The long-term goal of this research is to understand the molecular mechanisms of influenza pathogenesis. previous studies have demonstrated that the matrix (M) and neuraminidase (NA) genes play essential roles in the virulence of mammalian influenza viruses, but the molecular mechanisms of the these effects are largely unknown. Recent development of the reverse genetics technique now permits direct examination of the relevance of specific amino acid alterations to the functions of viral gene products. The molecular basis of the contribution of the M and NA gene products to influenza pathogenesis will be sought through the following specific aims: Aim 1. To elucidate the molecular mechanisms of virulence attributable to the M gene. This set of experiments tests the hypothesis that highly efficient viral replication, driven by the M gene, is a critical factor in the virulence of mammalian influenza A viruses. By exploiting the reverse genetics system to generate mutant viruses, it should then be possible to clarify how different M-gene mutations contribute to virulence. Aim 2. To determine the roles of the phosphorylated residues and the zinc-finger motif of the M1 protein, and the cytoplasmic tail of the m2 protein, in influenza pathogenesis. The M gene encodes two proteins; M1 and M2. By systematically analyzing the effects of mutations on these three structural features using reverse genetics, and then analyzing mutant M-gene products produced in an in vitro expression system, the contributions of M1 and M2 proteins to influenza pathogenesis will be identified. Aim 3. To identify the structural featrues of the NA protein required for viral morphogenesis. A previously unknown function of the NA molecule in viral morphogenesis was recently identified in the applicant's laboratory. To establish this role, it will be necessary to determine the NA molecular requirements for virion formation, using the complementation system for a temperature-sensitive NSA as well as reverse genetics. Knowledge generated by this proposal should increase understanding of the events that occur during primary replication, virus spread, and secondary replication, thus providing a new working model for influenza pathogenesis. Of the many viral mutants to be generated in the course of this project, some may grow in high titers, which could aid in vaccine production by providing an alternative high-growth master strain. Mutant viruses that replicate without producing serious clinical symptoms in animals can provide valuable information on mutations for use in preparation of live-attenuated vaccines. Finally, knowledge gained from studies on the structural features of the NA and M2 proteins required for incorporation into virions may be used to develop new antiviral therapeutics.