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W G Laver

Publications and source records attributed to W G Laver.

At least 73 records · Page 4Linked to original sources
PubMed1987

Crystals of antibodies complexed with influenza virus neuraminidase show isosteric binding of antibody to wild-type and variant antigens.

We describe here, for the first time, crystals of antibodies bound to a viral antigen which diffract X-rays to beyond 3 A. Crystals have been grown of Fab fragments of monoclonal antibody NC41, complexed with influenza virus neuraminidase (NA) of the N9 subtype and with a variant of N9 NA having a sequence change of Asn to Asp at position 331. This reduces, but does not abolish, the binding of NC41 antibody (in the case of another variant, Ser 371 to Leu, binding of NC41 antibody appears to be abolished). We are presenting data on the three-dimensional structure of these two complexes which indicates that NC41 antibody binds isosterically to the wild type and variant neuraminidase molecules.

Antibodies, Monoclonal↗
PubMed1987

Antigenic structure and variation in an influenza virus N9 neuraminidase.

We previously determined, by X-ray crystallography, the three-dimensional structure of a complex between influenza virus N9 neuraminidase (NA) and the Fab fragments of monoclonal antibody NC-41 [P. M. Colman, W. G. Laver, J. N. Varghese, A. T. Baker, P. A. Tulloch, G. M. Air, and R. G. Webster, Nature (London) 326:358-363, 1987]. This antibody binds to an epitope on the upper surface of the NA which is made up of four polypeptide loops over an area of approximately 600 A2 (60 nm2). We now describe properties of NC-41 and other monoclonal antibodies to N9 NA and the properties of variants selected with these antibodies (escape mutants). All except one of the escape mutants had single amino acid sequence changes which affected the binding of NC-41 and which therefore are located within the NC-41 epitope. The other one had a change outside the epitope which did not affect the binding of any of the other antibodies. All the antibodies which selected variants inhibited enzyme activity with fetuin (molecular weight, 50,000) as the substrate, but only five, including NC-41, also inhibited enzyme activity with the small substrate N-acetylneuramin-lactose (molecular weight, 600). These five probably inhibited enzyme activity by distorting the catalytic site of the NA. Isolated, intact N9 NA molecules form rosettes in the absence of detergent, and these possess high levels of hemagglutinin activity (W.G. Laver, P.M. Colman, R.G. Webster, V.S. Hinshaw, and G.M. Air, Virology 137:314-323, 1984). The enzyme activity of N9 NA was inhibited efficiently by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, whereas hemagglutinin activity was unaffected. The NAs of several variants with sequence changes in the NC-41 epitope lost hemagglutinin activity without any loss of enzyme activity, suggesting that the two activities are associated with separate sites on the N9 NA head.

Antibodies, Monoclonal↗
PubMed1986

Electron and X-ray diffraction studies of influenza neuraminidase complexed with monoclonal antibodies.

Complexes of influenza virus neuraminidase both with antigen-binding (Fab) fragments and with whole monoclonal antibody molecules have been crystallized. Uniformly thin platelet microcrystals suitable for structure analysis by electron diffraction, yielding reflections to approximately 4.3 A resolution, have been grown from one neuraminidase-Fab complex, that of N9 neuraminidase with 32/3 Fab, and thicker crystals of a second neuraminidase-Fab complex (N9 neuraminidase-NC35 Fab) diffract X-rays to approximately 4.0 A resolution. Electron microscope lattice images of microcrystals both of Fab and of immunoglobulin G complexed with neuraminidase have been interpreted in terms of negatively stained images of the respective individual complex protomers. The sites of binding of the antibodies to the antigen are consistent with the notion that single amino acid changes observed in monoclonal variants of neuraminidase occur in binding epitopes for the antibody used for their selection.

Antibodies, Monoclonal↗
PubMed1985

An 18-amino acid deletion in an influenza neuraminidase.

The genes coding for the neuraminidase (NA) enzyme in antigenic variants of influenza virus X-7(F1) (which contains the NA of A/RI/5+/57) are shorter than other N2 neuraminidase genes. When the parent X-7(F1) virus was cloned at limiting dilution prior to selection of variants with monoclonal antibodies, a minor component with a shorter neuraminidase gene was cloned out, and this deletion was retained in all the variants. Sequence analysis has shown that there is a single deletion, 54 nucleotides long, which occurs in the coding region of the NA. The 18 deleted amino acids are from the stalk region of the protein, which is a thin structure containing the four polypeptide chains of the tetrameric enzyme which separates the enzymatically and antigenically active "head" from the hydrophobic sequence embedded in the viral membrane. The deletion can be seen as a shortened stalk when rosettes of detergent-released NA are examined by electron microscopy, which confirms that the NA stalk is a highly extended structure. Although the more abundant hemagglutinin (HA) would probably extend beyond the shortened NA molecule on the surface of the virus, the NA head is still accessible to all of the available monoclonal antibodies to the different antigenic sites on the molecule. Enzymatic activity toward a small substrate molecule, N-acetyl neuraminyl lactose, is the same for both the parental X-7(F1) virus and the strain having the deleted NA; however, the latter virus is slower to cleave a large substrate, fetuin, and elutes from red cells at a reduced rate.

