PROJECT SUMMARY/ABSTRACT Influenza A viruses (IAV) kill most of the cell types in which they replicate, both in cell culture and in infected lungs in vivo. While regulated cell death represents a host defense mechanism that limits both virus spread and host immunopathology early in an infection, unbridled cell death, particularly necrosis, can lead to severe degradation of bronchioalveolar epithelia and consequent mortality despite control of virus replication in vivo. Indeed, severe illness following infection with highly pathogenic strains of IAV is well-correlated with widespread pulmonary epithelial cell death and bronchioalveolar tissue damage in humans. Despite this, remarkably little is known of the molecular mechanisms by which IAV activates cell death in relevant lung cell types. Thus (1) understanding the mechanisms by which IAV triggers cell death, (2) determining the identity and importance of lung cell types that die by these mechanisms during IAV infection in vivo; and (3) determining if pharmacological manipulation of cell death represents a new therapeutic entry-point for respiratory IAV are each important unmet objectives. We have recently discovered a mechanism of cell death that appears to account for almost all IAV- activated death in infected airway epithelial cells. This pathway is initiated when the protein DAI senses IAV genomic RNA and nucleates the kinase RIPK3. RIPK3 then activates parallel pathways of programmed necrosis (necroptosis), as well as apoptosis. Necroptosis downstream of RIPK3 relies on MLKL and apoptosis on FADD, such that deletion of DAI, RIPK3, or MLKL+FADD renders mice extraordinarily susceptible to respiratory IAV replication and lethality. Remarkably, eliminating MLKL alone has no discernible effect, demonstrating that the FADD apoptosis axis can fully compensate for loss of MLKL and necroptosis. To our knowledge, these findings represent the first description of a dedicated IAV activated cell death pathway, the first implication of DAI as a sensor of RNA viruses, and the first identification of a virus that triggers both apoptosis and necroptosis downstream of RIPK3. The redundancy of necroptosis with apoptosis to IAV clearance also provides an unexpected therapeutic opportunity in cases where necrotic death is implicated in IAV pathogenesis. Based on these and other observations, the goals of this proposal are to: (1) identify the molecular mechanisms by which the DAI-RIPK3 axis recognizes IAV and activates cell death; (2) employ cutting-edge mouse reporter models to isolate and identify lung cell types that succumb to IAV by RIPK3-driven apoptosis versus necroptosis, and determine in which of these cell types is RIPK3 signaling important for virus control; and (3) test if selective blockade of necroptosis will have clinical benefit following infection with highly-pathogenic strains of IAV. Successful completion of these Aims has the potential to transform our understanding of IAV pathogenesis, with immediate clinical ramifications.