Identification of Haemagglutinin-Neuraminidase Antibody Binding Sites by Western Blot Analysis of Antibody-resistant Mutants and Partial Digest Fragments of Newcastle Disease Virus

Samson, A. C. R.; Nesbit, Mark E.; Lyon, A. M.; Meulemans, G.

doi:10.1099/0022-1317-69-2-473

Cited by 9 publications

(7 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2, lanes 2 and 9). This peptide is also found in HN of NDV strains B1, AV, Italien, D26 and Ulster 2C (Jorgensen et al, 1987;McGinnes et al, 1987;Wemers et al, 1987;Sato et al, 1987;Millar et al, 1988), and is within the region identified previously (Samson et al, 1988). In contrast, recombinants with the oligonucleotide encoding the peptide YKRYNDTCP were negative on Western blots (Fig.…”

mentioning

confidence: 81%

“…MAb-resistant mutants have been isolated from the Beaudette C strain of NDV (Samson et al, 1985). Mutants raised against any of the antibodies that recognized reduced denatured HN were cross-resistant to the other antibodies that recognized reduced denatured HN (Samson et al, 1988). This suggests that the epitopes for these MAbs may comprise parts of one of the major antigenic sites on the HN protein detected by others (Iorio & Bratt, 1983;Nishikawa et al, 1983;Le Long et al, 1986).…”

mentioning

confidence: 99%

“…Several of these bind HN in Western blots when the structure of HN has been disrupted by boiling with SDS and 2-mercaptoethanol (Le Long et al, 1986;Samson, 1986;Samson et al, 1988). These antibodies may, therefore, recognize epitopes that are continuous or are readily regenerated by refolding after denaturation.…”

mentioning

confidence: 99%

“…The cloning and expression proceeded rapidly once the use of the ~-peptide as a carrier had been developed, and most DNA manipulations were performed on small scale plasmid preparations. The plasmids constructed to map the epitope for MAb 14 will be used to map epitopes of other MAbs to the NDV HN protein (Samson et al, 1988). This approach could, in theory, be applied to the location of continuous epitopes in any polypeptide and is not limited to analysing MAbs that neutralize viruses.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Location of a Neutralizing Epitope for the Haemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus

Chambers¹,

Nesbit

Yusoff³

et al. 1988

Journal of General Virology

Self Cite

View full text Add to dashboard Cite

SUMMARYThe binding site of a monoclonal antibody to the haemagglutinin-neuraminidase (HN) polypeptide of Newcastle disease virus (NDV) has been located. Complementary DNA or synthetic oligonucleotides corresponding to portions of the HN gene were cloned into the Escherichia coli vector pUC19 and fragments of the HN protein were thereby fused to the ~-peptide of fl-galactosidase. Western blot analysis of E. coli lysates containing expressed fragments of the HN cDNA or synthetic oligonucleotides identified an antibody-binding peptide (Asp-Glu-Gln-Asp-Tyr-Gln-Ile-Arg; amino acid residues 346 to 353). Nucleotide sequence analysis of an antibody-resistant mutant of NDV revealed a Glu (wild-type) to Lys (mutant) substitution within the above sequence. The methods described could be useful for the location of continuous epitopes of other polypeptides.

show abstract

mentioning

confidence: 81%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Location of a Neutralizing Epitope for the Haemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus

Chambers¹,

Nesbit

Yusoff³

et al. 1988

Journal of General Virology

Self Cite

View full text Add to dashboard Cite

show abstract

“…In addition, in this extended SDS-PAGE run, the HN protein of the MAb NHN-l-resistant mutant, Z18, migrated slightly faster than that of the wild-type virus (Mr 71K, lane 8), indicating a loss of a glycosylation site at epitope C1 (otherwise known as site IV;Gotoh et al, 1988). That the site of extra glycosylation in the F5 mutant is between amino acid residues 130 and 385 in the HN glycoprotein has been confirmed by chemical cleavage of the Asp-Probonds of the HN protein, followed by Western blotting against MAb 14 (Piszkiewicz et al, 1970;Samson et al, 1988) which shows that all partial digests of the HN protein from mutant F5 which contain the 130 to 385 fragment are shifted up in size compared with that of the wild-type (R. Prince, unpublished data).…”

mentioning

confidence: 95%

Effect of Amino Acid Substitutions on Glycosylation of the Haemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus Strain Beaudette C

