Search PubMed⌕ Search

Biomedical subjects

W G Laver

Publications and source records attributed to W G Laver.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1990

Mechanism of antigenic variation in an individual epitope on influenza virus N9 neuraminidase.

Monoclonal antibodies which inhibit influenza virus neuraminidase (NA) and which therefore indirectly neutralize virus infectivity bind to epitopes located on the rim of the active-site crater. The three-dimensional structure of one of these epitopes, recognized by monoclonal antibody NC41, has previously been determined (W. R. Tulip, J. N. Varghese, R. G. Webster, G. M. Air, W. G. Laver, and P. M. Colman, Cold Spring Harbor Symp. Quant. Biol. 54:257-263, 1989). Nineteen escape mutants of influenza virus A/tern/Australia/G70c/75 (N9) NA selected with NC41 were sequenced. A surprising restriction was seen in the sequence changes involved. Ten mutants had a Ser-to-Phe change at amino acid 372, and six others had mutations at position 367. No escape mutants with changes at 369 or 370 were found, although these mutations were selected with other antibodies and rendered the epitope unrecognizable by antibody NC41. Another N9 NA, from A/ruddy turnstone/NJ/85, which differs by 14 amino acids from the tern virus NA, still bound antibody NC41. Epitope mapping by selecting multiple escape mutants with antibody NC41 thus identified only three of the five polypeptide loops on NA that contact the antibody. Escape mutants selected sequentially with three different monoclonal antibodies showed three sequence changes in two loops of the NC41 epitope. The multiple mutants were indistinguishable from wild-type virus by using polyclonal rabbit antiserum in double immunodiffusion tests, but NA inhibition titers were fourfold lower. The results suggest that although the NC41 epitope contains 22 amino acids, only a few of these are so critical to the interaction with antibody that a single sequence change allows selection of an escape mutant. In that case, the variety of amino acid sequence changes which can lead to polyclonal selection of new epidemic viruses during antigenic drift might be very limited.

Amino Acid Sequence↗
PubMed1989

Three-dimensional structures of influenza virus neuraminidase-antibody complexes.

X-ray diffraction analysis of crystals of a monoclonal Fab fragment NC41 bound to a viral antigen, influenza virus neuraminidase, shows an epitope involving five surface loops of the antigen. In addition it reveals an unusual pairing pattern between the domains of light and heavy chains in the variable module of the antibody. We interpret this result to imply that association with antigen can induce changes in the quaternary structure of the Fab, through a sliding of domains at the variable light/variable heavy chains (VL-VH) interface. In addition, Fab binding has altered the conformation of some of the surface loops of the antigen. The structure of the NC10 Fab-neuraminidase complex has now also been solved. It binds an epitope that overlaps the NC41 epitope. In this structure, there is no electron density for the C-module of the Fab fragment, implying it is disordered in the crystal lattice. The implications of these, and other antibody-antigen structures, for immune recognition are discussed.

Antigen-Antibody Complex↗
PubMed1989

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.

Binding Sites↗
PubMed1989

Sialic acid is cleaved from glycoconjugates at the cell surface when influenza virus neuraminidases are expressed from recombinant vaccinia viruses.

Three different influenza virus neuraminidase (NA) genes have been subcloned into the vector pSC11 and expressed from the recombinant vaccinia viruses. These genes are from influenza viruses A/Tokyo/3/67 (N2); A/tern/Australia/G70c/75 (N9); and B/Hong Kong (HG)(NA of B/Lee/40). Cells infected with recombinants containing the NA gene express enzymatically active NA on the cell surface. The expressed protein results in the infected cells beings stripped of sialic acid, the receptor for influenza virus. This is not due to cleavage by NA from detached cells since at low multiplicity of infection only cells present at plaques are devoid of sialic acid. Thus NA is able to cleave sialic acid from neighboring glycoconjugates on the same membrane.

Cell Adhesion↗
PubMed1989

Crystallization of Sendai virus HN protein complexed with monoclonal antibody Fab fragments.

The hemagglutinin-neuraminidase (HN) protein of Sendai virus has been isolated from virus particles in a biologically active soluble form after removal by proteolytic digestion of the hydrophobic amino-terminal anchor sequence (S. D. Thompson, W. G. Laver, K. G. Murti, A. Portner, J. Virol., 62, 4653-4660, 1988). The soluble HN exists as both dimers and tetramers, and crystallization trials with each of these forms have so far yielded amorphous material. Dimers complexed with Fab fragments of a monoclonal antibody formed long needle crystals. So far, these are not suitable for X-ray diffraction analysis but the results suggest that HN molecules from paramyxoviruses, even if not crystallizable, may, when complexed with Fab fragments, in some cases yield crystals suitable for X-ray diffraction analysis.

Antibodies, Monoclonal↗
PubMed1988

Crystallization and preliminary X-ray analyses of two neuraminidases from influenza B virus strains B/Hong Kong/8/73 and B/Lee/40.

Crystals of neuraminidase heads from two different influenza B virus strains have been grown. Neuraminidase crystals of influenza B/Hong Kong/8/73 were grown from solutions of potassium phosphate. The crystals are tetragonal prisms, space group I422; the axes are a = 123 A and c = 165 A. Influenza B/Lee/40 neuraminidase crystals were grown from solutions of polyethylene glycol 4000. The crystals are tetragonal pyramids, space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2; the axes are a = 125 A and c = 282 A.

Crystallization↗
PubMed1988

Structure of an escape mutant of glycoprotein N2 neuraminidase of influenza virus A/Tokyo/3/67 at 3 A.

