Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase.

A model is proposed for the three-dimensional structure of the paramyxovirus hemagglutinin-neuraminidase (HN) protein. The model is broadly similar to the structure of the influenza virus neuraminidase and is based on the identification of invariant amino acids among HN sequences which have counterparts in the enzyme-active center of influenza virus neuraminidase. The influenza virus enzyme-active site is constructed from strain-invariant functional and framework residues, but in this model of HN, it is primarily the functional residues, i.e., those that make direct contact with the substrate sialic acid, which have identical counterparts in neuraminidase. The framework residues of the active site are different in HN and in neuraminidase and appear to be less strictly conserved within HN sequences than within neuraminidase sequences.     

Action of ortho- and paramyxovirus neuraminidase on gangliosides. Hydrolysis of ganglioside GM1 by Sendai virus neuraminidase

The action of neuraminidase of influenza A virus, Sendai virus and Newcastle disease virus particles on bovine brain ganglioside GM1 and the properties of Sendai virus neuraminidase for GM1 were studied. With Sendai virus, GM1 was hydrolyzed to asialo-GM1 (GA1) and N-acetylneuraminic acid even in the absence of surfactant or other additives, while the hydrolysis of GM1 by Newcastle disease virus or influenza A virus was very low or undetectable under the same conditions. The formation of GA1 by Sendai virus neuraminidase was confirmed by thin-layer chromatography and immunodiffusion test using anti-GA1 antiserum. The apparent Km of Sendai virus neuraminidase for GM1 hydrolysis was found to be 2.67 x 10(-4) M and the optimum pH was 5.6. GM3, GM2 and oligosaccharide of GM1 were hydrolyzed more effectively than GM1 in the absence of surfactant (GM3 greater than GM2 greater than oligosaccharide of GM1 greater than GM1). The hydrolysis of GM1 by the Sendai virus enzyme was stimulated by the addition of sodium cholate or sodium taurocholate, but was inhibited by divalent cations (10 mM), Ca2+, Mg2+, ZN2+, Fe2+ and CU2+. In the absence of the surfactant, Sendai virus neuraminidase hydrolyzed GM1 more efficiently than Arthobacter ureafaciens neuraminidase which has been reported recently as being an adequate enzyme to hydrolyze ganglioside GM1 as a substrate.     

Enzymological characteristics of avian influenza A virus neuraminidase

Neuraminidases of 18 strains of avian influenza A virus were examined by both colorimetric and fluorometric assays using fetuin and 4-methylumbelliferyl-N-Ac-alpha-D-neuraminide as substrates, respectively, to compare them with those of human influenza A and B viruses. The ratios of the neuraminidase activity of avian influenza virus measured by the colorimetric assay method to that measured by the fluorometric assay were distributed in the range of 2.4-20.3. The enzyme of avian influenza virus showed calcium-ion dependence in both assay methods. These results suggest that neuraminidase of avian influenza A virus is varies greatly from one strain to another in substrate specificity as compared with those of human influenza A and B viruses, and that some strains of avian influenza A virus have a neuraminidase with unique enzymological characteristics different from that of human influenza A virus as well as that of influenza B virus.     

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.     

The neuraminidase of influenza virus

It is the enzyme neuraminidase, projecting from the surface of influenza virus particles, which allows the virus to leave infected cells and spread in the body. Antibodies which inhibit the enzyme limit the infection, but antigenic variation of the neuraminidase renders it ineffective in a vaccine. This article describes the crystal structure of influenza virus neuraminidase, information about the active site which may lead to development of specific and effective inhibitors of the enzyme, and the structure of epitopes (antigenic determinants) on the neuraminidase. The 3-dimensional structure of the epitopes was obtained by X-ray diffraction methods using crystals of neuraminidase complexed with monoclonal antibody Fab fragments. Escape mutants, selected by growing virus in the presence of monoclonal antibodies to the neuraminidase, possess single amino acid sequence changes. The crystal structure of two mutants showed that the change in structure was restricted to that particular sidechain, but the change in the epitope was sufficient to abolish antibody binding even though it is known in one case that 21 other amino acids on the neuraminidase are in contact with the antibody.     

Inhibitor selectivity of a new class of oseltamivir analogs against viral neuraminidase over human neuraminidase enzymes

The viral neuraminidase enzyme is an established target for anti-influenza pharmaceuticals. However, viral neuraminidase inhibitors could have off-target effects due to interactions with native human neuraminidase enzymes. We report the activity of a series of known inhibitors of the influenza group-1 neuraminidase enzyme (N1 subtype) against recombinant forms of the human neuraminidase enzymes NEU3 and NEU4. These inhibitors were designed to take advantage of an additional enzyme pocket (known as the 150-cavity) near the catalytic site of certain viral neuraminidase subtypes (N1, N4 and N8). We find that these modified derivatives have minimal activity against the human enzymes, NEU3 and NEU4. Two compounds show moderate activity against NEU3, possibly due to alternative binding modes available to these structures. Our results reinforce that recognition of the glycerol side-chain is distinct between the viral and human NEU enzymes, and provide experimental support for improving the selectivity of viral neuraminidase inhibitors by exploiting the 150-cavity found in certain subtypes of viral neuraminidases.     

