The long-term goal of this research is to understand the assembly of influenza viruses at the molecular level. To this end, we propose to elucidate mechanism(s) by which influenza virus RNA segments are incorporated into virions, and to assess the contribution of selective viral (v) RNA incorporation to the generation of new reassortant influenza viruses. A plasmid-based reverse genetics system, developed in the applicant's laboratory, allows influenza virus to be produced entirely from cloned cDNA. This technology was used to identify segment-specific regions that mediate vRNA virion incorporation, providing the first direct evidence for a selective rather than random mechanism of vRNA packaging. Aim 1 seeks to refine the packaging signals within these regions, while Aim 2 determines if interactions among these signals mediate the assembly of sets of eight different vRNA segments. Aim 3 attempts to elucidate the mechanism(s) by which these sets of vRNA segments are incorporated into budding virions. The virion incorporation of vRNAs appears to involve viral structural proteins that sequester the viral genome segments into budding particles. Candidate mediators of this interaction (from the virion shell) are the three viral transmembrane proteins HA, NA, and ion channel protein (M2), which may execute this function through their cytoplasmic tails; candidates, from the viral ribonucleoprotein complex associated with each vRNA segment include the three polymerase proteins (PB2, PBI, PA). Aim 4 will test the hypothesis that selective vRNP incorporation plays a significant role in the reassortment and generation of new influenza virus strains. Indeed, the 1957 and 1968 pandemic influenza strains, as well as a triple reassortant that appeared in the North American pig population in 1998, were characterized by the introduction of a PB1 segment, in addition to HA and NA segments or an HA segment, into a new host. Thus, interactions between the PB1 vRNA and other vRNA segments (e.g., HA) may govern their preferential incorporation into virions. The proposed experiments will provide new fundamental insights into the life cycle of influenza viruses, in particular the mechanisms for organized recruitment of multiple vRNA segments into budding virions. If selective vRNA incorporation contributes significantly to the generation of novel reassortant viruses, new avenues will be opened for the development of antiviral interventions, including compounds that interfere with efficient vRNA virion incorporation or perhaps live vaccines that do not reassort with field strains due to altered incorporation sequences.