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PubMed1964

NEURAMINIDASES AND INFLUENZA VIRUS INFECTION IN EMBRYONATED EGGS.

Highly purified neuraminidase from influenza virus prevents embryonated eggs from infection by Lee-B influenza virus. The degree of prevention afforded is equal to that of neuraminidases from Vibrio cholerae and from Clostridium perfringens when assayed at the same enzyme activity. The preventive effect is much lower against PR-8 virus. The results support the concept that the intact "receptor" containing sialic acid is required for the binding and infection of cells by influenza viruses.

Animals↗

PubMed1964

ENZYMATIC VARIANTS OF INFLUENZA VIRUS. III. FUNCTION OF NEURAMINIDASE IN THE VIRAL GROWTH CYCLE.

Padgett, Billie L. (University of Wisconsin, Madison), and Duard L. Walker. Enzymatic variants of influenza virus. III. Function of neuraminidase in the viral growth cycle. J. Bacteriol. 87:363-369. 1964.-Multiplication of a slowly reacting enzymatic variant of influenza B virus, strain LEE, was compared with that of parent virus in ovo. At 35 C, although their final yields were equal, variant virus reached its peak concentration in the allantoic fluids later than parent virus. At 39 C, multiplication of both viruses was slower, parent virus requiring 4 hr and variant virus 8 hr longer to reach infectivity levels comparable with those at 24 hr at 35 C. Variant enzyme activity in vitro can be controlled by altering the temperature and calcium concentration. Growth curves of these viruses in pieces of chorioallantoic membrane (CAM) in culture under conditions in which the variant should be as active as the parent revealed only minor differences between them. Under conditions in which variant enzyme activity would be much slower than the parent, the release of variant virus from the CAM was delayed and the rate of release was slower. Under the most adverse conditions, 39 C and no calcium, formation of infectious variant virus ceased after 5 hr, and the hemagglutination inhibitor in the cells was not degraded although hemagglutinins were produced. These findings are discussed in relation to the function of neuraminidase during viral multiplication.

Calcium↗

PubMed2002

The S128R polymorphism of E-selectin mediates neuraminidase-resistant tethering of myeloid cells under shear flow.

E-selectin mediates the rolling of circulating leukocytes on vascular endothelial cells. A polymorphism, in which serine is substituted for arginine at position 128 (S128R) in the EGF domain, has been associated with both early-onset atherosclerosis and SLE. We investigated whether the substitution alters the ligand-binding properties of E-selectin under shear flow by studying the capacity of Chinese hamster ovary cell transfectants expressing wild type (WT) or S128R E-selectin to support interactions of neutrophils, K562 cells or HL60 cells. We initially chose to study non-fucosylated K562 cells. No interactions were observed on WT E-selectin, whereas S128R supported a transient tethering interaction of K562 cells, which was resistant to digestion with either neuraminidase or O-sialoglycoprotein endopeptidase, and, in turn, could result in firm adhesion in the presence of a beta2-integrin. HL60 cells exhibited increased rolling on S128R E-selectin. Although neuraminidase treatment inhibited all HL60 interactions with WT E-selectin, it unmasked transient tethers on S128R. We further observed that S128R recruited significantly more neutrophils than WT E-selectin, without affecting neutrophil rolling velocity. This polymorphism may therefore amplify leukocyte-endothelial cell interactions and may be a factor linking the S128R polymorphism to vascular disease.

Animals↗

PubMed1988

Inherited lysosomal storage disease associated with deficiencies of beta-galactosidase and alpha-neuraminidase in sheep.

Histopathologic, ultrastructural and Golgi impregnation studies disclosed lesions characteristic of a neuronal lysosomal storage disease in related sheep with onset of neurologic signs at 4-6 months. Biochemical and enzymatic evaluation disclosed storage of GM1 ganglioside, asialo-GM1, and neutral long chain oligosaccharides in brain, urinary excretion of neutral long chain oligosaccharides, and deficiencies of lysosomal beta-galactosidase and alpha-neuraminidase. Retrospective and limited prospective genetic studies suggested autosomal recessive inheritance. A gene-dosage effect on beta-galactosidase levels was documented in fibroblasts from putative heterozygous sheep. Fibroblasts from affected sheep did not have increased beta-galactosidase activity after incubation with the protease inhibitor, leupeptin. In some aspects this disease is similar to GM1 gangliosidosis, but is unique in that a genetic defect in lysosomal beta-galactosidase may cause the deficiency of lysosomal alpha-neuraminidase.

