Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds.
The oligomeric structure of the influenza A virus M2 integral membrane protein was determined. On SDS-polyacrylamide gels under nonreducing conditions, the influenza A/Udorn/72 virus M2 forms disulfide-linked dimers (30 kDa) and tetramers (60 kDa). Sucrose gradient analysis and chemical cross-linking analysis indicated that the oligomeric form of M2 is a tetramer consisting of either a pair of disulfide-linked dimers or disulfide-linked tetramers. In addition, a small amount of a cross-linked species of 150-180,000 kDa, which the available data suggest contains only M2 polypeptides, was observed. The role of M2 cysteine residues in disulfide bond formation and their role in forming oligomers were examined by converting each of the two extracellular and single cytoplasmic cysteine residues to serine residues and expressing the altered M2 proteins in eukaryotic cells. Removal of either one of the N-terminal cysteines at residues 17 or 19 indicated that tetramers formed that consisted of a pair of noncovalently associated disulfide-linked dimers, suggesting that each of the cysteine residues is equally competent for forming disulfide bonds. When both cysteine residues were removed from the M2 N-terminal domain, no disulfide-linked forms were observed. When solubilized in detergent this double-cysteine mutant lost reactivity with a M2-specific mAb and exhibited an altered sedimentation pattern on sucrose gradients. However, chemical cross-linking of this double-cysteine mutant in membranes indicated that it can form tetramers. Taken together, these data suggest that disulfide bond formation, although not essential for oligomeric assembly, stabilizes the M2 tetramer from disruption by detergent solubilization.
Holsinger
L J
LJ
Department of Biochemistry, Molecular and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500.
Lamb
R A
RA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000502:physiology; D004220:Disulfides / Q000737:chemistry*; D005455:Fluorescent Antibody Technique; D009980:Influenza A virus / Q000032:analysis* / Q000235:genetics / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000086082:Viroporin Proteins
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D003545:Cysteine; D004220:Disulfides; D014763:Viral Matrix Proteins; C031540:M-protein, influenza virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins