Binding affinity of GM3 lactone for influenza virus

We investigated the interaction of GM3 lactone with influenza virus. The specific bindings of influenza virus and its hemagglutinin to GM3 lactone-containing mixed monolayers were studied by using a quartz-crystal microbalance. It has been known that gangliosides as receptors for influenza virus are also substrates for virus neuraminidase. GM3 lactone, however, was found to bind to influenza virus hemagglutinin, but not to be substrate for virus neuraminidase.     

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.     

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.     

Novel aromatic inhibitors of influenza virus neuraminidase make selective interactions with conserved residues and water molecules in the active site.

The active site of type A or B influenza virus neuraminidase is composed of 11 conserved residues that directly interact with the substrate, sialic acid. An aromatic benzene ring has been used to replace the pyranose of sialic acid in our design of novel neuraminidase inhibitors. A bis(hydroxymethyl)pyrrolidinone ring was constructed in place of the N-acetyl group on the sialic acid. The hydroxymethyl groups replace two active site water molecules, which resulted in the high affinity of the nanomolar inhibitors. However, these inhibitors have greater potency for type A influenza virus than for type B influenza virus. To resolve the differences, we determined the X-ray crystal structure of three benzoic acid substituted inhibitors bound to the active site of B/Lee/40 neuraminidase. The investigation of a hydrophobic aliphatic group and a hydrophilic guanidino group on the aromatic inhibitors shows changes in the interaction with the active site residue Glu275. The results provide an explanation for the difference in efficacy of these inhibitors against types A and B viruses, even though the 11 active site residues of the neuraminidase are conserved.     

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.     

Cells Persistently Infected with Newcastle Disease Virus III. Thermal Stability of Hemagglutinin and Neuraminidase of a Mutant Isolated from Persistently Infected L Cells

Data were obtained which indicated the possible cause of the defective elution from erythrocytes of the mutant virus (NDV(pi)) isolated from L cells persistently infected with the Herts strain of Newcastle disease virus (NDV(o)). The chicken erythrocyte receptors for the mutant and wild-type viruses were equally sensitive to the action of Vibrio cholera filtrate neuraminidase; this suggests that the failure of NDV(pi) to elute from chicken erythrocytes is not due to a specific neuraminidase-resistant receptor for this virus on the erythrocyte membrane. There was no difference in the enzyme content of the intact virions of NDV(o) and NDV(pi) when tested with a soluble substrate, indicating that the inefficient elution of NDV(pi) was not due to a reduced enzyme content. The neuraminidase activity of intact NDV(pi) virions was significantly more stable at 55 C than the enzyme of NDV(o) virions, whereas the dissociated enzymes of the two viruses were inactivated at the same rate. On the basis of these findings, it seems likely there is a structural difference between the two viruses. The neuraminidase protein of the mutant NDV(pi) may be incorporated into the viral envelope in such a manner that it is prevented from reacting with the substrate in the erythrocyte membrane, although it can react with a soluble substrate. The hemagglutinin activity of both intact and disrupted NDV(pi) was significantly more resistant to thermal inactivation than that of the wild-type NDV(o). This finding suggests a genetic difference in the hemagglutinin protein of the two viruses.     

Oseltamivir resistance depends on the position 273 amino acid of neuraminidase of the type A influenza virus (H1N1), circulating in human population

The structure of neuraminidase of the type A influenza virus (H1N1) spreading in the human population was analyzed. The obtained results indicate a significant correlation between the oseltamivir sensitivity and the nature of the amino acid localized not only to neuraminidase position 274, but also to position 273 of this protein. Phenylalanine at position 273 in neuraminidase indicates a higher propensity to influenza virus mutation H274Y, leading to the appearance of resistant strains. It is suggested that the mutation at position 273 may be one of the characteristics allowing type A influenza virus to be ascribed to a pandemic or a seasonal type.     

Influenza Hemagglutinin and Neuraminidase Membrane Glycoproteins

Considerable progress has been made toward understanding the structural basis of the interaction of the two major surface glycoproteins of influenza A virus with their common ligand/substrate: carbohydrate chains terminating in sialic acid. The specificity of virus attachment to target cells is mediated by hemagglutinin, which acquires characteristic changes in its receptor-binding site to switch its host from avian species to humans. Anti-influenza drugs mimic the natural sialic acid substrate of the virus neuraminidase enzyme but utilize the much tighter binding of the drugs for efficacy. Resistance to one of the two main antiviral drugs is differentially acquired by the two distinct subsets of neuraminidase as a consequence of structural differences in the enzyme active site between the two phylogenetic groups.     