The three-dimensional structure of the membrane glycoprotein neuraminidase of an escape mutant of the influenza virus strain A/Tokyo/3/67 has been determined to 3 A (1 A = 0.1 nm) resolution by X-ray diffraction. The mutant virus, selected by growing the virus in the presence of a monoclonal antibody to the neuraminidase, is shown to have undergone a single amino acid change of lysine to glutamic acid at residue 368. The three-dimensional structure of the neuraminidase is identical with that reported for A/Tokyo/3/67, except for a purely local adjustment of the structure at position 368.

Amino Acid Sequence↗
PubMed1988

Protection against lethal influenza with neuraminidase.

The role of the neuraminidase in eliciting protection against a lethal influenza A virus [A/Ck/Penn/1370/83 (H5N2)] infection was investigated in chickens. Isolated N2 neuraminidase administered in adjuvant did not prevent infection but did prevent systemic spread of virus and death of chickens. N2 expressed in a recombinant vaccinia virus protected chickens when administered in adjuvant but was less effective when allowed to replicate and produce pox on the chicken's comb. Chickens vaccinated with isolated N2 in adjuvant or with inactivated H5N2 influenza virus were protected from clinical signs and death after challenge with A/Ck/Penn/1370/83 influenza virus. However, these animals were completely susceptible and died of infection with a heterologous subtype (H7N7) of influenza virus. The role of the different antigenic determinants of the N2 NA was investigated in chickens by passive transfer of monoclonal antibodies. Antibodies to antigenic determinants rimming the enzyme active center reduced disease signs in approximately half of the birds but did not significantly reduce virus levels. Antibodies to one of the two independent antigenic determinants that are distant from the enzyme active center were most effective at reducing virus replication and disease signs. This is surprising because antigenic variants could not be selected in vitro with these antibodies and suggests that they may facilitate clearance of virus. Antibodies to the other determinant that is located distally to the enzyme site were ineffective at providing protection.

Animals↗
PubMed1988

Purification, thioredoxin renaturation, and reconstituted activity of the three subunits of the influenza A virus RNA polymerase.

The virion-associated RNA polymerase and the structural nucleoprotein of influenza A virus were separated by sodium dodecyl sulfate/PAGE, electroblotted to a polyvinylidine membrane, and eluted with good recovery from the membrane. After renaturation by incubating with Escherichia coli thioredoxin, these proteins were active in a reconstituted in vitro transcription reaction with purified genomic RNAs. All four proteins (i.e., the three subunits of the RNA polymerase as well as the structural nucleoprotein) were required for activity. The RNA products were plus-strand, mRNA-sized species.

Bacterial Proteins↗
PubMed1988

Isolation of a biologically active soluble form of the hemagglutinin-neuraminidase protein of Sendai virus.

As a first step in establishing the three-dimensional structure of the Sendai virus hemagglutinin-neuraminidase (HN), we have isolated and characterized a potentially crystallizable form of the molecule. The sequence of HN, a surface glycoprotein, predicts a protein with an uncharged hydrophobic region near the amino terminus which is responsible for anchorage in the viral envelope. To avoid rosette formation (aggregation), which would preclude crystallization, this hydrophobic tail was removed from a membrane-free form of HN by proteolytic digestion. This digestion resulted in a single product with a molecular weight of about 10,000 less than native HN. N-terminal amino acid sequence analysis of cleaved HN (C-HN) indicated a single cleavage site at amino acid residue 131, resulting in a product consisting of the carboxyl-terminal 444 amino acids of HN. Functional analyses revealed that C-HN retained full neuraminidase activity and was able to bind erythrocytes, indicating that the N-terminal 131 residues were not necessary for these biological activities. Furthermore, this cleavage product retained the antigenic structure of intact HN, since monoclonal antibodies still bound to C-HN in enzyme-linked immunosorbent assay and Western (immuno-) blot analysis. Viewed by electron microscopy, the dimeric and tetrameric forms of intact HN form rosettes while C-HN maintains the oligomeric structure but no longer aggregates. Furthermore, the electron micrographs revealed a C-HN tetramer strikingly similar to the influenza virus neuraminidase in both size and gross structural features.

Animals↗
PubMed1988

Crystallization and preliminary X-ray analysis of type B influenza virus neuraminidase complexed with antibody Fab fragments.

Fab fragments from four different monoclonal antibodies have been complexed with influenza B virus neuraminidase (B/Lee/40) and the complexes have been crystallized. Three of the complex crystals are, so far, not suitable for X-ray diffraction studies, but the fourth (B/Lee/40 NA-B1Fab) forms large crystals which diffract X-rays to 3.0 A resolution. The crystals have a space group of F432, a = 441.21 A. Vm calculations show that the asymmetric unit contains two monomeric complexes.

Antibodies, Monoclonal↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1987

Distribution of sequence differences in influenza N9 neuraminidase of tern and whale viruses and crystallization of the whale neuraminidase complexed with antibodies.

Neuraminidase genes from A/tern/Australia/G70C/75 (H11N9) and A/whale/Maine/1/84 (H13N9) influenza viruses have been sequenced. Seventy-two nucleotide changes were found, 17 of which result in changes in the amino acid sequence of the neuraminidase; 3 in the stalk region and 14 in the heads. To our surprise, all of the sequence changes in the head region are located on the base of the neuraminidase tetramer, resulting in conservation of antigenic sites on top of the neuraminidase which vary extensively in human influenza virus neuraminidase. Whale N9 neuraminidase, like tern N9 neuraminidase, possesses high levels of hemagglutinating activity but, unlike the tern neuraminidase, failed to form large well-ordered crystals. However, when the neuraminidase was complexed with Fab fragments of monoclonal antibodies, which were made against the tern N9 neuraminidase, large crystals of the complexes were obtained which diffract X-rays to beyond 3 A.

Amino Acid Sequence↗