Influenza continues to be a regular epidemic, and occasionally pandemic, disease which ranks among the leading causes of morbidity and mortality throughout the world, particularly in the elderly and in individuals with heart and lung disease. Influenza viruses encode two small integral membrane proteins of similar and unusual structure, the M2 protein of influenza A virus and the NB glycoprotein of influenza B virus. The proposed research is based upon our demonstration that the M2 protein forms an ion channel and our finding that ion channel activity is associated with the NB protein. (1) We propose to study the structure- function relationship of the M2 protein by making measurements of ion channel activity (including specific activity) of site-directed mutants modified to render them insensitive to the antiviral drug amantadine or lacking in certain post-translational modifications. We also propose to create an active ion channel of the minimal size and to reconstitute ion channel activity in lipid bilayers. (2) The role of the ion channel activity of the M2 protein in the life-cycle of the influenza A virus will be characterized by measuring the pH-activation of the ion channel in mammalian cells that express the protein, comparing the pH-activation of the M2 proteins of various strains of influenza A virus having hemagglutinin (HA) molecules which undergo conformational changes to the acid state at different values of pH and by comparing the ion channel properties of M2 proteins derived from """"""""high yield"""""""" strains of influenza A virus. (3) The structure-function relationship of the NB-associated ion channel activity will be studied. We will compare the properties of the ion channel activity associated with the wild-type and various mutant forms of the NB protein to determine if the NB protein is itself an ion channel. The roles of carbohydrate addition, Ca2+ regulation and the polar residues in the transmembrane domain in the function of the NB protein will be investigated using analysis of site-specific mutants. The specific activity of the NB-associated ion channel activity will be measured, the minimal protein needed for channel activity will be determined, and the channel activity reconstituted in lipid bilayers. These studies will provide essential information about the role of the M2 and NB proteins that may help target them for drug action and will provide basic information upon which future structural (e.g., NMR and crystallographic) studies of these minimal channels can be based.
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