The long-term goal of this research is to understand the pathogenesis of viral diseases at the molecular level. Studies in the principal investigator's laboratory identified the structural features of avian influenza virus hemagglutinin (HA) that determine its cleavage and established that a single point mutation in the HA could alter virulence of influenza viruses. Knowledge gained from these studies has made it possible to pursue four specific aims directly related to the molecular pathogenesis of human influenza. 1. Elucidation of the structural features required for HA cleavage of mammalian Influenza viruses. The structural features required for HA cleavage in mammalian influenza viruses will be identified by selection of HA mutants and by in vitro mutagenesis performed on the cloned HA genes. This information should provide the molecular basis for the enzyme-substrate interaction underlying HA cleavage. 2. Determination of the intracellular location of HA cleavage. The site of HA cleavage within cells will be sought by use of markers of protein transport and monoclonal or polyclonal antibodies specific for uncleaved or cleaved HA. These studies should disclose the cellular organelle where the cleavage enzyme resides. 3. Purification and characterization of the cleavage enzyme for HAs with multiple basic amino acids at the cleavage site. Isolation and characterization of the cleavage enzyme will be critical in studies to establish the basis of cleavage activation of the HA. 4. Determination of the tissue distribution of the cleavage enzyme in mice. Genetic and immunological probes will be used to directly establish the relative amounts of cleavage enzyme in different tissue sites, as a means of proposed studies should lead to a better understanding of the pathogenesis of influenza by establishing the molecular basis of cleavage activation of the HA. Because there are many viral glycoproteins with multiple basic amino acids at the cleavage site, the pathogenetic mechanism identified in this proposal may also apply to other viral diseases. It may be possible to use the findings of this research to develop inhibitors of the HA cleavage enzyme, which could lead to a suitable chemotherapeutic approach to the control of influenza due to highly virulent viruses.
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