Role of individual cysteine residues in the processing and antigenicity of the measles virus haemagglutinin protein

Abstract

The haemagglutinin (H) protein is the dominant envelope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide bonds impaired the capacity of the protein to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of individual cysteine residues to the folding of H protein into its native conformation. Site-directed oligonucleotide mutagenesis was used to create 13 mutants, each with a serine replacing a cysteine. The mutated genes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic structure and intracellular processing properties of the mutant proteins reveal the following outcome. (i) Replacements of cysteine residues 139, 154, 188,386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a mutant with a replaced cysteine residue 154 displayed modified migration properties. (ii) Alterations of cysteine residues 381 or 494 displayed a moderate effect on H protein properties. The two mutants expressed discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex and failed to be transported to the cell surface. (iii) Substitutions of cysteine residues 287, 300, 394, 579 or 583 resulted in a complete loss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epitope. No dimeric form of the proteins was observed and only high mannose oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not oligomerize and were retained in the endoplasmic reticulum. In conclusion, cysteine residues 287, 300, 381,394, 494, 579 and 583 appear to play a particularly critical role in the antigenic structure and processing of the H molecules and they probably participate in the inter-or intramolecular disulphide bonding.

Characterization of a Region of the Measles Virus Hemagglutinin Sufficient for Its Dimerization

“…A mutational analysis in which each of the 13 cysteine residues in the MV H protein was individually replaced with serine was interpreted as suggesting that cysteines 287, 300, 381, 394, 494, 579, and 583 may be important for dimerization (14). However, the H proteins mutated in these cysteine residues lost all ability to bind conformation-dependent antibodies, indicating that a loss in overall conformational structure, rather than loss of a specific intermolecular interaction, may account for their inability to dimerize.…”

Genetic Characterization of the Hemagglutinin Genes of Wild-Type Measles Virus Circulating in China, 1993–2009

“…The H protein contains 13 cysteine residues, seven of which are located at amino acid positions 287, 300, 381, 394, 494, 579, 583. These amino acids play a critical role in maintaining the antigenic structure of the H protein [18]. A few linear neutralizing epitopes have been identified using monoclonal antibodies [19], [20].…”

Molecular Characterization of Wild Type Measles Virus from Adult Patients in Northern China, 2014

“…30 Seven of the 13 cysteine residues (amino acid positions 287, 300, 381, 394, 494, 579 and 583) play a pivotal role in maintaining the antigenic structure of the H protein. 31 Additionally, the H protein contains the binding sites for MeV cellular receptors including Signaling Lymphocyte Activation Molecule (SLAM, CD150), CD46 and nectin-4. [32][33][34][35] The HNE epitope has been shown to be highly stable in both vaccine as well as wild type strains.…”