Structural and functional relationship between the receptor recognition and neuraminidase activities of the Newcastle disease virus hemagglutinin-neuraminidase protein: receptor recognition is dependent on neuraminidase activity

The terminal globular domain of the paramyxovirus hemagglutinin-neuraminidase (HN) glycoprotein spike has a number of conserved residues that are predicted to form its neuraminidase (NA) active site, by analogy to the influenza virus neuraminidase protein. We have performed a site-directed mutational analysis of the role of these residues in the functional activity of the Newcastle disease virus (NDV) HN protein. Substitutions for several of these residues result in a protein lacking both detectable NA and receptor recognition activity. Contribution of NA activity, either exogenously or by coexpression with another HN protein, partially rescues the receptor recognition activity of these proteins, indicating that the receptor recognition deficiencies of the mutated HN proteins result from their lack of detectable NA activity. In addition to providing support for the homology-based predictions for the structure of HN, these findings argue that (i) the HN residues that mediate its NA activity are not critical to its attachment function and (ii) NA activity is required for the protein to mediate binding to receptors.     

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.     

Influenza virus hemagglutinin and neuraminidase cytoplasmic tails control particle shape.

The cytoplasmic tails of the influenza virus glycoproteins hemagglutinin (HA) and neuraminidase (NA) are highly conserved in sequence for all virus subtypes and it is believed that assembly of this enveloped virus depends on interactions of these domains with cytoplasmic viral components. However, it is possible to rescue altered influenza viruses lacking either the HA or NA cytoplasmic tails. We have obtained an influenza virus that lacks both the cytoplasmic tail of HA and NA. Particle production is reduced approximately 10-fold but these particles, although having a fairly normal protein composition, are greatly elongated and of extended irregular shape. We propose a model in which the interactions of the cytoplasmic tails of HA and NA with an internal viral component are so important for spherical virion shape that there is dual redundancy in the interactions.     

The neuraminidase yield-reduction bioassay of human other interferons.

The production of neuraminidase by the recombinant influenza virus X7(F1) in human monkey, rabbit, hamster, mouse, and chicken cell cultures is inhibited by interferon. Described is a new enzyme assay for neuraminidase that can be applied to the bioassay of interferons. The advantages of this interferon bioassay are its sensitivity, reproducibility, rapidity, and convenience.     

The anti-influenza virus agent 4-GU-DANA (zanamivir) inhibits cell fusion mediated by human parainfluenza virus and influenza virus HA

4-GU-DANA (zanamivir) (as well as DANA and 4-AM-DANA) was found to inhibit the neuraminidase activity of human parainfluenza virus type 3 (HPF3). The viral neuraminidase activity is attributable to hemagglutinin-neuraminidase (HN), an envelope protein essential for viral attachment and for fusion mediated by the other envelope protein, F. While there is no evidence that HN's neuraminidase activity is essential for receptor binding and syncytium formation, we found that 4-GU-DANA prevented hemadsorption and fusion of persistently infected cells with uninfected cells. In plaque assays, 4-GU-DANA reduced the number (but not the area) of plaques if present only during the adsorption period and reduced plaque area (but not number) if added only after the 90-min adsorption period. 4-GU-DANA also reduced the area of plaques formed by a neuraminidase-deficient variant, confirming that its interference with cell-cell fusion is unrelated to inhibition of neuraminidase activity. The order-of-magnitude lower 50% inhibitory concentrations of 4-GU-DANA (and also DANA and 4-AM-DANA) for plaque area reduction and for inhibition in the fusion assay than for reducing plaque number or blocking hemadsorption indicate the particular efficacy of these sialic acid analogs in interfering with cell-cell fusion. In cell lines expressing influenza virus hemagglutinin (HA) as the only viral protein, we found that 4-GU-DANA had no effect on hemadsorption but did inhibit HA2b-red blood cell fusion, as judged by both lipid mixing and content mixing. Thus, 4-GU-DANA can interfere with both influenza virus- and HPF3-mediated fusion. The results indicate that (i) in HPF3, 4-GU-DANA and its analogs have an affinity not only for the neuraminidase active site of HN but also for sites important for receptor binding and cell fusion and (ii) sialic acid-based inhibitors of influenza virus neuraminidase can also exert a direct, negative effect on the fusogenic function of the other envelope protein, HA.     