Animals↗

PubMed1990

Combined deficiency of beta-galactosidase and neuraminidase: natural history of the disease in the first 18 years of an American patient with late infantile onset form.

We describe the clinical findings over the first 18 years of a patient with a novel phenotype for galactosialidosis, the storage disease produced by the combined deficiency of beta-galactosidase and neuraminidase. Clinical findings in the first few months included somewhat unusual appearance and hepatosplenomegaly. Dysostosis multiplex was evident by age 2 1/2 years. Mitral and aortic valvular disease appeared over the next few years and cardiac disease has become the most important clinical problem. Foam cells were present in the bone marrow, and vacuolated lymphocytes were present in the peripheral blood smear. The patient had no neurological symptoms, cherry red spots, or intellectual deterioration during the first 18 years. Evidence presented elsewhere indicates that the basic defect in this late infantile form of galactosialidosis (as is thought to be true for the other forms of galactosialidosis) is a reduced amount of the 32 kDa phosphoglycoprotein which associates with beta-galactosidase and alpha-neuraminidase in lysosomes.

Aortic Valve Insufficiency↗

PubMed1979

Adult type neuronal storage disease with neuraminidase deficiency.

We describe a patient with adult-onset neuronal storage disease characterized by myoclonus, cerebellar ataxia, convulsive seizures, cherry-red spots, skeletal dysplasia, mild gargoyle features, inguinal hernia, and angiokeratoma. Cytoplasmic inclusions consistent with lysosomal storage disease were demonstrated in neurons of the autonomic nervous system. Accumulation of GM3 and GM2 gangliosides was found in sympathetic ganglia but a catabolic disturbance of these gangliosides was ruled out by normal levels of GM3 ganglioside sialidase and N-acetyl-beta-hexosaminidase A activities. beta-Galactosidase activity was decreased in leukocytes and fibroblasts, but not in serum. GM1 gangliosidosis was ruled out by lipid analyses, and mucopolysaccharidosis by normal excretion of mucopolysaccharide in urine. Sialyl oligosaccharides were increased in urine and alpha-neuraminidase was deficient in fibroblasts. This disorder is considered to be an inherited metabolic disorder of sialyl glycoproteins and oligosaccharides due to deficiency of an alpha-neuraminidase.

Galactosidases↗

PubMedDate not supplied

ZstatFlu-II test: a chemiluminescent neuraminidase assay for influenza viral diagnostics.

The ZstatFlu-II test is a highly sensitive, specific, rapid, point-of-care chemiluminescent diagnostic test for influenza infection. Influenza viral neuraminidase-specific substrate, spiroadamantyl-1,2-dioxetane-4,7-dimethoxy-N-acetyl-neuraminic acid, is at the core of the ZstatFlu-II Test. The enzymatic reaction was carried out at 25 degrees C and neutral pH, representing the optimum assay conditions for influenza types A and B viral neuraminidases. The results were outputted on a Polaroid trade mark High Speed Detector Film. Positive results appeared as a '+'-shaped white film image; negative results produced no image. The 'glow' kinetics, facilitated by a unique combination of light enhancers, also 'tuned' the wavelength of emission to match the spectral properties of the film. The substrate hydrolysed non-enzymatically at acid pH or at temperatures above 25 degrees C. In order to minimize false positives, the ZstatFlu-II Test was formatted with 0.3-0.4 K(m) substrate and freezing the test kit until use. The pH optimization of the ZstatFlu-II test is discussed with reference to model compounds of sialyl-glycosides. A nucleophilic attack or an electrostatic stabilization of a developing carbonium ion under the influence of the adjacent carboxyl group was probably responsible for non-enzymatic hydrolysis of the substrate. Intramolecular general acid catalysis is proposed as a mechanism for the lability of the O-glycosidic linkage of the substrate.

Heterocyclic Compounds↗

PubMed1991

Allo-cross-reactivity of a human neuraminidase-specific T cell clone dependent on presentation of an endogenous B cell-specific antigen.

