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

Abstract

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