Influenza virus neuraminidase: structure, antibodies, and inhibitors.

The determination of the 3-dimensional structure of the influenza virus neuraminidase in 1983 has served as a platform for understanding interactions between antibodies and protein antigens, for investigating antigenic variation in influenza viruses, and for devising new inhibitors of the enzyme. That work is reviewed here, together with more recent developments that have resulted in one of the inhibitors entering clinical trials as an anti-influenza virus drug.     

Mechanism of interference mediated by human parainfluenza virus type 3 infection

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine whether this resistance is attributable to the viral neuraminidase, we used a cloned variant HPF3 HN that has two amino acid alterations in HN leading to the loss of detectable neuraminidase activity. Cells expressing the neuraminidase-deficient variant HN-GFP were not protected from infection, despite expressing HN on their surface at levels even higher than the wild-type cell clones. Our results demonstrate that the HPF3 HN-mediated interference effect can be attributed to the presence of an active neuraminidase enzyme activity and provide the first definitive evidence that the mechanism for attachment interference by a paramyxovirus is attributable to the viral neuraminidase.     

Specific Nucleoprotein Residues Affect Influenza Virus Morphology

Influenza virus strains are often pleiomorphic, a characteristic that is largely attributed to specific residues in matrix protein 1 (M1). Although the mechanism by which M1 controls virion morphology has not yet been defined, it is suggested that the M1 interaction with other viral proteins plays an important role. In this study, we rescued recombinant virus WSN-AichiM1 containing the spherical A/WSN/33 (WSN) backbone and the M1 protein from A/Aichi/2/68 (Aichi). Aichi M1 differs from WSN M1 by 7 amino acids but includes those identified to be responsible for filamentous virion formation. Interestingly, Aichi virus produced spherical virions, while WSN-AichiM1 exhibited a long filamentous morphology, as detected by immunofluorescence and electron microscopy. Additional incorporation of Aichi nucleoprotein (NP) but not the hemagglutinin (HA), neuraminidase (NA), or M2 gene to WSN-AichiM1 abrogated filamentous virion formation, suggesting that specific M1-NP interactions affect virion morphology. Further characterization of viruses containing WSN/Aichi chimeric NPs identified residues 214, 217, and 253 of Aichi NP as necessary and sufficient for the formation of spherical virions. NP residues 214 and 217 localize at the minor groove between the two opposite-polarity NP helical strands of viral ribonucleocapsids, and residue 253 also localizes near the surface of the groove. These findings indicate that NP plays a critical role in influenza virus morphology, possibly through its interaction with the M1 layer during virus budding.     

Three-dimensional structure of neuraminidase of subtype N9 from an avian influenza virus

Neuraminidases from different subtypes of influenza virus are characterized by the absence of serological cross-reactivity and an amino acid sequence homology of approximately 50%. The three-dimensional structure of the neuraminidase antigen of subtype N9 from an avian influenza virus (A/tern/Australia/G70c/75) has been determined by X-ray crystallography and shown to be folded similarly to neuraminidase of subtype N2 isolated from a human influenza virus. This result demonstrates that absence of immunological cross-reactivity is no measure of dissimilarity of polypeptide chain folding. Small differences in the way in which the subunits are organized around the molecular fourfold axis are observed. Insertions and deletions with respect to subtype N2 neuraminidase occur in four regions, only one of which is located within the major antigenic determinants around the enzyme active site.     

Inhibitory potency of flavonoid derivatives on influenza virus neuraminidase

The constant risk of emerging new influenza virus strains that are resistant to established inhibitors like oseltamivir leaves influenza neuraminidase (NA) a prominent target for drug design. The inhibitory activity of several flavonoid derivatives was experimentally tested in comparison to oseltamivir for the NA expressed by the seasonal influenza virus strains A/California/7/09 (A(H1N1)pdm09), A/Perth/16/09 (A(H3N2)), and B/Brisbane/60/08. IC₅₀ values of polyphenols confirmed moderate inhibition in the μM range. Structurally, the amount and site of glycosylation of tested flavonoids have no significant influence on their inhibitory potency. In a pharmacophore-based docking approach the structure–activity relationship was evaluated. Molecular dynamics simulations revealed highly flexible parts of the enzyme and the contribution of salt bridges to the structural stability of NA. The findings of this study elucidate the impact of flavonoids on viral neuraminidase activity and the analysis of their modes of action provide valuable information about the mechanism of NA inhibition.     