A screening assay for neuraminidase inhibitors using neuraminidases N1 and N3 from a baculovirus expression system

Context: Development of inexpensive and safe enzymatic assays to screen for putative neuraminidase inhibitors. Objective: Validate the use of recombinant neuraminidase expressed in baculovirus located on the viral surface capsule to develop a neuraminidase inhibitor screening assay. Materials and methods: Recombinant baculovirus particles displaying neuraminidase N1 and N3 were used as enzyme sources. The assay set-up required the use of 2'-(4-methylumbelliferyl)-α-D-acetyl neuraminic acid as substrate and oseltamivir carboxylate as benchmark inhibitor. Results: The assay was set up in a standard 96-well plate. The within- and between-assay coefficients of variation were, on average, less than 10%. The 50% inhibitory concentration values of the inhibitor were in good agreement with those determined by independent kinetic experiments. Discussion and conclusions: The assay showed satisfactory within- and between-assay repeatability. The obtained results suggest that recombinant baculovirus expressing neuraminidase located on the virus membrane capsule can be used to set up affordable and reliable neuraminidase inhibitors screening assays.     

Virulence factors of influenza A viruses: WSN virus neuraminidase required for plaque production in MDBK cells.

The genetic basis for the distinctive capacity of influenza A/WSN/33 (H0N1) virus (WSN virus) to produce plaques on bovine kidney (MDBK) cells was found to be related to virus neuraminidase. Recombinant viruses that derived only the neuraminidase of WSN virus were capable of producing plaques, whereas recombinant viruses identical to WSN except for neuraminidase did not produce plaques. With viruses that do not contain WSN neuraminidase, infectivity of virus yields from MDBK cells was increased approximately 1,000-fold after in vitro treatment with trypsin. In contrast, no significant increase in infectivity was observed after trypsin treatment of viruses containing WSN neuraminidase. In addition, polyacrylamide gel analysis of proteins of WSN virus obtained after infection of MDBK cells demonstrated that hemagglutinin was present in the cleaved form (HA1 + HA2), whereas only uncleaved hemagglutinin was obtained with a recombinant virus that derived all of its genes from WSN virus except its neuraminidase. These data are in accord with the hypothesis that neuraminidase may facilitate production of infectious particles by removing sialic acid residues and exposing appropriate cleavage sites on hemagglutinin.     

Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes.

The reverse genetics system has made it possible to modify the influenza virus genome. By this method, we were able to assess influenza virus as a vaccine vector for protecting BALB/c mice against otherwise lethal lymphocytic choriomeningitis virus (LCMV) infection. A single dose of influenza virus [A/WSN/33 (H1N1)] bearing a cytotoxic T-lymphocyte-specific epitope of the LCMV nucleoprotein (residues 116 to 127) in the neuraminidase stalk protected mice against LCMV challenge for at least 4 months. The immunity was mediated by cytotoxic T lymphocytes and was haplotype specific, indicating that the observed protective response was solely a consequence of prior priming with the H-2d LCMV nucleoprotein epitope expressed in the recombinant influenza virus. We also found that as many as 58 amino acids could be inserted into the neuraminidase stalk without loss of viral function. These findings demonstrate the potential of influenza virus as a vaccine vector, with the neuraminidase stalk as a repository for foreign epitopes.     

Characterization of influenza virus neuraminidase with hemagglutinin activity and its comparison with that of viral neuraminidase

The neuraminidase associated with the bifunctional protein, hemagglutinin-neuraminidase, of influenza virus has been characterized. The enzyme has a pH optimum of 4.5, does not require Ca2+ and is inactivated (98%) by incubation at 50 degrees C. The enzyme has a Km of 2.00 X 10(-3) M and 0.06 X 10(-3) M with the substrates 2-(3-methoxyphenyl)-N-acetylneuraminic acid and fetuin, respectively. The Ki is 400 X 10(-6) with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. The incorporation of labeled cysteine, valine and leucine in the hemagglutinin-neuraminidase protein is different from that of viral neuraminidase. A comparison of the properties of the neuraminidase associated with protein hemagglutinin-neuraminidase with that of viral neuraminidase or sialidase showed that the former is biochemically different and an antigenically distinct enzyme. The unique feature of the new enzyme is that it has the hemagglutinin activity as well. The two biological activities could not be separated from each other in all systems used. Apparently, protein hemagglutinin-neuraminidase is genetically transferable and it is detectable in a laboratory recombinant virus E-2971 (H3 Aichi X N7). These results suggest that protein hemagglutinin-neuraminidase is a unique surface protein of the influenza virus A/Aichi/2/68 (H3N2).     

