The sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza C virus CM2 protein

The sites for fatty acylation, disulphide bond formation and phosphorylatio n of influenza C virus CM2 were investigated by site-specific mutagenesis. Cysteine 65 in the cytoplasmic tail was identified as the site for palmitoy lation. Removal of one or more of three cysteine residues in the ectodomain showed that all of cysteines 1, 6 and 20 can participate in the formation of disulphide-linked dimers and/or tetramers, although cysteine 20 may play the most important role in tetramer formation. Furthermore, it was found t hat serine 78, located within the recognition motifs for mammary gland case in kinase and casein kinase I, is the predominant site for phosphorylation, although serine 103 is phosphorylated to a minor extent by proline-depende nt protein kinase. The effects of acylation and phosphorylation on the form ation of disulphide-linked oligomers were also studied. The results showed that, while palmitoylation has no role in oligomer formation, phosphorylati on accelerates tetramer formation without influencing dimer formation. CM2 mutants defective in acylation, phosphorylation or disulphide bond formatio n were all transported to the cell surface, suggesting that none of these m odifications is required for proper oligomerization. When proteins solubili zed in detergent were analysed on sucrose gradients, however, the mutant la cking cysteines 1, 6 and 20 sedimented as monomers, raising the possibility that disulphide bond formation, although not essential for proper oligomer ization, may stabilize the CM2 multimer. This was supported by the results of chemical cross-linking analysis, which showed that the triple-cysteine m utant can form multimers.