Suppression of influenza A virus nuclear antigen production and neuraminidase activity by a nutrient mixture containing ascorbic acid, green tea extract and amino acids

Influenza, one of the oldest and most common infections, poses a serious health problem causing significant morbidity and mortality, and imposing substantial economic costs. The efficacy of current drugs is limited and improved therapies are needed. A unique nutrient mixture (NM), containing ascorbic acid, green tea extract, lysine, proline, N-acetyl cysteine, selenium among other micronutrients, has been shown to exert anti-carcinogenic and anti-atherogenic activity both in vitro and in vivo. Many of the constituents of NM have been shown to have an inhibitory effect on replication of influenza virus and HIV. This prompted us to study the effect of NM on influenza A virus multiplication in infected cells and neuraminidase activity (NA) in virus particles. Addition of NM to Vero or MDCK cells post infection resulted in dose-dependent inhibition of viral nucleoprotein (NP) production in infected cells. NM-mediated inhibition of viral NP was selective and not due to cytotoxicity towards host cells. This antiviral effect was enhanced by pretreatment of virus with the nutrient mixture. Individual components of NM, namely ascorbic acid and green tea extract, also blocked viral NP production, conferring enhanced inhibition when tested in combination. Incubation of cell-free virus with NM resulted in dose-dependent inhibition of associated NA enzyme activity. In conclusion, the nutrient mixture exerts an antiviral effect against influenza A virus by lowering viral protein production in infected cells and diminishing viral enzymatic activity in cell-free particles.     

Influenza virus neuraminidase activates latent transforming growth factor beta.

Transforming growth factor beta (TGF-beta) is a family of proteins secreted by virtually all cells in a biologically inactive form. TGF-beta levels increase during many pathophysiological situations, including viral infection. The mechanism for increased TGF-beta activity during viral infection is not understood. We observed an increase in active TGF-beta levels within 1 day in mice infected with influenza virus. Further studies showed that the neuraminidase glycoprotein of influenza A and B viruses directly activates latent TGF-beta in vitro. There are sufficient levels of TGF-beta activated by virus to induce apoptosis in cells. In addition, influenza virus-induced apoptosis is partially inhibited by TGF-beta-specific antibodies. These novel findings suggest a potential role for activation of TGF-beta during the host response to influenza virus infection, specifically apoptosis. This is the first report showing direct activation of latent TGF-beta by a viral protein.     

The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid.

Influenza virus neuraminidase catalyses the cleavage of terminal sialic acid, the viral receptor, from carbohydrate chains on glycoproteins and glycolipids. We present the crystal structure of the enzymatically active head of influenza B virus neuraminidase from the strain B/Beijing/1/87. The native structure has been refined to a crystallographic R-factor of 14.8% at 2.2 A resolution and its complex with sialic acid refined at 2.8 A resolution. The overall fold of the molecule is very similar to the already known structure of neuraminidase from influenza A virus, with which there is amino acid sequence homology of approximately 30%. Two calcium binding sites have been identified. One of them, previously undescribed, is located between the active site and a large surface antigenic loop. The calcium ion is octahedrally co-ordinated by five oxygen atoms from the protein and one water molecule. Sequence comparisons suggest that this calcium site should occur in all influenza A and B virus neuraminidases. Soaking of sialic acid into the crystals has enabled the mode of binding of the reaction product in the putative active site pocket to be revealed. All the large side groups of the sialic acid are equatorial and are specifically recognized by nine fully conserved active site residues. These in turn are stabilized by a second shell of 10 highly conserved residues principally by an extensive network of hydrogen bonds.     

Anomeric specificity and protein-substrate interactions support the 3D model for the hemagglutinin-neuraminidase from sendai virus.

The 3D structure of paramyxovirus hemagglutinin-neuraminidase has not yet been resolved; however, a theoretical model has been built by using influenza virus and bacterial neuraminidases as template [V. C. Epa (1997) Proteins Struct. Funct. Gen. 29, 264-281]. Two common features of the catalytic mechanism of the neuraminidases of known 3D structure are the anomeric specificity and the involvement of a tyrosine residue in the stabilization of the transition state. These key features have been investigated on the water-soluble ectodomain of the hemagglutinin-neuraminidase from Sendai virus (cHN). The anomeric specificity of the hydrolysis of the substrate by cHN has been investigated by NMR spectroscopy. The immediate product of the reaction was the alpha-anomer, meaning that cHN belongs between glycohydrolases retaining anomeric configuration like influenza virus neuraminidase. Measurements of the UV difference spectrum upon binding of the substrate analogue 2,3-dehydro 2-deossi N-acetyl neuraminic acid indicate the ionization of a tyrosine residue and decreased polarity in the environment of a tryptophan residue. Functional significance of the spectral data was derived from the known structure of influenza neuraminidase, where a tyrosinate ion is involved in the stabilization of the transition-state carbonium ion, and a tryptophan residue is involved in the binding of the acetyl moiety of the substrate. The data give experimental support to the 3D model of paramyxovirus neuraminidase.