Amino Acid Residues Contributing to the Substrate Specificity of the Influenza A Virus Neuraminidase

Influenza A viruses possess two glycoprotein spikes on the virion surface: hemagglutinin (HA), which binds to oligosaccharides containing terminal sialic acid, and neuraminidase (NA), which removes terminal sialic acid from oligosaccharides. Hence, the interplay between these receptor-binding and receptor-destroying functions assumes major importance in viral replication. In contrast to the well-characterized role of HA in host range restriction of influenza viruses, there is only limited information on the role of NA substrate specificity in viral replication among different animal species. We therefore investigated the substrate specificities of NA for linkages between N-acetyl sialic acid and galactose (NeuAcalpha2-3Gal and NeuAcalpha2-6Gal) and for different molecular species of sialic acids (N-acetyl and N-glycolyl sialic acids) in influenza A viruses isolated from human, avian, and pig hosts. Substrate specificity assays showed that all viruses had similar specificities for NeuAcalpha2-3Gal, while the activities for NeuAcalpha2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcalpha2-3Gal), as represented by swine and more recent N2 human viruses. Using site-specific mutagenesis, we identified in the earliest human virus with a detectable increase in NeuAcalpha2-6Gal specificity a change at position 275 (from isoleucine to valine) that enhanced the specificity for this substrate. Valine at position 275 was maintained in all later human viruses as well as swine viruses. A similar examination of N-glycolylneuraminic acid (NeuGc) specificity showed that avian viruses and most human viruses had low to moderate activity for this substrate, with the exception of most human viruses isolated between 1967 and 1969, whose NeuGc specificity was as high as that of swine viruses. The amino acid at position 431 was found to determine the level of NeuGc specificity of NA: lysine conferred high NeuGc specificity, while proline, glutamine, and glutamic acid were associated with lower NeuGc specificity. Both residues 275 and 431 lie close to the enzymatic active site but are not directly involved in the reaction mechanism. This finding suggests that the adaptation of NA to different substrates occurs by a mechanism of amino acid substitutions that subtly alter the conformation of NA in and around the active site to facilitate the binding of different species of sialic acid.     

Study of specific oligosaccharide structures related with swine flu (H1N1) and avian flu, and tamiflu as their remedy

The infection of pandemic influenza viruses such as swine flu (H1N1) and avian flu viruses to the host cells is related to the following two factors: First, the surface protein such as HA (hemagglutinin) and NA (neuraminidase) of the influenza virus. Second, the specific structure of the oligosaccharide [sialic acid(alpha2-6) galactose(beta1-4)glucose or sialic acid(alpha2-3)galactose(beta1-4)glucose] on the host cell. After recognizing the specific structure of the oligosaccharide on the surface of host cells by the surface protein of the influenza virus, the influenza virus can secrete sialidase and cleave the sialic acid attached on the final position of the specific structure of the oligosaccharide on the surface of host cells. Tamiflu (oseltamivir), known as a remedy of swine flu, has a saccharide analog structure, especially the sialic acid analog. Tamiflu can inhibit the invasion of influenza viruses (swine flu and avian flu viruses) into the host cells by competition with sialic acid on the terminal position of the specific oligosaccharide on the surface of the host cell. Because of the emergence of Tamiflu resistance, the development of new potent anti-influenza inhibitors is needed. The inhibitors with positive-charge groups have potential as antiviral therapeutics, and the strain specificity must also be resolved.     

