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

• 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

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.

研究了由RNA段6（M Gene）的未贴合的mRNA产生CM2的机制。突变的引入。设置位于核苷酸732和749之间的推定的三个起始密码子，表明三个AUG密码子都没有用于启动CM2合成。我们最近确定了一种374-氨基酸蛋白（指定的p42），该蛋白是从未填充的mRNA.p42中翻译的。P42是一种积分的膜蛋白包含两个内部疏水结构域，其中一个（残基241至252）（残基241至252）随后是两个序列（252 ile-thr-thr-thr-thr-ser-thr-ser-ser-ser-ser-sera-sera sera-sera sera-sera sera-sera sera fere pect in firmitions），这是一个良好的pecteration。信号肽酶的基序可防止CM2蛋白在转染的气体细胞和体外翻译中同时表达在微粒体膜的情况下。从这些结果中，我们得出结论，Ala残基259后信号肽酶对p42的切割产生由C末端115氨基酸组成的CM2和由N末端259氨基酸组成的M1组成，以证明CM2的离子通道活性。 程序。发现在超极化卵母细胞膜上诱导了内流电流。在超极化脉冲期间，电流的幅度随时间缓慢增加，并且电流显示出向内的整流特性，表明CM2形成了电压激活的离子通道。我们还获得了数据，表明与M2相关的离子通道相比，与CM2相关的离子通道对pH的变化不太敏感，并且通过抗Imfluenza A抗盐酸盐抗体阻滞了病毒药物盐酸盐。

项目成果

Hongo S.: "Characterization of a second protein (CM2) encoded by RNA segment 6 of influenza C virus." J Virol. 71・4. 2786-2792 (1997)

Hongo S.：“丙型流感病毒 RNA 片段 6 编码的第二种蛋白质 (CM2) 的特征”，J Virol 71·4 (1997)。

Kimura H.: "Interspecies transmission of influenza C virus between humans and pigs." Virus Res. 48・1. 71-79 (1997)

Kimura H.：“人类和猪之间的丙型流感病毒的跨物种传播”，病毒研究 48・1（1997）。

Tada Y.: "Phosphorylation of influenza C virus CM2 protein" Virus Res. 58・1. 65-72 (1998)

Tada Y.：“丙型流感病毒CM2蛋白的磷酸化”病毒研究58・1（1998）。

Hongo S.: "Identification of a 374 amino acid protein encoded by RNA segment 6 of influenza C virus" J Gen Virol. 79・9. 2207-2213 (1998)

Hongo S.：“丙型流感病毒RNA片段6编码的374个氨基酸的蛋白质的鉴定”J Gen Virol 79·9（1998）。

Hongo S.: "Influenza C virus CM2 protein is produced from a 374-amino-acid protein (P42) by signal peptidase cleavage" J Virol. 73・1. 46-50 (1999)

Hongo S.：“丙型流感病毒 CM2 蛋白是通过信号肽酶切割从 374 个氨基酸的蛋白（P42）中产生的”J Virol 73·1（1999）。