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RNA第6片段（M基因）未剪接mRNA产生CM2的机制。将突变引入到位于核苷酸732和749之间的假定的三个起始密码子中，表明这三个AUG密码子都不用于CM2合成的起始。我们最近发现了一个由未剪接的mRNA翻译而来的374个氨基酸的蛋白（命名为P42）。P42是一种完整的膜蛋白，包含两个内部疏水结构域，其中一个（残基241 - 252）后面有两个有利于信号肽酶切割的序列（252 Ile-Thr-Ser和257 Ala-Ser-Ala）。信号肽酶的第二个识别基序的消除阻止了CM2蛋白在转染的Gas细胞中表达和在微粒体膜存在下的体外翻译。根据这些结果，我们得出结论，信号肽酶在Ala残基259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，我们在非洲爪蟾卵母细胞中表达了CM2蛋白，并通过双电极电压钳法测量了全细胞电流。发现卵母细胞膜超极化后，向内电流被诱导。在超极化脉冲过程中，电流幅值随时间缓慢增加，电流表现出向内整流特征，表明CM2形成了电压激活离子通道。我们还获得的数据表明，cm2相关离子通道对pH变化的敏感性低于m2相关离子通道，并且抗抗甲型流感病毒药物盐酸金刚烷胺阻断。

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）。

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）。

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.: "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）。