Influenza contributes substantially to worldwide morbidity and mortality, and it has been estimated that the next influenza pandemic will result in over 80,000 deaths, 300,000 hospitalizations and 18 million outpatient visits in the United States. The M2 ion channel protein of influenza A virus is the target of the antiviral drug rimantadine, but its usefulness is limited by the formation of rimantadine-resistant escape mutations within a few days of treatment. In order to develop more effective antiviral drugs, more information is needed about the M2 protein, especially the structure of its transmembrane pore and cytoplasmic domains and the mechanism by which it is activated. The following individual aims are designed to elucidate important features of the structure and function of the M2 protein. 1. Tryptophan 41 in the transmembrane pore is essential for activation of the M2 ion channel protein; we will perform functional and structural experiments to ascertain the role of tryptophan 41 in activation of the M2 protein. 2. The function of the M2 protein depends on the integrity of its cytoplasmic tail. Fluorescence and electroparamagnetic resonance (EPR) spectroscopy experiments will be performed to determine whether the cytoplasmic tail of the M2 protein forms a rosette lying on the membrane surface or forms a helix extending into the cytoplasm. 3. Key features of the structure of the pore and the cytoplasmic tail of the M2 ion channel will be determined using (EPR) spectroscopy. 4. In order to understand better the mechanism for resistance and to identify which functional properties of the channel are essential for its role in the virus life cycle, the functional properties of amantadine-resistant mutant M2 proteins will be characterized. 5. In a separate set of experiments we will use a sensitive method to test the NB protein of influenza B virus for ion channel activity. ? ? ?
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