THE CM2 PROTEIN OF INFLUENZA-C VIRUS IS AN OLIGOMERIC INTEGRAL MEMBRANE GLYCOPROTEIN STRUCTURALLY ANALOGOUS TO INFLUENZA-A VIRUS M-2 AND INFLUENZA-B VIRUS NB PROTEINS

We have undertaken a characterization of the CM2 protein of influenza C virus. The CM2 coding region of RNA segment 6 (nucleotides 731-1147) was cloned from two strains of influenza C virus and expressed using t he vaccinia virus-bacteriophage T7 RNA polymerase (vac-T7) system. An antiserum raised to a C-terminal peptide in the CM2 open reading frame recognized the CM2 protein in influenza C virus-infected cells and af ter vac-T7 expression of the CM2 open reading frame. CM2 is posttransl ationally modified by addition of high-mannose carbohydrate chains (M- r similar to 18 kDa) and by further addition of polylactosaminoglycans (M-r similar to 21-35 kDa). The available data indicate that CM2 has a cleavable signal peptide at the N-terminus of the protein. Site-dire cted mutagenesis eliminated the single potential N-linked carbohydrate attachment site on CM2 and indicated that the protein has an NoutCin orientation in membranes. Nonreducing SDS-PAGE indicated that the prot ein was expressed as disulfide-linked dimers and tetramers. Cell surfa ce biotinylation and indirect immunogluorescence showed the protein to be expressed at the cell surface. Elimination of the N-linked carbohy drate attachment site and addition of a C-terminal HA epitope tag did not adversely affect surface expression of CM2. The NoutCin membrane o rientation of CM2, the size of the ectodomain and cytoplasmic tail of CM2, and its ability to form disulfide-linked oligomers are reminiscen t of the structural properties of influenza A virus M-2 and influenza B virus NE proteins. (C) 1997 Academic Press.