Studies on the ion channel activity of influenza C virus CM2 protein

采用定点突变的方法对丙型流感病毒CM 2的脂肪酰化、二硫键形成和磷酸化位点进行了研究。胞质尾区的半胱氨酸65被确定为棕榈酰化位点。去除胞外域中三个半胱氨酸残基中的一个或多个表明，所有半胱氨酸1、6和20都可以参与二硫键连接的二聚体和/或四聚体的形成，尽管半胱氨酸20可能在四聚体形成中发挥最重要的作用。此外，发现位于乳腺酪蛋白激酶和酪蛋白激酶I的识别基序内的丝氨酸78是磷酸化的主要位点，尽管丝氨酸103在较小程度上被脯氨酸依赖性蛋白激酶磷酸化。酰化和磷酸化对二硫键连接的寡聚体的形成的影响也进行了研究。结果表明，虽然棕榈酰化在低聚物形成中没有作用，但磷酸化加速了四聚体的形成，而不影响二聚体的形成。CM 2突变体在酰化、磷酸化或二硫键形成方面有缺陷，都被转运到细胞表面，这表明这些修饰中没有一个是适当寡聚化所必需的。当蛋白质溶解在洗涤剂上分析蔗糖梯度，然而，缺乏半胱氨酸1，6和20的突变体作为单体沉降，提高了二硫键形成的可能性，虽然不是必要的适当的寡聚化，可能稳定的CM 2多聚体。化学交联分析的结果支持了这一点，表明三重半胱氨酸突变体可以形成多聚体。

The sites for fatty acylation, disulfide bond formation and phosphorylation of influenza C virus CM2 were investigated by site-specific mutagenesis. Cysteine 65 in the cytoplasmic tail was identified as the site for palmitoylation. 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 disulfide- linked dimers and/or tetramers, although cysteine 20 may play the most important role in tetramer formation. Furthermore, it was found that serine 78, located within the recognition motifs for mammary gland casein kinase and casein kinase I, is the predominant site for phosphorylation, although serine 103 is phosphorylated to a minor extent by proline -dependent protein kinase. The effects of acylation and phosphorylation on the formation of disulfide-linked oligomers were also studied. The results showed that, while palmitoylation has no role in oligomer formation, phosphorylation accelerates tetramer formation without influencing dimer formation. CM2 mutants defective in acylation, phosphorylation or disulfide bond formation were all transported to the cell surface, suggesting that none of these modifications is required for proper oligomerization. When proteins solubilized in detergent were analysed on sucrose gradients, however, the mutant lacking cysteines 1, 6 and 20 sedimented as monomers, raising the possibility that disulfide bond formation, although not essential for proper oligomerization, may stabilize the CM2 multimer. This was supported by the results of chemical cross-linking analysis which showed that the triple cysteine mutant can form multimers.