T cells specific for foreign antigen recognize a complex of peptides and self-major histocompatibility complex (MHC) molecules and can also cross-react with allo-MHC molecules. It remains controversial, however, what alloreactive T cells exactly recognize. It has been proposed that alloreactive T cells recognize endogenous peptides presented by allo-MHC molecules. To test this hypothesis, we examined an influenza virus-specific T cell clone (6H5), specific for neuraminidase N2 and restricted by HLA-DR1. In the absence of influenza virus, this clone cross-reacted with HLA-DR1Dw1+ but not with HLA-DR1Dw20+ Epstein-Barr virus-transformed lymphoblastoid cells (B-LCL). Cold target inhibition experiments and the rearrangement pattern of the T cell receptor beta chain indicated that 6H5 was a monoclonal T cell population most likely using the same T cell receptor for both responses. To determine whether determinants other than HLA-DR1Dw1+ B-LCL or activated B cells, but, surprisingly, not to other cell types expressed HLA-DR1Dw1, including monocytes and transfected L cells. These experiments further support the concept that recognition of allogeneic MHC (in this case HLA-DR1Dw1) may result from a cross-reactivity of T cells specific for a complex of foreign antigen and self-MHC (neuraminidase N2 and HLA-DR1Dw20). Furthermore, allorecognition of T cell clone 6H5 appears to depend upon the recognition of a complex of allogeneic MHC and a cell-type specific endogenous peptide presented by activated B cells.

Antigens, Surface↗

PubMedDate not supplied

Functional balance between haemagglutinin and neuraminidase in influenza virus infections.

Influenza A and B viruses carry two surface glycoproteins, the haemagglutinin (HA) and the neuraminidase (NA). Both proteins have been found to recognise the same host cell molecule, sialic acid. HA binds to sialic acid-containing receptors on target cells to initiate virus infection, whereas NA cleaves sialic acids from cellular receptors and extracellular inhibitors to facilitate progeny virus release and to promote the spread of the infection to neighbouring cells. Numerous studies performed recently have revealed that an optimal interplay between these receptor-binding and receptor-destroying activities of the surface glycoproteins is required for efficient virus replication. An existing balance between the antagonistic HA and NA functions of individual viruses can be disturbed by various events, such as reassortment, virus transmission to a new host, or therapeutic inhibition of neuraminidase. The resulting decrease in the viral replicative fitness is usually overcome by restoration of the functional balance due to compensatory mutations in HA, NA or both proteins.

Hemagglutinin Glycoproteins, Influenza Virus↗

PubMed1996

The structures of Salmonella typhimurium LT2 neuraminidase and its complexes with three inhibitors at high resolution.

The structure of Salmonella typhimurium LT2 neuraminidase (STNA) is reported here to a resolution of 1.6 angstroms together with the structures of three complexes of STNA with different inhibitors. The first is 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (Neu5Ac2en or DANA), the second and third are phosphonate derivatives of N-acetyl-neuraminic acid (NANA) which have phosphonate groups at the C2 position equatorial (ePANA) and axial (aPANA) to the plane of the sugar ring. The complex structures are at resolutions of 1.6 angstroms, 1.6 angstroms and 1.9 angstroms, respectively. These analyses show the STNA active site to be topologically inflexible and the interactions to be dominated by the arginine triad, with the pyranose rings of the inhibitors undergoing distortion to occupy the space available. Solvent structure differs only around the third phosphonate oxygen, which attracts a potassium ion. The STNA structure is topologically identical to the previously reported influenza virus neuraminidase structures, although very different in detail; the root-mean-square (r.m.s) deviation for 210 C alpha positions considered equivalent is 2.28 angstroms (out of a total of 390 residues in influenza and 381 in STNA). The active site residues are more highly conserved, in that both the viral and bacterial structures contain an arginine triad, a hydrophobic pocket, a tyrosine and glutamic acid residue at the base of the site and a potential proton-donating aspartic acid. However, differences in binding to O4 and to the glycerol side-chain may reflect the different kinetics employed by the two enzymes.

Binding Sites↗

PubMed2001

Comparison of structural changes of cell surface carbohydrates in the eustachian tube epithelium of chinchillas infected with a Streptococcus pneumoniae neuraminidase-deficient mutant or its isogenic parent strain.

