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Characterization of a protein candidate for the ion channel of influenza C virus and its expression.

丙型流感病毒离子通道候选蛋白的表征及其表达。

基本信息

批准号：
07670337
负责人：
HONGO Seiji
金额：
$ 1.41万
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：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670337/
关键词：
Influenza C virus M gene CM2 ion channel

项目摘要

The biochemical properties of a second protein (CM2) encoded by RNA segment 6 of influenza C virus were investigated.1.Three forms of CM2 with different electrophoretic mobilities (CM2_0, CM2a and CM2b) were detected in infected cells by immunoprecipitation with antiserum to GST-CM2 fusion protein. 2.A mannose-rich oligosaccharide core is added to unglycosylated CM2_0 (Mr-16,000) to form CM2a (Mr-18,000) and the processing of the carbohydrate chain from high mannose type to complex one converts CM2a into CM2b heterogeneous in electrophoretic mobility (Mr-22,000 to 30,000). 3.Labeling of infected cells with [^3H] palmitic acid showed that CM2 is fatty acylated. The fatty acid bond was sensitive to treatment with hydroxylamine and mercaptoethanol, which indicates a labile thioester-type linkage. 4.The CM2 protein was also found to form disulfide-linked dimers and tetramers on SDS-PAGE under nonreducing conditions. 5.Trypsin treatment of infected cell surface as well as that of microsome vesicles from infected cells followed by immunoprecipitation with antiserum to the GST fusion protein containing the 56 C-terminal amino acid residues of CM2 suggested that this domain is intracellular and exposed on the cytoplasmic side of microsomes. 6.A small amount of CM2 is incorporated into progeny virus particles.These results, altogether, suggest that CM2 is an integral membrane protein with biochemical properties similar to those of influenza A virus M2 and influenza B virus NB proteins.

研究了丙型流感病毒RNA第6片段编码的第二蛋白（CM2）的生化特性。采用GST-CM2融合蛋白抗血清免疫沉淀法在感染细胞中检测到具有不同电泳迁移率的3种CM2 （CM2_0、CM2a和CM2b）。2.在未糖基化的CM2_0 （Mr-16,000）中加入一个富含甘露糖的低聚糖核，形成CM2a (Mr-18,000)，碳水化合物链从高甘露糖型到复合型的加工将CM2a转化为电泳迁移率不均匀的CM2b （Mr-22,000至30,000）。3.用[^3H]棕榈酸标记感染细胞显示CM2是脂肪酰化的。脂肪酸键对羟胺和巯基乙醇敏感，表明是一种不稳定的硫酯型键。4.在非还原条件下，还发现CM2蛋白在SDS-PAGE上形成二硫化物连接的二聚体和四聚体。5.用胰蛋白酶处理受感染细胞表面和受感染细胞的微粒体囊泡，然后用抗血清免疫沉淀GST融合蛋白，其中含有CM2的56个c端氨基酸残基，表明该结构域位于细胞内，暴露在微粒体的细胞质侧。6.少量的CM2被纳入子代病毒颗粒。综上所述，CM2是一种完整的膜蛋白，具有与甲型流感病毒M2和乙型流感病毒NB蛋白相似的生化特性。