Influenza A viruses lacking sialidase activity can undergo multiple cycles of replication in cell culture, eggs, or mice

Influenza A viruses possess both hemagglutinin (HA), which is responsible for binding to the terminal sialic acid of sialyloligosaccharides on the cell surface, and neuraminidase (NA), which contains sialidase activity that removes sialic acid from sialyloligosaccharides. Interplay between HA receptor-binding and NA receptor-destroying sialidase activity appears to be important for replication of the virus. Previous studies by others have shown that influenza A viruses lacking sialidase activity can undergo multiple cycles of replication if sialidase activity is provided exogenously. To investigate the sialidase requirement of influenza viruses further, we generated a series of sialidase-deficient mutants. Although their growth was less efficient than that of the parental NA-dependent virus, these viruses underwent multiple cycles of replication in cell culture, eggs, and mice. To understand the molecular basis of this viral growth adaptation in the absence of sialidase activity, we investigated changes in the HA receptor-binding affinity of the sialidase-deficient mutants. The results show that mutations around the HA receptor-binding pocket reduce the virus's affinity for cellular receptors, compensating for the loss of sialidase. Thus, sialidase activity is not absolutely required in the influenza A virus life cycle but appears to be necessary for efficient virus replication.     

Receptor binding profiles of avian influenza virus hemagglutinin subtypes on human cells as a predictor of pandemic potential

The host adaptation of influenza virus is partly dependent on the sialic acid (SA) isoform bound by the viral hemagglutinin (HA). Avian influenza viruses preferentially bind the α-2,3 SA and human influenza viruses the α-2,6 isoform. Each isoform is predominantly associated with different surface epithelial cell types of the human upper airway. Using recombinant HAs and human tracheal airway epithelial cells in vitro and ex vivo, we show that many avian HA subtypes do not adhere to this canonical view of SA specificity. The propensity of avian viruses to adapt to human receptors may thus be more widespread than previously supposed.     

Influenza A virus neuraminidase limits viral superinfection

Enveloped viruses use multiple mechanisms to inhibit infection of a target cell by more than one virion. These mechanisms may be of particular importance for the evolution of segmented viruses, because superinfection exclusion may limit the frequency of reassortment of viral genes. Here, we show that cellular expression of influenza A virus neuraminidase (NA), but not hemagglutinin (HA) or the M2 proton pump, inhibits entry of HA-pseudotyped retroviruses. Cells infected with H1N1 or H3N2 influenza A virus were similarly refractory to HA-mediated infection and to superinfection with a second influenza A virus. Both HA-mediated entry and viral superinfection were rescued by the neuraminidase inhibitors oseltamivir carboxylate and zanamivir. These inhibitors also prevented the removal of alpha-2,3- and alpha-2,6-linked sialic acid observed in cells expressing NA or infected with influenza A viruses. Our data indicate that NA alone among viral proteins limits influenza A virus superinfection.     

Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus

The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.     

Role of receptor binding specificity in influenza A virus transmission and pathogenesis

The recent emergence of a novel avian A/H7N9 influenza virus in poultry and humans in China, as well as laboratory studies on adaptation and transmission of avian A/H5N1 influenza viruses, has shed new light on influenza virus adaptation to mammals. One of the biological traits required for animal influenza viruses to cross the species barrier that received considerable attention in animal model studies, in vitro assays, and structural analyses is receptor binding specificity. Sialylated glycans present on the apical surface of host cells can function as receptors for the influenza virus hemagglutinin (HA) protein. Avian and human influenza viruses typically have a different sialic acid (SA)‐binding preference and only few amino acid changes in the HA protein can cause a switch from avian to human receptor specificity. Recent experiments using glycan arrays, virus histochemistry, animal models, and structural analyses of HA have added a wealth of knowledge on receptor binding specificity. Here, we review recent data on the interaction between influenza virus HA and SA receptors of the host, and the impact on virus host range, pathogenesis, and transmission. Remaining challenges and future research priorities are also discussed.     