Amino Acid Sequence↗
PubMed1985

Immunochemistry of variants of influenza virus hemagglutinin and neuraminidase.

Influenza is impossible to control by vaccination because of variation in the two surface antigens of the virus, the hemagglutinin (HA) and neuraminidase (NA). This variation is caused by sequence changes in the genes coding these antigens and this article summarizes recent information on the structure of the HA and NA, the way in which these glycoproteins vary and the effects of the changes on the antigenic properties of the virus.

Amino Acid Sequence↗
PubMed1985

Location of antigenic sites on the three-dimensional structure of the influenza N2 virus neuraminidase.

Sequence analysis of the neuraminidase (NA) genes of influenza virus X-7(F1) and of 12 variants selected with monoclonal antibodies has been used to define in physical terms the antigenic structure of this NA, which was operationally established by R. G. Webster, L. E. Brown, and W. G. Laver (1984, Virology 135, 30-42). X-7(F1) is a reassortant virus containing the NA of the early Asian (H2N2) isolate A/RI/5+/57, and the results of antigenic and sequence analysis of X-7(F1) and of variants selected with monoclonal antibodies have been combined with a similar analysis of the A/Tokyo/3/67 NA (H2N2, M. R. Lentz, G. M. Air, W. G. Laver, and R. G. Webster (1984), Virology 135, 257-265) to obtain a model of antibody binding to N2 NAs. The selection process was biased, however, since only those monoclonal antibodies which inhibited NA activity could be used to select variants. Most of the changes in the variants selected with monoclonal antibodies occur in those parts of the polypeptide chain which encircle the enzyme active site pocket in the three-dimensional structure (P. M. Colman, J. N. Varghese, and W. G. Laver (1983), Nature (London) 303, 41-44). The results suggest that in general the antibody binds to a site on the NA which includes those amino acid side chains which are altered in monoclonal variants. There are, however, several aspects of the antigen-antibody interaction which are not easily explained, and which will probably only be fully elucidated by X-ray crystallographic analysis of NA-antibody complexes.

Amino Acid Sequence↗
PubMed1985

Gene and protein sequence of an influenza neuraminidase with hemagglutinin activity.

An influenza virus neuraminidase (NA) of the N9 subtype also has hemagglutinin (HA) activity (W. G. Laver, P. M. Colman, R. G. Webster, V. S. Hinshaw, and G. M. Air (1984), Virology 137, 314-323). To determine sequence relationships between this NA and other known NA and HA subtype sequences, and as a necessary step toward a complete structure determination, we have cloned a full-length copy of the coding sequence of the N9 NA of influenza virus A/tern/Australia/G70C/75 into the plasmid pUC9 using SalI linkers. The gene was sequenced by directed subcloning into the single-stranded phage vectors M13mp19 and M13mp18 and use of the dideoxy procedure. Most of the NA sequence was also obtained by direct protein sequencing of tryptic peptides. The N9 NA has 43 and 44% homology when compared to N1 or N2 sequences, respectively. There is no significant homology to any known HA sequence, or to the HN protein of the paramyxovirus SV5. Like the other NA molecules, the N9 NA is anchored in the membrane by an N-terminal hydrophobic region, from which biologically active heads can be released by pronase.

Amino Acid Sequence↗
PubMed1985

Recognition of influenza virus hemagglutinin by subtype-specific and cross-reactive proliferative T cells: contribution of HA1 and HA2 polypeptide chains.