High-efficiency formation of influenza virus transfectants.

cDNA-derived RNAs were introduced into the genomes of influenza viruses by using an improved ribonucleoprotein (RNP) transfection protocol. Up to 10(5) viral transfectants with a novel neuraminidase gene could be obtained by using a 35-mm dish (10(6) cells) for RNP transfection. In addition to genes coding for surface proteins (hemagglutinin and neuraminidase), we also exchanged a gene coding for nonsurface proteins. The cDNA-derived influenza A/PR/8/34 virus NS gene was introduced into a temperature-sensitive mutant with a defect in this gene. We suggest that the term influenza virus transfectant be used for those viruses which are made by RNP transfection with cDNA-derived RNA.     

Prediction of epitopes in hemagglutinin and neuraminidase proteins of influenza A virus H5N1 strain: a clue for diagnostic and vaccine development

The avian influenza is an important infectious disease of birds. The genome of influenza A virus was segmented, single-stranded, negative-sense RNA molecules, which encodes many proteins. The significant surface proteins are hemagglutinin and neuraminidase for the pathogenicity of birds to humans. The prediction of epitopes in protein provides a suitable primary immunodiagnostic antigen for the detection of the influenza A virus H5N1. It was further used in the development and approval of epitopes, which were used as antigens, and the peptides can be used for vaccines in the potential control of an emerging pandemic of this pathogen. The conserved epitopes may be useful for the diagnosis of animals infected with the influenza virus. These might be helpful to prevent the spreading of influenza in animal to animal and also in the prevention and monitoring of its spread in the newer region. The epitopes provide the support for serodiagnosis or as a protective immunogen in novel vaccines. In this study, the preliminary data from the in silico analysis of hemagglutinin and neuraminidase was done to find potential T-cell epitopes. The determined peptides were beneficial for vaccine development, as they can reduce time by minimizing the number of required tests to find the possible selected epitopes, which target for vaccine development. T-cell recognition of the peptide-major histocompatible complex (MHC) is a prerequisite for cellular immunity. This work examines existing computational strategies for the study of peptide-MHC interactions. We have also provided guidelines for predicting antigenic peptides based on the availability of existing experimental data.     

Comparison of Two Methods for Enumeration of Anaerobe Numbers on Forages and Evaluation of Ethylene Oxide Treatment for Forage Sterilization

Experiments were conducted to (i) compare most-probable-number (MPN) procedures with roll tube procedures for enumeration of forage anaerobic bacteria and (ii) evaluate the efficacy of using ethylene oxide to sterilize wet herbage. Alfalfa, corn, and alfalfa-orchardgrass silages and alfalfa and orchardgrass herbages were analyzed for total anaerobic bacteria (medium pH, 6.8) and acid-tolerant anaerobic bacteria (medium pH, 4.5) by both roll tube and MPN procedures. No difference was found between the roll tube and MPN procedures for total bacteria; however, higher counts were obtained for acid-tolerant bacteria when the MPN procedure was used. Although MPN procedures require less time to obtain an estimate of bacterial numbers, isolation and identification of the microbial population is not possible. Alfalfa herbage was treated with ethylene oxide for 12, 24, or 36 h, incubated for 7 days at 37°C with or without addition of a bacterial inoculant, and analyzed for total bacteria by MPN procedures. Microbial growth after inoculation of ethylene oxide-treated herbage indicated that there was insufficient residual ethylene oxide to inhibit subsequent microbial growth. The results also indicated that 24 h was required to adequately sterilize fresh herbage. Thus, ethylene oxide can be used to sterilize wet herbage for use as a substrate for pure cultures of silage bacteria.     

Sialidase activity in rimantadine-resistant and -sensitive influenza A viruses

Rimantadine-resistant and -sensitive influenza A variants were assayed for their sialidase (neuraminidase, EC 3.2.1.18) activity. The kinetic parameters determined (pH optimum, stability against different pH values, thermal stability, activity on methylumbelliferyl-alpha-D-N-acetylneuraminic acid, N-acetylneuraminyl-lactose, fetuin and bovine submandibular gland mucin as substrates, Km with the former substrate, inhibition by two competitive inhibitors, and behavior towards amantadine) revealed the same results for both variants of the virus. Thus, it can be deduced that resistance to rimantadine does not influence the sialidase activity of influenza A virus.     

