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

丙型流感病毒CM2蛋白的生物合成机制及其离子通道活性

基本信息

批准号：
09670307
负责人：
HONGO Seiji
金额：
$ 1.79万
依托单位：
YAMAGATA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670307/
关键词：
Influenza C virus M gene CM2 ion channel amantadine シグナルペプチダーゼ

项目摘要

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.

研究了RNA片段6（M基因）的未剪接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证明这三个AUG密码子中没有一个用于起始CM2合成。我们最近鉴定了一种由未剪接的 mRNA 翻译而来的 374 个氨基酸蛋白（命名为 P42）。P42 是一种完整膜蛋白，包含两个内部疏水结构域，其中一个（残基 241 至 252）后面跟着两个序列（残基 252）。 Ile-Thr-Ser 和 257 Ala-Ser-Ala) 有利于信号肽酶的切割。消除第二个识别基序信号肽酶阻止 CM2 蛋白在转染的气细胞中表达以及在存在微粒体膜的体外翻译中表达。从这些结果中，我们得出结论，信号肽酶在 Ala 残基 259 之后切割 P42，产生由 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，我们表达非洲爪蟾卵母细胞中的这种蛋白质，并通过两电极电压钳程序测量了全细胞电流。研究发现，卵母细胞膜超极化时会诱发内向电流。超极化脉冲期间电流幅度随时间缓慢增加，且电流呈现内向整流特性，表明CM2形成电压激活离子通道。我们还获得的数据表明，CM2 相关离子通道对 pH 值变化的敏感性低于 M2 相关离子通道，并且对抗甲型流感病毒药物盐酸金刚烷胺的阻断具有抵抗力。