Mechanisms of how the influenza A virus hemoagglutinin (HA) glycoprotein mediates both cellular-receptor binding and the membrane fusion that effects entry of viral nucleocapsids into the cytoplasm are being developed. The results are of specific importance to understanding viral infections and of general importance to understanding protein-ligand interactions, inhibitor design, and membrane fusion. Membrane fusion is a poorly understood but fundamental cellular process critical to many viral infections (including HIV-1) and numerous biological phenomena including intracellular vesicle trafficking, synaptic transmission, fertilization, and muscle development. Proposed studies on receptor binding include crystallographic analysis of receptor specificity, analysis of binding energetics by mutagenesis, and the design of high affinity ligands. The latter based on finding a second binding site and by combining strategies for increasing affinity discovered in the previous grant period. Proposed studies on membrane fusion seek to determine the relationship of the low pH fusion active conformation of HA in solution to that recently determined for a soluble, crystalline low pH HA fragment. Mutagenesis, semi-synthesis, and NMR experiments are proposed to study parts of the structure thought to disorder during fusion activity. X-ray studies on HA from a 1957 pandemic influenza A strain and the HEF (Hemoagglutinin-esterase-membrane) protein of influenza C virus are proposed to define structural bases for their functions and to provide a framework for designing experiments on puzzling differences in their membrane fusion activities. Structural information about M2, an influenza virus membrane ion channel, and its interaction with the anti-viral inhibitor amantadine will be sought by crystallography, NMR, and studies on a synthetic transmembrane fragment.
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