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Biomedical subjects

G M Air

Publications and source records attributed to G M Air.

At least 37 records · Page 2Linked to original sources
PubMed1995

Red cells bound to influenza virus N9 neuraminidase are not released by the N9 neuraminidase activity.

Influenza virus neuraminidase (NA) of the N9 subtype also possesses hemagglutinin activity and the hemagglutinating, or hemabsorbing (HB), site is distinct from the catalytic site. Previous results suggested that the NA was binding to sialic acid on the red cell surface, but we now report that the HB receptor is not sensitive to N9 influenza neuraminidase activity. Cell lines that constitutively express N9 or N2 neuraminidase have been used to further investigate the specificity of red blood cell binding to the HB site. The results suggest that the ligand is N-acetylneuraminic acid in a linkage or environment that is not sensitive to influenza virus neuraminidase, but which is released by the broadly specific bacterial sialidases from Micromonospora viridifaciens or Arthrobacter ureafaciens.

Animals↗
PubMed1995

Benzoic acid inhibitors of influenza virus neuraminidase.

A strategy was developed to design non-carbohydrate inhibitors of influenza virus neuraminidase. Using an iterative cycle of modeling, synthesis, biological testing and X-ray crystallography structure determination, a series of inhibitors based on benzoic acid were produced. The refined structures of three compounds complexed with neuraminidase are reported. The results demonstrate the success of this structure-based drug-design strategy.

Journal Article↗
PubMed1995

Structures of aromatic inhibitors of influenza virus neuraminidase.

Neuraminidase (NA), a surface glycoprotein of influenza virus, is a potential target for design of antiinfluenza agents. The crystal structure of influenza virus neuraminidase showed that in the active site 11 residues are universally conserved among all strains known so far. Several potent inhibitors based on the carbohydrate compound 2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (DANA) have been shown to bind to the conserved active site and to reduce virus infection in animals when administered by nasal spray. Inhibitors of this type are, however, rapidly excreted from physiological systems and may not be effective in order to provide long-time protection. A new class of specific NA inhibitors, which are benzoic acid derivatives, has been designed on the basis of the three-dimensional structure of the NA-DANA complex and modeling of derivatives of 4-(acetylamino)benzoic acid in the NA active site. Intermediates were synthesized and were shown to moderately inhibit the NA activity and to bind to the NA active site as predicted. These rudimentary inhibitors, 4-(acetylamino)-3-hydroxy-5-nitrobenzoic acid, 4-(acetylamino)-3-hydroxy-5-aminobenzoic acid, and 4-(acetylamino)-3-aminobenzoic acid, and their X-ray structures in complexes with N2 (A/Tokyo/3/67) and B/Lee/40 neuraminidases have been analyzed. The coordinates of such inhibitors complexed with NA were used as the starting model for further design of more potent benzoic acid inhibitors. Because the active site residues of NA are invariant, the designed aromatic inhibitors have the potential to become an antiviral drug against all strains of influenza virus.

Aminobenzoates↗
PubMed1995

A sialic acid-derived phosphonate analog inhibits different strains of influenza virus neuraminidase with different efficiencies.

A phosphonate analog of N-acetyl neuraminic acid (PANA) has been designed as a potential neuraminidase (NA) inhibitor and synthesized as both the alpha (ePANA) and beta (aPANA) anomers. Inhibition of type A (N2) and type B NA activity by ePANA was approximately a 100-fold better than by sialic acid, but inhibition of type A (N9) NA was only ten-fold better than by sialic acid. The aPANA compound was not a strong inhibitor for any of the NA strains tested. The crystal structures at 2.4 A resolution of ePANA complexed to type A (N2) NA, type A (N9) NA and type B NA and aPANA complexed to type A (N2) NA showed that neither of the PANA compounds distorted the NA active site upon binding. No significant differences in the NA-ePANA complex structures were found to explain the anomalous inhibition of N9 neuraminidase by ePANA. We put forward the hypothesis that an increase in the ePANA inhibition compared to that caused by sialic acid is due to (1) a stronger electrostatic interaction between the inhibitor phosphonyl group and the active site arginine pocket and (2) a lower distortion energy requirement for binding of ePANA.

Binding Sites↗
PubMed1995

Probing the structure of influenza B hemagglutinin using site-directed mutagenesis.

The crystal structure of the hemagglutinin (HA) of influenza virus A/Aichi/68 (H3N2) from the X-31 reassortant virus was reported in 1981, but as yet there are no X-ray diffraction structures for hemagglutinins of other types or even subtypes of influenza virus. We have used site-directed mutagenesis to probe the structure of the hemagglutinin of influenza B/Hong Kong/8/73. We investigated a region in the globular head domain that is helical in the influenza A HA structure, targeting sidechains that in the H3 HA point toward solvent (Thr196) or into the receptor-binding pocket (Gln197). None of the mutations affected hemagglutination activity, but mutations T196P or Q1971 eliminated binding of a monoclonal antibody. The data suggest that this region of the influenza B HA forms a surface structure different from the alpha-helix of the influenza A HA structure and that it accounts for much of the antigenic activity of influenza B HA.

