of human-type substrates by resolving steric conflicts of the Neu5Ac2-6Gal moiety with the bulky side chain of Y347, decreasing the free energy of substrate binding, and increasing the solvation of the Neu5Ac2-6Gal bond. Y347 was conserved in all N1 NA sequences of avian influenza viruses in the GISAID EpiFlu database with two exceptions. First, the Y347F substitution was present in the NA of a specific H6N1 poultry virus lineage and was associated with the substitutions G228S and/or E190V/L in the receptor-binding site (RBS) of the hemagglutinin (HA). Second, the highly pathogenic avian H5N1 viruses of the Gs/Gd lineage contained sporadic variants with the NA substitutions Y347H/D, which were frequently associated with substitutions in the HA RBS. The Y347N substitution occurred following the introductions of avian precursors into humans and pigs with N/D347 conserved during virus circulation in these hosts. Comparative evolutionary analysis of site 347 revealed episodic positive selection across the entire tree and negative selection within most host-specific groups of viruses, suggesting that substitutions at NA position 347 occurred during host switches and remained under pervasive purifying selection thereafter. Our results elucidate the role of amino acid 347 in NA recognition of sialoglycan substrates and emphasize the significance of substitutions at position 347 as a marker of host range and adaptive evolution of influenza viruses.

Dataset Information

An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity.

Publications

An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets