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

采用定点诱变方法研究了C型流感病毒CM2的脂肪酰化位点、二硫键形成位点和磷酸化位点。细胞质尾部的半胱氨酸65被确定为棕榈酰化位点。去除三种半胱氨酸残基中的一种或多种，表明所有的半胱氨酸1、6和20都可以参与二硫连接二聚体和/或四聚体的形成，尽管半胱氨酸20可能在四聚体的形成中起最重要的作用。此外，发现丝氨酸78位于乳腺酪蛋白激酶和酪蛋白激酶I的识别基序内，是磷酸化的主要位点，尽管丝氨酸103被脯氨酸依赖性蛋白激酶磷酸化的程度较小。还研究了酰基化和磷酸化对二硫连接低聚物形成的影响。结果表明，棕榈酰化在低聚物的形成中没有作用，而磷酸化在不影响二聚体形成的情况下加速了四聚体的形成。在酰化、磷酸化或二硫键形成方面有缺陷的CM2突变体都被转运到细胞表面，这表明这些修饰都不需要适当的寡聚化。然而，当溶解在洗涤剂中的蛋白质在蔗糖梯度上分析时，缺乏半胱氨酸1、6和20的突变体作为单体沉积，这提高了二硫键形成的可能性，尽管对适当的寡聚化不是必需的，但可以稳定CM2多聚体。化学交联分析结果表明，三倍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.