Influenza viruses, a group of major human pathogens, are responsible for 10,000-20,000 deaths and economic loss of $10-20 billions/yr. Influenza viruses asemble and bud from the plasma membrane, specifically from the apical side of polarized epithelial cells. Long term goal of this project is to elucidate the processes involved in polarized transport of viral proteins and assembly and budding of virus particles. Specific objectives are to: (i) define the apical determinants of HA and NA, the envelope viral proteins, (ii) define the interactions of HA and NA with M1, (iii) define the role of envelope proteins, HA and NA determining the apical vs. basolateral budding. We shall use chimeric constructions, site-specific mutations as well as reverse genetics to define the function of these proteins in virus assembly and budding. We have discovered a novel apical signal in the transmembrane domain (TMD) of apical NA and HA proteins. We will dissect and define the sequences and requirements of apical signal in the TMD of HA and NA. Using the floatation gradient analysis of Triton X-100 detergent-treated membranes, we will dissect the sequences in the TMD and cytoplasmic tail of HA and NA required for specific interaction with M1. Using reverse genetics, we will determine the role TMD and cytoplasmic tail of HA and NA in virus biology. Finally, using basolaterally targeted HA and NA in transfectant viruses we will examine if HA and NA determine the budding site (apical vs. basolatral) of influenza viruses in polarized MDCK cells. Assembly and budding of influenza viruses are critical for growth, replication and consequently in pathogenesis of influenza viruses. A detailed understanding of these processes will facilitate the rational development of antiviral agents which could interfere with one or more steps in virus assembly.
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