The influenza pandemics have long been a serious global health threat responsible for approximately 20,000 deaths per year in the US. It has been established that small H+ selective channels M2 and BM2 from influenza A and B virus play a critical role in viral replication and thus are effective targets for anti-influenza therapy. Amantadine is a licensed anti-influenza A drug that functions by inhibiting the pH activation of the M2 channel. However a number of M2 variants, as well as the BM2 channel of influenza B can escape the effect of amantadine, leading to the amantadine-resistance of these viruses. We propose to provide the atomic details of H+ gating and drug interaction of these small channels for understanding the mechanisms by which some influenza strains are resistant to amantadine. Initially we will solve the high resolution structure of the M2 H+ channel of influenza A virus in the closed state at pH 7.2. A new method has been devised to facilitate structure determination by solution NMR, which incorporates the dipolar couplings for acquiring orientation parameters and lipid/detergent bilayered micelles to enable characterization of atomic structures in a near lipid-bilayer environment. We will also solve the channel structure in the open state at pH 5.2 to delineate changes in conformation and dynamics that are coupled to low-pH activation. To understand the mechanism of amantadine inhibition, we will solve the channel structures, both closed and open, in the presence of drug binding. The sites of amantadine interaction in the channel will also be located. In parallel, the structures of the known amantadine-resistant variants of M2 will be examined for direct comparison to elucidate mechanisms of channel blocking. Similar studies will be done for the BM2 channel, which functions similarly to M2 but is resistant to amantadine. Structural details of the potential amantadine-BM2 interaction that does not block the channel will also be explored. The results will lay a solid foundation for our long- range goal of finding a true blocker of H+ channels for anti-influenza treatment.
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