PROJECT SUMMARY/ABSTRACT Influenza A virus (IAV) triggers the death of most cell types in which it replicates, both in culture and in vivo. In 2016, we described an IAV-activated cell death pathway which accounts for almost all IAV-activated death in infected pulmonary cells. Over that past funding cycle, we showed that this pathway is initiated when the host sensor protein ZBP1 detects viral Z-RNAs and activates RIPK3 kinase. RIPK3 then triggers parallel, redundant pathways of programmed necrosis (necroptosis) and apoptosis. These studies have outlined the dominant cell death pathway activated by IAV in infected lungs. They have also demonstrated that ZBP1 is an RNA sensing protein, and that the RNA structures it recognizes are Z-RNAs, which are unique, left-handed double-helical RNA species that had not previously been thought to occur in nature. We have now made several intriguing new discoveries that set the stage for this application. First, we found that the Z-RNAs produced during IAV infections are not just viral in origin, but host cell-derived as well. Second, we observed that in alveolar macrophages (AMs), unlike in all other lung cell types tested, ZBP1 activation does not result in cell death, but instead in a non-cytolytic transcriptional program essential for protection against IAV lethality. Third, we found that necroptosis during IAV infections is activated not in the cytoplasm, but in the nucleus, resulting in nuclear rupture. Such `nuclear necroptosis' is highly inflammatory, and is a major driver of the pathogenesis during severe influenza. Finally, we have obtained important new insight into the coevolution of IAV with the necroptosis machinery in birds and bats, the two major natural hosts of these viruses. While birds simply do not express ZBP1, we have found that bats possess a single amino acid alteration in RIPK3 which selectively abrogates necroptosis (but not apoptosis) signaling, potentially explaining how these organisms tolerate such a large diversity of IAV subtypes. To our knowledge, these discoveries provide the first evidence that endogenous Z-RNAs may function as innate ligands for ZBP1 in anti-IAV immunity, that ZBP1 has non-cytolytic functions during IAV infections, and that a IAV activates a unique, hyper-inflammatory form of necroptosis from the nucleus. They also provide fresh evolutionary insight into how IAV strains are tolerated in their natural reservoirs, but can activate pathogenic necroptosis in humans. Based these and other observations, the goals of this proposal are to (1) to identify and characterize endogenous Z-RNA ligands for ZBP1, and to determine their importance to ZBP1 activated cell death in IAV-infected cells; (2) to delineate the mechanism and function of non-cytolytic ZBP1 signaling in AMs; and (3) to understand the role of nuclear necroptosis in influenza pathogenesis and in evolution. These studies unite two labs with expertise in ZBP1 signaling (Balachandran) and IAV pathogenesis (T...