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

对丙型流感病毒RNA片段6编码的第二种蛋白(CM2)的生化性质进行了研究。1.用GST-CM2融合蛋白抗血清免疫沉淀，在感染细胞中检测到三种不同电泳率的CM2(CM2_0、CM2a和CM2b)。2.在未糖化的CM2_0(MR-16,000)上加一个富含甘露糖的低聚糖核，形成CM2a(MR-18,000)，糖链从高甘露糖型向复合型转变，使CM2a转变为凝胶迁移率不均一的CM2b(MR-22,000~30,000)。3.用[~3H]棕榈酸标记感染细胞，结果表明CM2是脂肪酰化的。脂肪酸键对羟胺和巯基乙醇处理敏感，这表明它是一个不稳定的硫代酯型键。4.在非还原条件下，SDS-PAGE显示CM2蛋白可形成二硫键连接的二聚体和四聚体。5.用胰酶处理感染细胞表面和感染细胞的微泡，然后用抗血清免疫沉淀含有CM2的56个C-末端氨基酸残基的GST融合蛋白，表明该结构域位于细胞内，暴露在微粒体的细胞质一侧。6.在子代病毒颗粒中存在少量的CM2，这些结果表明CM2是一种完整的膜蛋白，其生化性质与甲型流感病毒M2和乙型流感病毒NB蛋白相似。

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.