ROLE OF INDIVIDUAL CYSTEINE RESIDUES IN THE PROCESSING AND ANTIGENICITY OF THE MEASLES-VIRUS HEMAGGLUTININ PROTEIN

The haemagglutinin (H) protein is the dominant envelope glycoprotein o f measles virus. The protein contains 13 cysteine residues among its 6 17 amino acids and all are located in its ectodomain. In previous stud ies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown t hat the absence of disulphide bonds impaired the capacity of the prote in to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of indi vidual cysteine residues to the folding of H protein into its native c onformation. Site-directed oligonucleotide mutagenesis was used to cre ate 13 mutants, each with a serine replacing a cysteine. The mutated g enes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic str ucture and intracellular processing properties of the mutant proteins reveal the following outcome. (i) Replacements of cysteine residues 13 9, 154, 188, 386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a m utant with a replaced cysteine residue 154 displayed modified migratio n properties. (ii) Alterations of cysteine residues 381 or 494 display ed a moderate effect on H protein properties. The two mutants expresse d discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex a nd failed to be transported to the cell surface. (iii) Substitutions o f cysteine residues 287, 300, 394, 579 or 583 resulted in a complete l oss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epit ope. No dimeric form of the proteins was observed and only high mannos e oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not oligomerize and were retained in the en doplasmic reticulum. In conclusion, cysteine residues 287, 300, 381, 3 94, 494, 579 and 583 appear to play a particularly critical role in th e antigenic structure and processing of the H molecules and they proba bly participate in the inter- or intramolecular disulphide bonding.