Residue Y161 of influenza virus hemagglutinin is involved in viral recognition of sialylated complexes from different hosts

Influenza A virus glycoprotein hemagglutinin (HA) binds to host cell surface sialic acid (SA)-terminated sugars in glycoproteins to initiate viral entry. It is thought that avian influenza viruses preferentially bind to N-acetylneuraminic acid α3 (NeuAcα3) sugars, while human influenza viruses exhibit a preference for NeuAcα6-containing sugars. Thus, species-specific SA(s) is one of the determinants in viral host tropism. The SA binding pocket of the HA1 subunit has been extensively studied, and a number of residues important for receptor binding have been identified. In this study, we examined the potential roles of seven highly conserved HA surface-located amino acid residues in receptor binding and viral entry using an H5 subtype. Among them, mutant Y161A showed cell-type-dependent viral entry without obvious defects in HA protein expression or viral incorporation. This mutant also displayed dramatically different ability in agglutinating different animal erythrocytes. Oligosaccharide binding analysis showed that substituting alanine at Y161 of HA changed the SA binding preference from NeuAc to N-glycolylneuraminic acid (NeuGc). Rescued mutant Y161A viruses demonstrated a 5- to 10-fold growth defect, but they were robust in viral replication and plaque forming ability. Our results demonstrate that Y161 is a critical residue involved in recognition of different SA species. This residue may play a role in determining influenza virus host tropism.     

Sequence analysis and receptor specificity of the hemagglutinin of a recent influenza H2N2 virus isolated from chicken in North America

Influenza viruses bind host cells following an interaction between the viral hemagglutinin (HA) protein and host cell sialylated glycoproteins and glycolipids. Differences in binding affinities of the HAs for different types of sialic acid linkages (α2-3 vs. α2-6) contribute to determining the host range of an influenza virus. The ability of an avian influenza virus HA to bind the human form of the receptor may be one requirement for an avian virus to propagate in the human population. In this paper, we describe the characterization of the HA from an H2N2 virus isolated from a Pennsylvania chicken farm in 2004. Sequence analysis revealed that this HA is a member of the Eurasian clade, and receptor binding studies show that it maintains its specificity for the avian influenza virus α2-3 linked sialic acid receptor.     

Biophysical Measurement of the Balance of Influenza A Hemagglutinin and Neuraminidase Activities

The interaction of influenza A viruses with the cell surface is controlled by the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). These two glycoproteins have opposing activities: HA is responsible for binding the host receptor (sialic acid) to allow infection, and NA is responsible for cleaving the receptor to facilitate virus release. Several studies have demonstrated that compatible levels of HA and NA activity are required for a virus to replicate efficiently. This is consequently of great interest for determining virus transmissibility. The concurrent role of these two proteins in receptor binding has never been directly measured. We demonstrate a novel biophysical approach based on bio-layer interferometry to measure the balance of the activities of these two proteins in real time. This technique measures virus binding to and release from a surface coated with either the human-like receptor analog α2,6-linked sialic acid or the avian-like receptor analog α2,3-linked sialic acid in both the presence and absence of NA inhibitors. Bio-layer interferometry measurements were also carried out to determine the effect of altering HA receptor affinity and NA stalk length on receptor binding.     

Impact of host cell line adaptation on quasispecies composition and glycosylation of influenza A virus hemagglutinin

The genome of influenza A viruses is constantly changing (genetic drift) resulting in small, gradual changes in viral proteins. Alterations within antibody recognition sites of the viral membrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) result in an antigenetic drift, which requires the seasonal update of human influenza virus vaccines. Generally, virus adaptation is necessary to obtain sufficiently high virus yields in cell culture-derived vaccine manufacturing. In this study detailed HA N-glycosylation pattern analysis was combined with in-depth pyrosequencing analysis of the virus genomic RNA. Forward and backward adaptation from Madin-Darby Canine Kidney (MDCK) cells to African green monkey kidney (Vero) cells was investigated for two closely related influenza A virus PR/8/34 (H1N1) strains: from the National Institute for Biological Standards and Control (NIBSC) or the Robert Koch Institute (RKI). Furthermore, stability of HA N-glycosylation patterns over ten consecutive passages and different harvest time points is demonstrated. Adaptation to Vero cells finally allowed efficient influenza A virus replication in Vero cells. In contrast, during back-adaptation the virus replicated well from the very beginning. HA N-glycosylation patterns were cell line dependent and stabilized fast within one (NIBSC-derived virus) or two (RKI-derived virus) successive passages during adaptation processes. However, during adaptation new virus variants were detected. These variants carried "rescue" mutations on the genomic level within the HA stem region, which result in amino acid substitutions. These substitutions finally allowed sufficient virus replication in the new host system. According to adaptation pressure the composition of the virus populations varied. In Vero cells a selection for "rescue" variants was characteristic. After back-adaptation to MDCK cells some variants persisted at indifferent frequencies, others slowly diminished and even dropped below the detection limit.     

