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CM 2由RNA片段6（M基因）的未剪接mRNA产生的机制。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证明了三个AUG密码子中没有一个用于起始CM 2合成。我们最近发现了一种374氨基酸的蛋白质P42是一种完整的膜蛋白，含有两个内部疏水结构域，其中一（残基241至252）之后是两个序列（252 Ile-Thr-Ser和257 Ala-Ser-Ala）信号肽酶第二识别基序的消除阻止了CM 2蛋白在转染的Gas细胞和转染的Gas细胞中的表达。在微粒体膜存在下的体外翻译。从这些结果，我们得出结论，信号肽酶切割后的P42丙氨酸残基259产生CM 2的C-末端115个氨基酸组成的和M1'的N-末端259个氨基酸组成的。为了证明CM 2的离子通道活性，我们表达该蛋白在非洲爪蟾卵母细胞和全细胞电流的双电极电压钳程序。结果表明，超极化卵母细胞膜可诱导内向电流。在超极化脉冲过程中，电流幅度随时间缓慢增加，电流呈现内向整流特性，表明CM 2形成了电压激活的离子通道。我们还获得的数据表明，CM 2相关离子通道对pH变化的敏感性低于M2相关离子通道，并且对抗甲型流感病毒药物盐酸金刚烷胺的阻断具有抗性。

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）。