Yusoff

Nesbit

Samson

et al. 1989

Journal of General Virology

Self Cite

View full text Add to dashboard Cite

SUMMARYThe nucleotide sequences of two monoclonal antibody-resistant mutant viruses predict changes from the wild-type in the number of potential glycosylation sites (Asn-X-Thr/Ser) in the mutant haemagglutinin-neuraminidase (HN) glycoproteins of the Beaudette C strain of Newcastle disease virus. The HN glycoproteins of these mutants, F5 and Z18, migrate either slower (F5) or faster (Z18) than that of the wild-type in SDS-PAGE. HN proteins synthesized in chick embryo fibroblasts following infection by either mutant or wild-type virus in the presence of tunicamycin (an inhibitor of glycosylation), comigrate on SDS-PAGE. These results confirm that the HN protein of the mutant virus, F5, has gained a glycosylation site at Asn(323)-Ser-Ser and that the conserved potential glycosylation site at Asn(481)-His-Thr is indeed glycosylated in the HN protein of the wild-type Beaudette C strain of Newcastle disease virus but is lost in that of the mutant virus, Z18.Three antigenic sites on the haemagglutinin-neuraminidase (HN) glycoprotein of the Newcastle disease virus (NDV) strain Beaudette C have recently been identified on the basis of competitive binding assay experiments using anti-HN monoclonal antibodies (MAbs). Direct RNA sequencing of the HN genes of mutant viruses resistant to various neutralizing anti-HN MAbs and comparison of the nucleotide sequences with that of the corresponding wild-type virus has led to the mapping of seven neutralizing epitopes within the HN protein of NDV (Yusoff et al., 1988). A schematic representation of the location of these epitopes on the hydropathy profile of the HN protein is presented in Fig. 1.Here we report the effect of amino acid substitutions on potential glycosylation sites at two of the epitopes, B3 and C1. MAbs NHN-6 and NHN-1 recognize the epitopes B3 and C1, respectively. Both antibodies exhibit haemagglutination inhibition (HI) and neutralization (NT) functions but do not inhibit neuraminidase activity when N-acetylneuramin lactose is used as substrate. MAb NHN-6, however, has very low HI and NT titres, which are approximately 32-fold lower than MAb NHN-1 (Yusoff et al., 1988).Two NDV mutants which are resistant to MAb NHN-6, F5 and F6, each have a mutation changing amino acid Pro(325) to either Ser (F5) or Leu (F6) at the B3 epitope. Thus, the mutation in F5 appears to create an additional potential glycosylation site ] at that position on the HN protein primary sequence whereas there would be no predicted change in the number of potential glycosylation sites in the F6 mutant. The HN protein of F5 was observed to migrate more slowly than that of the wild-type virus on SDS-PAGE. Another mutant, Z18 (resistant to MAb NHN-1 which recognizes the C1 epitope), has a mutation that changes amino acid Asn(481) to Asp resulting in the loss of the potential glycosylation site, Asn-0000-8828 © 1989 SGM

show abstract

Newcastle Disease Virus Variations

Russell

Samson

Alexander

1990

Virus Variability, Epidemiology and Control

View full text Add to dashboard Cite

Identification of Haemagglutinin-Neuraminidase Antibody Binding Sites by Western Blot Analysis of Antibody-resistant Mutants and Partial Digest Fragments of Newcastle Disease Virus

Cited by 9 publications

References 19 publications

Location of a Neutralizing Epitope for the Haemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus

Location of a Neutralizing Epitope for the Haemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus

Effect of Amino Acid Substitutions on Glycosylation of the Haemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus Strain Beaudette C

Newcastle Disease Virus Variations

Contact Info

Product

Resources

About