NA Proteins of Influenza A Viruses H1N1/2009, H5N1, and H9N2 Show Differential Effects on Infection Initiation,Virus Release,and Cell-Cell Fusion

Two surface glycoproteins of influenza virus, haemagglutinin (HA) and neuraminidase (NA), play opposite roles in terms of their interaction with host sialic acid receptors. HA attaches to sialic acid on host cell surface receptors to initiate virus infection while NA removes these sialic acids to facilitate release of progeny virions. This functional opposition requires a balance. To explore what might happen when NA of an influenza virus was replaced by one from another isolate or subtype, in this study, we generated three recombinant influenza A viruses in the background of A/PR/8/34 (PR8) (H1N1) and with NA genes obtained respectively from the 2009 pandemic H1N1 virus, a highly pathogenic avian H5N1 virus, and a lowly pathogenic avian H9N2 virus. These recombinant viruses, rPR8-H1N1NA, rPR8-H5N1NA, and rPR8-H9N2NA, were shown to have similar growth kinetics in cells and pathogenicity in mice. However, much more rPR8-H5N1NA and PR8-wt virions were released from chicken erythrocytes than virions of rPR8-H1N1NA and rPR8-H9N2NA after 1 h. In addition, in MDCK cells, rPR8-H5N1NA and rPR8-H9N2NA infected a higher percentage of cells, and induced cell-cell fusion faster and more extensively than PR8-wt and rPR8-H1N1NA did in the early phase of infection. In conclusion, NA replacement in this study did not affect virus replication kinetics but had different effects on infection initiation, virus release and fusion of infected cells. These phenomena might be partially due to NA proteins’ different specificity to α2-3/2-6-sialylated carbohydrate chains, but the exact mechanism remains to be explored.     

Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities

Influenza A virus specificity for the host is mediated by the viral surface glycoprotein hemagglutinin (HA), which binds to receptors containing glycans with terminal sialic acids. Avian viruses preferentially bind to alpha2-3-linked sialic acids on receptors of intestinal epithelial cells, whereas human viruses are specific for the alpha2-6 linkage on epithelial cells of the lungs and upper respiratory tract. To define the receptor preferences of a number of human and avian H1 and H3 viruses, including the 1918 H1N1 pandemic strains, their hemagglutinins were analyzed using a recently described glycan array. The array, which contains 200 carbohydrates and glycoproteins, not only revealed clear differentiation of receptor preferences for alpha2-3 and/or alpha2-6 sialic acid linkage, but could also detect fine differences in HA specificity, such as preferences for fucosylation, sulfation and sialylation at positions 2 (Gal) and 3 (GlcNAc, GalNAc) of the terminal trisaccharide. For the two 1918 HA variants, the South Carolina (SC) HA (with Asp190, Asp225) bound exclusively alpha2-6 receptors, while the New York (NY) variant, which differed only by one residue (Gly225), had mixed alpha2-6/alpha2-3 specificity, especially for sulfated oligosaccharides. Only one mutation of the NY variant (Asp190Glu) was sufficient to revert the HA receptor preference to that of classical avian strains. Thus, the species barrier, as defined by the receptor specificity preferences of 1918 human viruses compared to likely avian virus progenitors, can be circumvented by changes at only two positions in the HA receptor binding site. The glycan array thus provides highly detailed profiles of influenza receptor specificity that can be used to map the evolution of new human pathogenic strains, such as the H5N1 avian influenza.     

Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors

No reliable cell culture assay is currently available for monitoring human influenza virus sensitivity to neuraminidase inhibitors (NAI). This can be explained by the observation that because of a low concentration of sialyl-alpha2,6-galactose (Sia[alpha2,6]Gal)-containing virus receptors in conventional cell lines, replication of human virus isolates shows little dependency on viral neuraminidase. To test whether overexpression of Sia(alpha2,6)Gal moieties in cultured cells could make them suitable for testing human influenza virus sensitivity to NAI, we stably transfected MDCK cells with cDNA of human 2,6-sialyltransferase (SIAT1). Transfected cells expressed twofold-higher amounts of 6-linked sialic acids and twofold-lower amounts of 3-linked sialic acids than parent MDCK cells as judged by staining with Sambucus nigra agglutinin and Maackia amurensis agglutinin, respectively. After transfection, binding of a clinical human influenza virus isolate was increased, whereas binding of its egg-adapted variant which preferentially bound 3-linked receptors was decreased. The sensitivity of human influenza A and B viruses to the neuraminidase inhibitor oseltamivir carboxylate was substantially improved in the SIAT1-transfected cell line and was consistent with their sensitivity in neuraminidase enzyme assay and with the hemagglutinin (HA) receptor-binding phenotype. MDCK cells stably transfected with SIAT1 may therefore be a suitable system for testing influenza virus sensitivity to NAI.     

The Surface Glycoproteins of H5 Influenza Viruses Isolated from Humans, Chickens, and Wild Aquatic Birds Have Distinguishable Properties

In 1997, 18 confirmed cases of human influenza arising from multiple independent transmissions of H5N1 viruses from infected chickens were reported from Hong Kong. To identify possible phenotypic changes in the hemagglutinin (HA) and neuraminidase (NA) of the H5 viruses during interspecies transfer, we compared the receptor-binding properties and NA activities of the human and chicken H5N1 isolates from Hong Kong and of H5N3 and H5N1 viruses from wild aquatic birds. All H5N1 viruses, including the human isolate bound to Sia2-3Gal-containing receptors but not to Sia2-6Gal-containing receptors. This finding formally demonstrates for the first time that receptor specificity of avian influenza viruses may not restrict initial avian-to-human transmission. The H5N1 chicken viruses differed from H5 viruses of wild aquatic birds by a 19-amino-acid deletion in the stalk of the NA and the presence of a carbohydrate at the globular head of the HA. We found that a deletion in the NA decreased its ability to release the virus from cells, whereas carbohydrate at the HA head decreased the affinity of the virus for cell receptors. Comparison of amino acid sequences from GenBank of the HAs and NAs from different avian species revealed that additional glycosylation of the HA and a shortened NA stalk are characteristic features of the H5 and H7 chicken viruses. This finding indicates that changes in both HA and NA may be required for the adaptation of influenza viruses from wild aquatic birds to domestic chickens and raises the possibility that chickens may be a possible intermediate host in zoonotic transmission.     

