PROJECT SUMMARY/ABSTRACT Influenza viruses induce significant morbidity and mortality in the human population every year. Despite the medical importance of influenza disease, the mechanisms underlying the viral pathogenesis are incompletely understood. After influenza viruses initiate infection, cellular pattern recognition receptors detect components of the virus and inflammatory cytokines such as type I interferon (IFN) are produced. While an important part of early control of viral replication and spread, excessive or prolonged IFN signaling is associated with immunopathology and more severe viral disease. We have previously shown that some respiratory epithelial cells can survive direct viral infection, display prolonged IFN signaling, and contribute to lung immunopathology likely by delaying epithelial regeneration. Thus, understanding how type I IFN is downregulated represents a critical aspect of influenza disease pathogenesis. Interestingly, however, we failed to detect a difference in any of the canonical IFN induction or suppression pathways in the cells that survived direct viral infection. We therefore hypothesized that there must be additional regulators of IFN signaling relevant for IFN control during the resolution of influenza virus-induced disease. By leveraging genome wide CRISPR activation screening approaches, we were able to identify a previously uncharacterized regulator of IFN signaling (the ETS- family transcription factor ETV7) which upon induction, non-uniformly suppressed the expression of individual interferon stimulated genes (ISGs). The major goal of this application is to define this novel, ETV7 mediated, IFN regulatory mechanism and understand its physiological importance during influenza viral disease in vivo. In Aim 1, we will perform high-throughput biochemical analysis of ETV7 binding across all ISG promoters and then define how intrinsic ETV7 affinities for primary promoter sequences affect transcription factor competition. This work will reveal the first regulatory pathway capable of differential suppression of individual ISGs. In Aim 2, we will focus on understanding the cell type-specific importance of ETV7 regulation during respiratory epithelial innate immune responses and tissue regeneration after infection. These experiments will establish a new mechanism for how the balance between the antiviral signaling and regenerative capability in the lung is maintained. Finally, in Aim 3, we will explore how dysregulation of ETS-family transcription factors affects viral pathogenesis and recovery from infection in vivo. Successful completion of these studies will increase our understanding of the mechanisms that regulate IFN signaling during influenza virus infection and potentially inform new interventions to limit the severity of viral disease.