Six different lectin probes were used to examine alterations of the cell surface carbohydrates in the chinchilla eustachian tube (ET) lumen subsequent to the intranasal (i.n.) challenge with the Streptococcus pneumoniae parent strain, D39, or its isogenic derivative, DeltaNA1, which is deficient in neuraminidase NanA. The labelling pattern revealed that the binding of wheat germ agglutinin (WGA), Erythrina cristagalli lectin (ECL), peanut agglutinin (PNA), Bandeiraea simplicifolia lectin II (BSL II) and succinylated wheat germ agglutinin (SWGA) were increased in the lumenal surface of the ET in the D39 inoculated cohort compared to the uninfected control, which indicated that N-acetylglucosamine (GlcNAc) and D-galactose residues were exposed. Concurrently, decreased labelling with Sambucus nigra agglutinin (SNA) indicated that there were few sialic acid residues remaining in the ET epithelium subsequent to i.n. inoculation with D39. The DeltaNA1 neuraminidase deficient mutant, however, did not induce any significant changes in the lectin labelling patterns, and was comparable to that of the control cohort. We propose that products of the nanA gene have a significant impact on the changes of the carbohydrate moieties in the ET epithelium and may be responsible for the previously reported increased ability of the D39 parent to colonize the nasopharynx and invade the middle ear.

Animals↗

PubMed1993

Comparison of structure and sequence of influenza B/Yamagata and B/Beijing neuraminidases shows a conserved "head" but much greater variability in the "stalk" and NB protein.

In 1988 the B/Yamagata/16/88 influenza virus appeared, which was antigenically quite different from the other B viruses circulating at that time. Neuraminidase heads of this strain were purified, crystallized, and analyzed by X-ray crystallography and compared with the structure of B/Beijing/1/87 neuraminidase. Only two point mutations could be detected in the "head" of the molecule leading to local rearrangement of amino acid side-chains. The nucleotide sequence, however, revealed significant differences in the stalk region and in the NB protein.

Amino Acid Sequence↗

PubMed1995

Neuraminidase is essential for fowl plague virus hemagglutinin to show hemagglutinating activity.

When hemagglutinin (HA) of fowl plague virus (FPV) was expressed in CV-1 cells by a simian virus 40 vector, hemadsorption was barely detectable, although HA was exposed at the cell surface. However, treatment of HA-expressing cells with Vibrio cholerae neuraminidase (VCNA) resulted in extensive hemadsorption. VCNA treatment enhanced the electrophoretic mobility of the HA1 subunit of HA, indicating the removal of sialic acid. When two oligosaccharides in the vicinity of the receptor binding site of FPV HA were deleted by site-specific mutagenesis, VCNA treatment was not required for hemadsorption. Mutants which retained one of these oligosaccharides and mutants in which oligosaccharides not adjacent to the receptor binding site were deleted needed VCNA treatment to show hemadsorption. VCNA treatment also enhanced hemadsorption of vector-expressed HA of the WSN strain, which had a complex-type oligosaccharide in the vicinity of the receptor binding site, but had no effect on hemadsorption of Hong Kong type HA, which has a high-mannose type oligosaccharide adjacent to the receptor binding site. These results indicate that sialic acid on oligosaccharides near the receptor binding site interferes with hemadsorption. Thus, the neuraminidase is essential for FPV HA to show hemagglutinating activity.

Glycosylation↗

PubMed1995

Inhibition of replication of avian influenza viruses by the neuraminidase inhibitor 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid.

The sialidase inhibitor 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-guanidino-Neu5Ac2en), designed with computer assistance and knowledge of the crystal structure of influenza virus neuraminidase, has shown antiviral effects in animal models of human influenza (M. von Itzstein et al., Nature, 363, 418-423, 1993). Here we demonstrate that the compound efficiently inhibits the enzyme activity of all nine subtypes of avian influenza A neuraminidase in vitro. When administered intranasally to chickens infected with lethal viruses, high doses of the compound (1000 micrograms/kg) protected 85% of birds harboring A/Chick/Victoria/1/85 (H7N7), a fowl plague virus, but not chickens infected with other highly virulent viruses of the N1, N2, or N3 subtype. This differential inhibitory effect was also seen in a plaque reduction assay with Madin-Darby canine kidney cells (MDCK), where 4-guanidino-Neu5Ac2en was more effective against A/Chick/Vic/85 (H7N7) than A/FPV/Rostock/34 (H7N1). In contrast to the substantial plaque reduction observed in MDCK cells, the drug failed to inhibit plaque formation in chicken embryo fibroblasts infected with either A/Chick/Vic/85 or A/FPV/Rostock/34, regardless of its concentration. The different levels of drug efficacy seen in two cell systems most likely reflect the location of virus budding and release in polarized versus nonpolarized cells, as well as the compound's mode of extracellular action.

Animals↗

PubMed1995

Cooperative neuraminidase activity in a paramyxovirus.