Human Cytotoxic T-Lymphocyte Repertoire to Influenza A Viruses

The murine CD8⁺ cytotoxic-T-lymphocyte (CTL) repertoire appears to be quite limited in response to influenza A viruses. The CTL responses to influenza A virus in humans were examined to determine if the CTL repertoire is also very limited. Bulk cultures revealed that a number of virus proteins were recognized in CTL assays. CTL lines were isolated from three donors for detailed study and found to be specific for epitopes on numerous influenza A viral proteins. Eight distinct CD8⁺ CTL lines were isolated from donor 1. The proteins recognized by these cell lines included the nucleoprotein (NP), matrix protein (M1), nonstructural protein 1 (NS1), polymerases (PB1 and PB2), and hemagglutinin (HA). Two CD4⁺ cell lines, one specific for neuraminidase (NA) and the other specific for M1, were also characterized. These CTL results were confirmed by precursor frequency analysis of peptide-specific gamma interferon-producing cells detected by ELISPOT. The epitopes recognized by 6 of these 10 cell lines have not been previously described; 8 of the 10 cell lines were cross-reactive to subtype H1N1, H2N2, and H3N2 viruses, 1 cell line was cross-reactive to subtypes H1N1 and H2N2, and 1 cell line was subtype H1N1 specific. A broad CTL repertoire was detected in the two other donors, and cell lines specific for the NP, NA, HA, M1, NS1, and M2 viral proteins were isolated. These findings indicate that the human memory CTL response to influenza A virus is broadly directed to epitopes on a wide variety of proteins, unlike the limited response observed following infection of mice.     

Comparative analysis of avian influenza virus diversity in poultry and humans during a highly pathogenic avian influenza A (H7N7) virus outbreak

Although increasing data have become available that link human adaptation with specific molecular changes in nonhuman influenza viruses, the molecular changes of these viruses during a large highly pathogenic avian influenza virus (HPAI) outbreak in poultry along with avian-to-human transmission have never been documented. By comprehensive virologic analysis of combined veterinary and human samples obtained during a large HPAI A (H7N7) outbreak in the Netherlands in 2003, we mapped the acquisition of human adaptation markers to identify the public health risk associated with an HPAI outbreak in poultry. Full-length hemagglutinin (HA), neuraminidase (NA), and PB2 sequencing of A (H7N7) viruses obtained from 45 human cases showed amino acid variations at different codons in HA (n=20), NA (n=23), and PB2 (n=23). Identification of the avian sources of human virus infections based on 232 farm sequences demonstrated that for each gene about 50% of the variation was already present in poultry. Polygenic accumulation and farm-to-farm spread of known virulence and human adaptation markers in A (H7N7) virus-infected poultry occurred prior to farm-to-human transmission. These include the independent emergence of HA A143T mutants, accumulation of four NA mutations, and farm-to-farm spread of virus variants harboring mammalian host determinants D701N and S714I in PB2. This implies that HPAI viruses with pandemic potential can emerge directly from poultry. Since the public health risk of an avian influenza virus outbreak in poultry can rapidly change, we recommend virologic monitoring for human adaptation markers among poultry as well as among humans during the course of an outbreak in poultry.     

A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity

The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the alpha2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the alpha2,6 and the alpha2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.     

Glycans as receptors for influenza pathogenesis

Influenza A viruses, members of the Orthomyxoviridae family, are responsible for annual seasonal influenza epidemics and occasional global pandemics. The binding of viral coat glycoprotein hemagglutinin (HA) to sialylated glycan receptors on host epithelial cells is the critical initial step in the infection and transmission of these viruses. Scientists believe that a switch in the binding specificity of HA from Neu5Acα2-3Gal linked (α2-3) to Neu5Acα2-6Gal linked (α2-6) glycans is essential for the crossover of the viruses from avian to human hosts. However, studies have shown that the classification of glycan binding preference of HA based on sialic acid linkage alone is insufficient to establish a correlation between receptor specificity of HA and the efficient transmission of influenza A viruses. A recent study reported extensive diversity in the structure and composition of α2-6 glycans (which goes beyond the sialic acid linkage) in human upper respiratory epithelia and identified different glycan structural topologies. Biochemical examination of the multivalent HA binding to these diverse sialylated glycan structures also demonstrated that high affinity binding of HA to α2-6 glycans with a characteristic umbrella-like structural topology is critical for efficient human adaptation and human-human transmission of influenza A viruses. This review summarizes studies which suggest a new paradigm for understanding the role of the structure of sialylated glycan receptors in influenza virus pathogenesis.