role of the individual gysteine residues in the formation of the mature, antigenic HN protein of Newcastel disease virus

The amino acid sequence of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV) has 14 cysteine residues, two of which are variably present in the sequences of the HN proteins of different strains of NDV while the rest are absolutely conserved. The role of each residue in the formation of the mature, oligomeric structure of the HN protein was assessed by characterizing proteins with mutations in each of the cysteine residues by Western analysis, immunoprecipitation with conformationally sensitive antibodies, immunofluorescence, and sedimentation on sucrose gradients. Proteins with mutations in the first cysteine (amino acid 6) or the second cysteine (amino acid 123), the nonconserved cysteine residues, formed antigenically mature oligomers which were transported to the cell surface like wild type. Protein with a mutation at cysteine 2 did not, however, form covalently linked oligomers demonstrating that it is this residue that is responsible for intermolecular disulfide bonds in the mature oligomer. Proteins with mutations in cysteine 3 (amino acid 172) or cysteine 5 (amino acid 196) formed proteins with all antigenic sites except one, site 23. These mutant proteins formed disulfide-linked dimers and were efficiently transported to the cell surface. They did not, however, sediment on gradients like the wild-type protein. They were also defective in the biological activities associated with the wild-type protein. Proteins with mutations in cysteines 4 (amino acid 186), 6 (amino acid 238), 7 (amino acid 247), 8 (amino acid 251), 13 (amino acid 531), or 14 (amino acid 542) contained no mature antigenic sites but formed noncovalently linked oligomers. Proteins with mutations in cysteines 9 (amino acid 344), 10 (amino acid 455), 11 (amino acid 461), or 12 (amino acid 465) formed proteins with antigenic site 4 but no other mature antigenic sites. These mutant proteins also formed noncovalently linked oligomers. These results suggest that mutations in different cysteine residues block the maturation of the HN protein at different stages.