The recognition of influenza virus hemagglutinin (HA) by T lymphocytes was examined by assaying the T cell proliferative response of influenza virus-primed T cells to purified HA of different influenza A subtypes or to isolated heavy (HA1) or light (HA2) polypeptide chains of the HA molecule. The proliferative response to HA was dependent on the activation of an Ly-1+2- subset of T cells and required the presence of nylon wool-adherent, radiation-resistant accessory cells. T cells from mice primed by infection with one strain of type A influenza virus cross-reacted with other purified HA not only of the same subtype as the priming virus but also of serologically distinct subtypes of influenza A (but not B) virus. The response of virus-primed T cells to the homologous HA or to HA of the same subtype was shown to involve recognition of determinants on both the HA1 and the HA2 chains. The recognition of HA of different subtype by cross-reactive T cells appeared to be directed predominantly to determinants on HA2. Because the antibody response to influenza virus HA is not cross-reactive between subtypes and is directed predominantly to determinants on HA1, the present results indicate that at least some of the determinants on HA recognized by T cells are different from those recognized by B cells and that the HA2 chain may be involved primarily in stimulation of T cell rather than B cell immunity.

Animals↗
PubMed1984

Antigenic and biological characterization of influenza virus neuraminidase (N2) with monoclonal antibodies.

Competitive radioimmunoassays using monoclonal antibodies established that the neuraminidase of A/RI/5+/57 (H2N2) influenza can be divided into four overlapping antigenic regions. Antigenic regions 1 and 4 are sufficiently far apart so that there was no competition between antibodies for these sites. Region 1 is conserved in neuraminidases from N2 viruses over a 10-year period, while the other regions changed antigenically during this time. The antibodies belonging to groups 2 and 3 completely inhibited catalytic activity on fetuin substrate, whereas antibodies in groups 1 and 4 inhibited weakly or not at all. Antigenic region 2 can be further divided into four overlapping areas (2a, 2b, 2c, and 2d) based on the reactivity patterns of monoclonal antibodies with antigenic variants, chemically modified neuraminidase, and the ability of the antibodies to inhibit enzyme activity of different molecular weight substrates. Previous studies [R. G. Webster, V. S. Hinshaw , and W. G. Laver (1982) Virology 117, 93-104; D. C. Jackson and R. G. Webster (1982) Virology 123, 69-77] characterized only region 2 of the neuraminidase molecule. Each of the monoclonal antibodies inhibited virus release from MDCK cells when incorporated in an agar overlay, and some antibodies in each group inhibited hemagglutination by intact virus, but only antibodies in group 2 neutralized virus in embryonated eggs and permitted selection of antigenic variants. The results indicate that antibodies to some antigenic sites on the neuraminidase may inhibit virus release more efficiently than others, depending on their relation to the enzyme active center. None of the monoclonal antibodies inhibited the hemolytic activity of viruses possessing N2. Based on antigenic mapping and biological properties of the monoclonal antibodies, a topographical map of the neuraminidase can be constructed. It is proposed that antigenic regions 1 and 4 are spacially separated and, based on their failure to inhibit biological activity, may be located on the bottom surface of the molecule; region 3 may be on the top surface of the molecule but at some distance from the catalytic center. Antigenic region 2 probably encompasses most of the top surface of the molecule; region 2d being closest to the enzyme center, with subregions 2a and 2b adjacent to it on the top surface. Chemical treatment of the neuraminidase with trinitrobenzenesulfonic acid (TNBS) causes modification of the 2b region, confirming the antigenic mapping results.

Animals↗
PubMed1984

Sequence of the neuraminidase gene of influenza virus A/Tokyo/3/67 and previously uncharacterized monoclonal variants.

A full-length cDNA copy of the neuraminidase (NA) gene of influenza strain A/Tokyo/3/67 was cloned into the plasmid pBR322, and the nucleotide sequence of the gene was determined. In addition, the sequence changes in six variants of A/Tokyo/3/67 selected with various monoclonal antibodies (Ab) to the NA were determined by dideoxy sequencing of the vRNA. In five of the monoclonal variants, a single change occurred, resulting in an amino acid substitution at residue 344. Arginine in the parent virus changed to every amino acid possible with a single nucleotide change. In another variant, arginine at position 253 changed to serine, a change that also occurred in field strains. All variants so far sequenced that were selected by monoclonal Ab to A/Tokyo/3/67 virus changed at position 344, except one which changed at residue 368. Both of these positions are in clusters of residues that vary considerably in field strains, the clusters being 344-347 and 368-370. Analysis of the three-dimensional crystal structure of the NA of A/Tokyo/3/67 shows that these clusters are directly adjacent on the protein, and likely comprise a single antigenic site. A total of three or four antigenic sites have been proposed for the NA protein, based on antigenic mapping with monoclonal Ab [R. G. Webster, V. S. Hinshaw , and W. G. Laver (1982) Virology 117, 93-104]. Variants selected by Ab to Tokyo/67 NA all change in this single antigenic site, whereas variants selected by Ab to other strains change in other regions. It is possible that, although there may be three or four antigenic sites on the NA molecule, there may be a single, dominant antigenic site for each strain.