Paramyxoviruses possess neuraminidase (NA) activity, i.e., the ability to cleave sialic acid from membrane-bound and soluble glycoconjugates. The activity is associated with a homotetrameric, surface glycoprotein spike, called the hemagglutinin-neuraminidase. This structure also mediates viral attachment to sialic acid-containing receptors and constitutes the major neutralizing antigen for the virus. Cooperativity has been demonstrated for the NA activity of an isolate of one of the paramyxoviruses, Newcastle disease virus. Although all known viral NA proteins are homooligomeric, this is the first demonstration of cooperativity in this family of proteins. A variant virus, selected by escape from neutralization by a monoclonal antibody thought to bind close to the NA active site, has lost cooperativity. Conversion to the noncooperative state correlates with increases in both avidity for cellular receptors and fusogenic activity.

Antibodies, Monoclonal↗

PubMed1997

A single sequence change destabilizes the influenza virus neuraminidase tetramer.

A single change (E119G) in the influenza A virus N9 neuraminidase (NA) results in resistance of the enzyme to the NA inhibitor 4-Guanidino-Neu5Ac2en (4-GuDANA). This change causes a salt link between Glu119, which sits in a pocket in the bottom of the active site of the enzyme, and the 4-guanidinium moiety of the inhibitor to be lost. NA "heads" of the resistant enzyme produced only a few small crystals under conditions in which the wild-type enzyme readily formed large crystals. These small crystals were of sufficient quality to yield X-ray crystallographic data which confirmed the E119G change and demonstrated the presence of electron density representing either a strong structural-water molecule or an anionic species in place of the glutamate carboxylate. NA heads of the resistant enzyme also have greatly reduced NA activity per milligram of total protein. We have now found that the mutant NA heads consist predominantly of monomers with a few dimers and tetramers, as determined by electron microscopic analysis of the protein. The low level of enzymatic activity as well as the small number of crystals obtained were probably from the few tetramers remaining intact in the preparation. The purified wild-type and 4-GuDANA-resistant enzymes were treated with the homobifunctional NHS-ester cross linker, DTSSP. SDS-PAGE analysis of the treated enzymes clearly revealed cross-linked dimers of the wild-type enzyme. In contrast, only a small proportion of the 4-GuDANA-resistant neuraminidase was cross-linked. An examination of the known X-ray crystallographic structure of the wild-type NA reveals a salt bridge between Glu119 and Arg156 of the same monomer. Arg156 is a conserved amino acid that is situated at the interface between monomers, and a salt link between this amino acid and Glu119 may contribute to the stability of enzyme tetramers. It is suggested that the E119G alteration in the 4-GuDANA-resistant NA leads to the abrogation of this interaction and thus to the instability of the NA tetramers.

Animals↗

PubMed1998

Substrate, inhibitor, or antibody stabilizes the Glu 119 Gly mutant influenza virus neuraminidase.

We have previously reported the isolation and characterization of an influenza virus variant with decreased sensitivity to the neuraminidase-specific inhibitor zanamivir. This variant, which has a mutation in the active site, Glu 119 Gly (E119G), has the same specific activity as the wild-type neuraminidase (NA), but is inherently unstable, as measured by loss of both enzyme activity and NC10 monoclonal antibody reactivity. However, despite the instability of the NA, replication of the virus in liquid culture is not adversely affected. We demonstrate here that in addition to enhanced temperature sensitivity the mutant NA was significantly more sensitive to formaldehyde and to specimen preparation for electron microscopy. Substrate, inhibitor, or monoclonal antibodies stabilized the NA against all methods of denaturation. These results suggest that the instability of the variant is primarily at the level of polypeptide chain folding rather than at the level of association of monomers into tetramers. Furthermore the presence of high levels of substrate, either cell or virus associated, may be sufficient to stabilize the NA during virus replication.

Animals↗

PubMed2000

Influenza A virus neuraminidase: regions of the protein potentially involved in virus-host interactions.

Phylogenetically informative amino acid positions (PIPs) were identified in influenza A neuraminidases of subtypes N1 and N2. Neuraminidase evolves in a lineage-specific way as the virus adapts to a new host or changes to evade the host's immune system. Thus, many PIPs undoubtedly identify positions involved in virus-host interactions. Phylogenetically important regions (PIRs) are defined as several PIPs near one another. There are 15 PIRs on N1 and 12 on N2, seven of which are shared between the two subtypes. Many PIRs are coincident with antigenic or glycosylation sites. Other PIRs may represent additional antigenic sites or may be involved in other aspects of virus-host biology.

Amino Acids↗