Emerging Infections: To study the mechanism by which influenza virus strains can emerge as infectious to humans, we plan to examine the X-ray structure and function of hemagglutinins from viral strains apparently limited to infecting animal reservoirs and for comparison human infectious strains from the major pandemics. Preferences for different sialoside linkages on cellular receptors correlate with the spread of infection in animals versus humans. One goal is to help explain observations like why outbreaks in the past two years in Hong Kong of avian virus infections in humans did not spread into the human population. Viral Entry Mechanisms: To investigate membrane fusion by influenza virus, we plan crystal structure studies of protein/detergent complexes of intact HA and HA2 and mechanistic studies of the interaction of HA with membranes and of intermediates in the fusion reaction. The hypothesis that HA2 in the low pH conformation observed by crystallography is membrane fusion active will also be tested with intact recombinant HA2 molecules transfected in cells suitable for membrane fusion assays. The hypothesis that the N- and C-terminal segments of HA1 plus all of HA2 (BHA's stem) was an ancestral membrane fusion protein will be tested by engineering such a protein, testing whether it can be proteolytically primed and activated by low pH, and whether the HA1 segments have a role, such as in the assembly of a putative multi-trimer containing pore. M2 Ion Channel: To generate structural information about the ion channel protein M2 of influenza virus we propose crystallization in detergent of bacterially expressed and refolded M2 tetramers that we have produced; and/or a tetramer of a channel-active synthetic transmembrane helix. Complexes with the inhibitory drug, amantadine, will also be studied.
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