Amino Acid Sequence↗
PubMed1995

A strategy for theoretical binding constant, Ki, calculations for neuraminidase aromatic inhibitors designed on the basis of the active site structure of influenza virus neuraminidase.

Neuraminidase (NA) is one of the two major surface antigens of influenza virus. It plays an indispensable role in the release and spread of progeny virus particles during infection. NA inhibitors reduce virus infection in animals. To improve the clinical efficacy of NA inhibitors, we have begun the design of non-carbohydrate inhibitors based on the active site structure of NA. The approach is an iterative process of ligand modeling and electrostatic calculations followed by chemical synthesis of compounds, biological testing, and NA-inhibitor complex structure determination by X-ray crystallography. A strategy has been developed to calculate Ki for newly designed inhibitors. The calculations using the DelPhi program were performed for carbohydrate inhibitors and three preliminary benzoic acid inhibitors of neuraminidase (BANA) that have been synthesized and shown to bind to the active site of NA in the crystal structure. The calculated Kis of these inhibitors have an enlightening agreement with their in vitro biological activities. This demonstrates that the calculations produce informative results on the affinity of modeled inhibitors. GRID maps were also calculated and several pockets were identified for accepting possible new ligands. The calculated Kis for newly designed ligands suggest that these potential compounds will have high inhibitory activities.

Aminobenzoates↗
PubMed1995

Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding.

We have used a neuraminidase-deficient influenza virus, NWS-Mvi, which was selected by supplying bacterial neuraminidase in the medium (C. Liu and G. M. Air, Virology 194:403-407, 1993), to define the role of neuraminidase in influenza virus replication. Electron microscopy showed that virions of the NWS-Mvi mutant assembled normally and formed large aggregates associated with cell surfaces. The NWS-Mvi virus grown in the absence of neuraminidase was able to carry out a second round of replication in MDCK cells without added neuraminidase, indicating that the virus particles contained in these aggregates were infectious. Aggregates of virus were also found in cytoplasmic vacuoles. When virus-infected cells were incubated in the presence of ferritin, such aggregates were found to be labeled with ferritin, indicating that they are derived from uptake at the cell surface. When the neuraminidase-deficient virus was administered intranasally to C57BL/6 mice, low titers of virus were recovered from the lungs and major histocompatibility complex class I-restricted cytotoxic T cells were generated: evidence that cells were infected in vivo. In C57BL/6 nu/nu mice, the low level of virus persisted for at least 28 days but never increased. These results suggest that neuraminidase is not required for influenza virus entry, replication, or assembly in cell culture or in mice.

Animals↗
PubMed1994

Structure of influenza virus neuraminidase B/Lee/40 complexed with sialic acid and a dehydro analog at 1.8-A resolution: implications for the catalytic mechanism.

Neuraminidase is one of the two glycoprotein spikes protruding from the influenza virus membrane. We have determined by X-ray crystallography the native structure of B/Lee/40 neuraminidase (NA) and the structures of its crystals soaked with a substrate, N-acetylneuraminyllactose (NANL), and an inhibitor, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) at 1.8-A resolution. NANL was hydrolyzed by the crystalline NA to generate the product N-acetylneuraminic acid (NANA, also known as sialic acid), which is still able to bind to NA. In the difference Fourier map of the presumed NA-NANA complex, the moiety bound in the active site had a distorted boat conformation of NANA, but there is no significant electron density for O2. The structure of the bound moiety is not identical to that of chemically synthesized DANA soaked into NA crystals. Prolonged incubation of NANA with NA in solution at room temperature produced only a trace amount of DANA as detected by NMR. On the basis of our studies, a mechanism is proposed for the enzymatic hydrolysis by influenza virus neuraminidase.

Binding Sites↗
PubMed1994

Expression of functional influenza virus A polymerase proteins and template from cloned cDNAS in recombinant vaccinia virus infected cells.

cDNAs containing the coding sequences of influenza type A virus polymerase proteins (PB1, PB2 and PA) and nucleoprotein (NP) have been expressed in mammalian cells by T7 polymerase provided by a recombinant vaccinia virus. The resulting proteins are able to form a complex that can copy a negative sense influenza-like RNA, transcribed from input DNA by the T7 polymerase, into a positive sense RNA that is translated into active chloramphenicol acetyltransferase (CAT). In this system there is no requirement for helper virus or purified viral core proteins, thus it will allow manipulation of all proteins as well as template for studies of replication in influenza virus.

Animals↗
PubMed1994

Defining the requirements for an antibody epitope on influenza virus neuraminidase: how tolerant are protein epitopes?

