: Influenza contributes substantially to worldwide morbidity and mortality. Influenza A virus has caused all human pandemics but influenza B virus infections occur annually and in some recent years they have constituted 50% of total influenza virus infections. The relative simplicity of using reverse genetics procedures to alter the genome of influenza A and B viruses to deliberately introduce pathogenicity determinants into the viral genome coupled with the aerosol transmission of influenza viruses, makes the viruses ideal candidates for use by bioterrorists to create epidemics and havoc. Recently, we discovered that the BM2 protein of influenza B virus is an oligomeric integral membrane protein with an ion channel activity. BM2 protein causes acidification of the virion interior that is required for virus uncoating in endosomes. The BM2 protein functions in a manner related to that of the influenza A virus M2 ion channel protein, the latter being the target of the anti-viral drug amantadine: the drug does not inhibit influenza B virus replication or the BM2 ion channel activity. The proposed experiments are designed to elucidate the important features of the structure and function of the BM2 protein and its role in the influenza B virus lifecycle. (1) We will characterize the BM2 protein using biochemistry, mutagenesis and reverse genetics. The phosphorylation state of the BM2 protein will be determined and the effect of specific mutations that affect BM2 function will be determined using reverse genetics procedures to introduce these mutations into influenza B virus. (2) The oligomeric state of the active form of the BM2 protein will be determined using mixtures of BM2 subunits with distinguishable properties. (3) The pore-lining residues of the BM2 channel will be identified using cysteine-scanning mutagenesis followed by biochemical and functional studies. (4) The mechanisms by which the activity of the BM2 protein is modulated will be studied. (5) We will develop systems for measuring the activity of recombinant BM2 protein in vitro in order to provide a useful means for the pharmaceutical industry to screen for antiviral compounds specific for BM2 and to use in biophysical experiments.
