SUMMARY Seasonal influenza viruses cause >600 million cases annually and hundreds of billions of dollars in losses. Pandemic and avian influenza viruses present an even greater threat because they can dysregulate our immune response and/or spread to and shut down multiple organs. Current evidence suggests that aberrant viral replication contributes to a dysregulating of the innate immune response and the emergence of highly pathogenic strains from low pathogenic precursors. The transition from a low to highly pathogenic virus involves insertion of multiple basic amino acids in the cleavage site of the viral hemagglutinin (HA) surface protein. In birds, this change in HA allows the virus to spread systemically, resulting in mortality rates of up to 100% in poultry. The molecular mechanism underlying insertion of amino acids in the cleavage site is not well understood, but it may involve stuttering of the RNA polymerase in the HA gene, resulting in nucleotide insertions. The viral RNA polymerase can also delete nucleotides from the viral genome, resulting in shorter aberrant RNAs. Recent studies have shown that pandemic and avian influenza A virus infections produce RNA molecules of about 56-125 nucleotides in length, called mini viral RNAs, and that their synthesis is correlated with the upregulation of disease markers. How the RNA polymerase makes such large deletions in the viral genome is not known. This project will use a novel method to stall the viral RNA polymerase during key steps of viral replication and aberrant RNA synthesis, and use state-of-the-art biochemical, biophysical, and structural approaches to reveal the steps of viral replication and genome encapsidation, as well as aberrant RNA synthesis. In doing so, this project will contribute to a complete mechanistic understanding of influenza replication and answer long-standing, fundamental questions about the emergence of highly pathogenic influenza viruses.