To determine the conformational requirements for antibody recognition and extent of flexibility within a protein epitope, a chimeric influenza A virus neuraminidase (NA) has been constructed in which five discontinuous polypeptide segments from a subtype N9 NA, which comprise the monoclonal antibody NC41 epitope, have been grafted onto a subtype N2 NA. The resulting chimeric NA was expressed, assembled as a tetramer, and transported to the cell surface, but was not recognized by NC41 in immunoprecipitation experiments or by surface immunofluorescence. Although the N2 and N9 protein folds are identical and this chimera contains all the antibody contacts as defined by the crystal structure of the complex, NC41 binding was not achieved. Modeling studies suggest that at least one polypeptide segment is displaced from its normal position which would account for the observed lack of enzyme activity as well as lack of antibody binding. This implies that in addition to the specific critical interactions between NA and Fab residues required for antibody binding, the overall arrangement of amino acids within an epitope must be in a specific orientation that is necessary for initial antibody recognition.

Amino Acid Sequence↗
PubMed1993

Three-dimensional structure of influenza A N9 neuraminidase and its complex with the inhibitor 2-deoxy 2,3-dehydro-N-acetyl neuraminic acid.

We present here the three-dimensional structure of neuraminidase (E.C. 3.2.1.18) from influenza virus A/Tern/Australia/G70c/75 (N9), determined by the method of multiple isomorphous replacement, and the structure of the neuraminidase complexed with an inhibitor, 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid (DANA). Native and inhibitor complex crystals are isomorphous and belong to space group I432 with unit cell dimensions of 183.78 A. The native enzyme structure and the inhibitor complex structure have been refined at 2.5 A and 2.8 A resolution, respectively, with crystallographic R-factor values of 0.193 for the native enzyme, and 0.179 for the inhibitor complex. The current enzyme model includes 387 amino acid residues which comprise the asymmetric unit. The root-mean-square deviation from ideal values is 0.013 A for bond lengths and 1.6 degree for bond angles. The neuraminidase (NA), as proteolytically cleaved from the virus, retains full enzymatic and antigenic activity, and is a box-shaped tetramer with edge lengths of 90 A and a maximal depth of 60 A. The NA tetramers are composed of crystallographically equivalent monomers related by circular 4-fold symmetry. Each monomer folds into six antiparallel beta-sheets of four strands. The secondary structure composition is 50% beta-sheet. The remaining 50% of the residues form 24 strand-connecting loops or turns. One of the loops contains a small alpha-helix. The structure of the complex of NA with DANA, a transition state analog, has enabled us to identify and characterize the site of enzyme catalysis. The center of mass of bound inhibitor is 32 A from the 4-fold axis of the tetramer, lodged at the end of a shallow crater of diameter 16 A with a depth of 8 to 10 A. There are 12 amino acid residues that directly bind DANA, with a further six conserved amino acids lining the active site pocket. The neuraminidase inhibitor complex provides a three-dimensional model which will be used to further the understanding of enzymatic hydrolysis and aid the design of specific, antineuraminidase antiviral compounds.

Binding Sites↗
PubMed1993

Identification of critical contact residues in the NC41 epitope of a subtype N9 influenza virus neuraminidase.

We have examined amino acids on influenza virus neuraminidase (NA) subtype N9 (A/tern/Australia/G70c/75) which are in contact with monoclonal antibody NC41 to analyze individual interactions important for antibody recognition. The crystal structure of NA complexed with NC41 Fab1 shows antibody contacts at 19 amino acid residues on the NA surface which are localized on five polypeptide loops surrounding the enzyme active site. Fifteen mutant NA genes were constructed to encode a protein which contained a single amino acid substitution and these were tested for effects of the replacement on NC41 binding. Our data revealed that NAs with changes at 368, 400, and 434 completely lost NC41 recognition. NAs with side chains replaced at residues 346 and 373 exhibited binding reduced to less than 50% of wild-type binding. Changes in seven other contacting residues, including substituted side chains which differed considerably from wild-type NA in size and charge, had no significant effect on NC41 binding. These results indicate that only a few of the many residues which make up an epitope are crucial for interaction and provide the critical contacts required for antibody recognition. This implies that antibody escape mutants are selected only if they contain changes at these crucial sites, or changes which introduce bulky side chains that sterically prevent antibody attachment.

Amino Acid Sequence↗
PubMed1993

Selection and characterization of a neuraminidase-minus mutant of influenza virus and its rescue by cloned neuraminidase genes.