Amino Acid Sequence↗
PubMed1984

Influenza virus neuraminidase with hemagglutinin activity.

Isolated intact influenza virus neuraminidase (NA) molecules of the N9 subtype have been found to possess hemagglutinin (HA) activity which, at equivalent protein concentration, was fourfold higher than that of isolated hemagglutinin molecules of the H3 subtype. The amino-terminal sequence of the N9 NA is the same as in neuraminidases of the eight other influenza A virus NA subtypes previously reported. Viruses possessing N9 NA therefore have two different HA activities and antibody to either HA or NA alone was incapable of inhibiting hemagglutination by the virus. However, antibody to the HA of an H1N9 virus neutralized its infectivity as effectively as it neutralized H1N1 or H1N2 viruses whose neuraminidases have no HA activity. (Antibodies to N9 NA did not neutralize the infectivity of viruses with N9 neuraminidase). 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid inhibited N9 NA activity but had no effect on the HA activity of the isolated N9 NA. One interpretation of this result would be that the HA and NA activities are located in separate sites. Pronase-released N9 NA heads form crystals suitable for X-ray diffraction studies and preliminary data to 2.9 A establish the space group as cubic, I432 with cell dimension a = 184 A. Data extend to beyond 1.9 A resolution, and these will be collected in the future.

Animals↗
PubMed1984

From the National Institutes of Health. Summary of a meeting on the origin of pandemic influenza viruses.

Influenza type A virus periodically undergoes major antigenic shifts in which the hemagglutinin (HAG) and sometimes the neuraminidase (NA) antigens are replaced by HAG and NA antigens of another subtype. Three such shifts have taken place since the virus was first isolated, and all appear to have occurred in China. The way in which these "new" influenza type A viruses suddenly appear (or reappear) in the human population is not known. At a meeting held in Beijing, China, on November 10-12, 1982, participants discussed the latest findings on the molecular biology of influenza viruses and on aspects of their ecology that may offer insight into the factors responsible for the origin of pandemic influenza viruses. Information obtained in earlier studies has provided some clues about how the antigenic shifts may occur. For example, the H3N2 virus has been found to be a recombinant deriving seven of its eight genes from an H2N2 strain and gene 4 (which encodes for the HAG) from some other virus, possibly an avian influenza virus of the H3 subtype [1-3]. In addition, studies of the genome of the H1N1 virus that appeared in Anshan, China, in 1977 have shown that this virus almost certainly underwent no replication for 27 years. This finding suggests that the virus existed in an animal reservoir during this period [4, 5].

Animals↗
PubMed1984

Antigenic variation and the structure of influenza virus glycoproteins.

Influenza is impossible to control by vaccination because of variation in the two surface antigens of the virus, the haemagglutinin (HA) and neuraminidase (NA). This variation is caused by sequence changes in the genes coding these antigens and this review summarizes recent information on the structure of the HA and NA, the way in which these glycoproteins vary and the effects of the changes on the antigenic properties of the virus.

Amino Acid Sequence↗
PubMed1983

Sequence of the hemagglutinin gene of influenza virus A/Memphis/1/71 and previously uncharacterized monoclonal antibody-derived variants.

A cDNA copy of the hemagglutinin (HA) gene of Mem/1/71HA Bel/42NA was cloned into the plasmid pBR322. The cloned gene contained the coding information for all of the HA except for the hydrophobic membrane insertion sequence of the C-terminus of HA2. The cloned HA DNA was used to make HA-specific primers from restriction fragments for internal priming on the viral RNA. These primers were used to sequence the complete HA gene of Mem/1/71, and to determine nucleotide sequence changes in four previously uncharacterized monoclonal antibody generated variants of Mem/1/71 (Webster, Laver, Air, Ward, Van Wyke, and Gerhard, Ann. N.Y. Acad. Sci. 354, 142-161, 1980).

Antibodies, Monoclonal↗
PubMed1983

The disulphide bonds of an Asian influenza virus neuraminidase.

The arrangement of the disulphide bonds in the pronase-released neuraminidase heads of the Asian influenza virus A/Tokyo/3/67 have been examined by cyanogen bromide fragmentation, enzymic digestion and diagonal peptide mapping. There are 9 intrachain disulphide bridges and one interchain bridge which links pairs of monomers at the distal end of the stalk region of the neuraminidase tetramer. The disulphide bond arrangements of the remaining 3 half-cystine residues in the membrane-embedded stalk region of the neuraminidase were not examined.

Amino Acid Sequence↗