The biosynthesis mechanism of influenza C virus CM2 protein and its ion channel activity

研究了RNA片段6(M基因)未剪接的mRNA产生CM2的机制。将突变引入位于核苷酸732和749之间的假定的三个起始密码子中，表明这三个AUG密码子中没有一个用于启动CM2的合成。我们最近发现了一个374个氨基酸的蛋白质(命名为P42)，它是由未剪接的mRNA翻译而来的。P42是一个完整的膜蛋白，含有两个内部疏水结构域，其中一个残基(241到252个残基)后面跟着两个序列(252个Ile-Thr-Ser-Ser和257个Ala-Ser-Ala)，有利于信号肽的切割。信号肽酶第二识别基序的消除阻止了CM2蛋白在转基因的Gas细胞中表达和在微体膜存在的情况下进行体外翻译。根据这些结果，我们得出结论，在ALA残基259之后，信号肽酶对P42的切割产生了由C端115个氨基酸组成的CM2和由N端259个氨基酸组成的M1‘。为了证明CM2的离子通道活性，我们在非洲爪哇卵母细胞中表达了CM2蛋白，并用双电极电压钳方法测量了整个细胞的电流。发现内向电流是在卵母细胞膜超极化时产生的。在超极化脉冲作用下，电流幅值随时间缓慢增加，呈现内向整流特征，提示CM2形成电压激活的离子通道。我们还获得的数据表明，CM2相关离子通道对pH变化的敏感性低于M2相关离子通道，并且不能被抗甲型流感病毒药物盐酸金刚烷胺阻断。

The mechanism by which CM2 is produced from unspliced mRNA of RNA segment 6(M gene) was investigated. The introduction of the mutations.into the putative three initiation codons located between nucleotides 732 and 749 demonstrated that none of the three AUG codons are used for the initiation of CM2 synthesis. We recently identified a 374-amino-acid protein (designated P42) which is translated from the unspliced mRNA.P42 is an integral membrane protein containig two internal hydrophobic domains, one of which (residues 241 to 252) is followed by two sequences (252 Ile-Thr-Ser and 257 Ala-Ser-Ala) favorable for cleavage by signal peptidase.The elimination of the second recognition motif for signal peptidase prevented CM2 protein from expressing both in the transfected Gas cells and in the translation in vitro in the presence of microsomal membranes. From these results, we conclude that cleavage of P42 by signal peptidase after Ala residue 259 produces CM2 composed of C-terminal 115 amino acids and M1' composed of N-terminal 259 amino acids.To demonstrate the ion channel activity of CM2, we expressed this protein in oocytes of Xenopus laevis and measured whole cell currents by a two-electrode voltage-clamp procedure. It was found that the inward currents were induced upon hyperpolarizing the oocyte membranes. The amplitude of the currents increased slowly with time during the hyperpolarizing pulse, and the currents showed inward rectification characteristics, suggesting that CM2 forms a voltage-activated ion channel. We also obtained data indicating that the CM2-associated ion channel is less sensitive to change in pH than the M2-associated ion channel, and is resistantto block by anti-influenza A virus drug amantadine hydrochloride.