A neuraminidase (NA)-deficient mutant, designated NWS-Mvi, of the reassortant influenza virus A/NWS/33HA-A/tern/Australia/G70c/75NA (H1N9), was selected by passaging virus in MDCK cells in a medium containing neuraminidase from the bacterium Micromonospora viridifaciens and polyclonal antiserum against the influenza NA. Growth of the resulting mutant virus is dependent on the addition of neuraminidase to the medium. Western blot analysis showed that the neuraminidase protein was absent from the mutant virus particles, and Northern hybridization showed that RNA segment 6, which contains the coding information for the NA, had undergone massive deletion. Viral protein synthesis in cells infected with the mutant virus was not dependent on the addition of neuraminidase. In the absence of a functional NA, the NWS-Mvi mutant virus can infect MDCK cells with normal cytopathic effects. This neuraminidase-minus influenza virus serves as an excellent source of parent virus for reverse genetics experiments involving genes that encode a functional neuraminidase.

Animals↗
PubMed1991

Transfer of the hemagglutinin activity of influenza virus neuraminidase subtype N9 into an N2 neuraminidase background.

It has previously been shown that influenza virus neuraminidase (NA) of the N9 subtype is unusual in that it possesses hemagglutinin activity as well as NA activity. Loss of red cell binding in certain escape mutants suggested that the hemagglutinating site is separate from the NA active site and involves at least two of the polypeptide loops found on the surface of the molecule (Webster et al., 1987. J. Virol. 61, 2910-2916). We have used site-directed mutagenesis to transfer the amino acids in these loops at positions 368-370 and 399-403 of N9 NA (A/tern/Australia/G70c/75), separately and together, into subtype N2 NA (A/Tokyo/3/67). The three mutant proteins were expressed from an SV40 transient expression system (Fuerst et al., 1986. Proc. Natl. Acad. Sci. USA. 83, 8122-8126). The mutant which contained both loops of N9 NA had acquired the hemagglutinin activity of N9. The agglutinated red cells are released by the enzyme activity of N9 NA, indicating that the agglutination involves binding to sialic acid in the same configuration as does the parental N9 NA, and an inhibitor of NA did not affect hemagglutination, indicating that this site is separate from the NA site as in parental N9.

Agglutination Tests↗
PubMed1990

New crystalline forms of neuraminidase of type B human influenza virus.

New crystalline forms of tetrameric neuraminidase heads from two strains (B/Lee/40 and B/mem/89) of type B human influenza virus were obtained and the crystals diffracted using X-rays to 2.5 A resolution without lattice disorder. The new B/Lee/40 crystalline form is tetragonal, space group P42(1)2, with unit cell dimensions a = 123.8 A, c = 71.8 A. The B/mem/89 crystalline form is also tetragonal, space group I422, with unit cell dimensions a = 122.9 A, c = 164.4 A. There is one neuraminidase monomer per asymmetric unit in both forms.

Crystallization↗
PubMed1990

Antigenic, sequence, and crystal variation in influenza B neuraminidase.

The neuraminidase (NA) genes of influenza B viruses B/Maryland/59, B/Hong Kong/8/73, B/Singapore/222/79, B/Oregon/5/80, B/USSR/100/83, B/Victoria/3/85, B/Leningrad/179/86, B/Memphis/6/86, and B/Memphis/3/89 have been sequenced. The deduced amino acid sequences show high variability in the stalk domain of the NA, but a surprising degree of sequence conservation in the head region which carries all the antigenic and enzyme activity. The variable region coding for the neuraminidase stalk also translates into a variable section in the overlapping NB polypeptide, which is coded from a second reading frame that overlaps the first 100 amino acids of NA. The influenza B NAs are antigenically distinguishable with monoclonal antibodies in neuraminidase-inhibition tests, even when there is only one amino acid sequence difference. However, seven of nine escape mutants selected with monoclonal antibodies were distinguished only by the antibody used for selection. When NA heads of influenza B viruses are crystallized, there are remarkable differences in crystal morphology between neuraminidases which have very few sequence changes.

Amino Acid Sequence↗
PubMed1990

Evolutionary changes in influenza B are not primarily governed by antibody selection.

Influenza B viruses evolve more slowly than human influenza A, but no reasons for the difference have been established. We have analyzed sequence changes in the hemagglutinin and neuraminidase of influenza B viruses (and have determined four hemagglutinin sequences, of B/Bonn/43, B/USSR/100/83, B/Victoria/3/85, and B/Memphis/6/86) in relation to antigenic properties and compared these with similar analyses of variation in influenza A antigens. Independent of the slower rate of change in influenza B antigens, only approximately 30% of nucleotide changes in either the hemagglutinin or neuraminidase gene sequence result in amino acid changes in the protein, whereas in influenza A 50% of nucleotide changes result in altered amino acids. Thus, there is less selection for change, or less tolerance to change, in the influenza B antigens. This is similar to findings with influenza C and findings with influenza A viruses that replicate in lower animals and birds and is closer to the type of variation found in other RNA viruses. We propose that human influenza A is unique in that it is the only virus group in which antibody selection dominates evolutionary change.

Antibodies, Viral↗