Development of a sensitive chemiluminescent neuraminidase assay for the determination of influenza virus susceptibility to zanamivir

Determination of the sensitivity of influenza viruses to neuraminidase (NA) inhibitors is presently based on assays of NA function because, unlike available cell culture methods, the results of such assays are predictive of susceptibility in vivo. At present the most widely used substrate in assays of NA function is the fluorogenic reagent 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (MUN). A rapid assay with improved sensitivity is required because a proportion of clinical isolates has insufficient NA to be detectable in the current fluorogenic assay, and because some mutations associated with resistance to NA inhibitors reduce the activity of the enzyme. A chemiluminescence-based assay of NA activity has been developed that uses a 1,2-dioxetane derivative of sialic acid (NA-STAR) as the substrate. When compared with the fluorogenic assay, use of the NA-STAR substrate results in a 67-fold reduction in the limit of detection of the NA assay, from 200 pM (11 fmol) NA to 3 pM (0.16 fmol) NA. A panel of isolates from phase 2 clinical studies of zanamivir, which were undetectable in the fluorogenic assay, was tested for activity using the NA-STAR substrate. Of these 12 isolates with undetectable NA activity, 10 (83%) were found to have detectable NA activity using the NA-STAR substrate. A comparison of sensitivity to zanamivir of a panel of influenza A and B viruses using the two NA assay methods has been performed. IC(50) values for zanamivir using the NA-STAR were in the range 1.0-7.5 nM and those for the fluorogenic assay in the range 1. 0-5.7 nM (n = 6). The NA-STAR assay is a highly sensitive, rapid assay of influenza virus NA activity that is applicable to monitoring the susceptibility of influenza virus clinical isolates to NA inhibitors.     

Protective Effects of Specific Immunity to Viral Neuraminidase on Influenza Virus Infection of Mice

Antibody specific for viral neuraminidase can be demonstrated in mice following (i) pulmonary infection with influenza virus, (ii) immunization with ultraviolet-in-activated influenza virus, (iii) immunization with isolated neuraminidase of influenza A(2) virus, and (iv) passive immunization with sera of rabbits immunized with isolated A(2) neuraminidase. Neuraminidase antibody produced by any of these methods exerts a profound inhibiting effect on virus replication in the lungs of mice challenged with strains of virus having homologous neuraminidase protein, even in the absence of hemagglutinating inhibiting antibody to the challenge virus, and results in markedly decreased pulmonary virus titers and diminished lung lesions. These observations suggest that antineuraminidase immunity may play a significant role in the protection against influenza virus challenge observed in mice after infection or artificial immunization.     

神经氨酸酶、碱性聚合酶2及非结构蛋白1影响A型流感病毒致病力的分子机制研究进展

随着研究的不断深入,血凝素(HA)之外的其他蛋白在影响A型流感病毒的致病力甚至宿主特异性方面的重要作用逐渐受到关注。本文对神经氨酸酶(NA)、碱性聚合酶2(PB2)及非结构蛋白1(NS1)的相关进展作了综述,以期进一步阐明流感病毒的致病分子基础,并藉此探讨可能的宿主范围限定因素。     

Sequence and structure alignment of Paramyxoviridae attachment proteins and discovery of enzymatic activity for a morbillivirus hemagglutinin.

On the basis of the conservation of neuraminidase (N) active-site residues in influenza virus N and paramyxovirus hemagglutinin-neuraminidase (HN), it has been suggested that the three-dimensional (3D) structures of the globular heads of the two proteins are broadly similar. In this study, details of this structural similarity are worked out. Detailed multiple sequence alignment of paramyxovirus HN proteins and influenza virus N proteins was based on the schematic representation of the previously proposed structural similarity. This multiple sequence alignment of paramyxovirus HN proteins was used as an intermediate to align the morbillivirus hemagglutinin (H) proteins with neuraminidase. Hypothetical 3D structures were built for paramyxovirus HN and morbillivirus H, based on homology modelling. The locations of insertions and deletions, glycosylation sites, active-site residues, and disulfide bridges agree with the proposed 3D structure of HN and H of the Paramyxoviridae. Moreover, details of the modelled H protein predict previously undescribed enzymatic activity. This prediction was confirmed for rinderpest virus and peste des petits ruminants virus. The enzymatic activity was highly substrate specific, because sialic acid was released only from crude mucins isolated from bovine submaxillary glands. The enzymatic activity may indicate a general infection mechanism for respiratory viruses, and the active site may prove to be a new target for antiviral compounds.