PROJECT SUMMARY/ABSTRACT Although inflammation is needed for the host to defend itself against influenza infection, too much inflammation is detrimental to the host, and contributes to morbidity and mortality. Yet, available therapeutics are solely antiviral and do not prevent inflammation-driven lung damage, mostly because how inflammation is regulated during influenza infection is not fully understood. As a result, influenza virus still kills tens of thousands of people every year in the US alone, and up to half a million worldwide. Since influenza itself has evolved mechanisms to regulate the host innate immune and inflammatory response, studying these mechanisms is one strategy to start designing new avenues of therapeutic intervention. Influenza A virus modulates host responses to infection in part through its virus-encoded ribonuclease (RNase) PA-X. Indeed, mutated PA-X- deficient viruses cause higher levels of inflammatory responses and increased mortality compared to wild-type viruses in animal models of infection. While PA-X globally degrades host mRNAs, how this activity specifically leads to modulation of the immune and inflammatory response is not known. Through transcriptomic analysis of infected and PA-X expressing cells, the Gaglia lab has found that PA-X actually targets specific subsets of RNAs, while sparing others. Importantly, innate immune genes are preferentially targeted by PA-X, consistent with its in vivo anti-inflammatory phenotype. Our RNAseq data also uncovered that spliced RNAs are more susceptible to PA-X degradation than intronless RNAs, a specificity that I confirmed using reporter constructs, suggesting a mechanistic link between PA-X and splicing. However, how this splicing based mechanism allows PA-X to modulate innate immunity and inflammation is unknown. In the proposed work, I will test the hypothesis that PA-X exploits RNA splicing to target nascent RNAs, allowing PA-X to down-regulate genes that are induced transcriptionally during infection and modulate the host innate immune response and inflammation. In Aim 1, I will study the role of specific splicing steps in recruiting PA-X to RNAs. In Aim 2, I will explore the link between PA-X targeting and transcription, and study the preferential targeting of nascent RNAs. In Aim 3, I will connect these findings to regulation of innate immunity and inflammation by PA-X in a biologically relevant 3D lung culture model. The Gaglia lab provides the best training environment for me to complete this work, as shown by my recent first-author publication, which expanded our understanding of the molecular mechanism of action of PA-X. I will acquire the technical and conceptual skills that are required for this project through my mentor’s comprehensive knowledge of viral control of host gene expression and high-throughput sequencing dataset analysis, my co-mentor’s extensive experience in RNA work and transcription, and our collaborators’ expertise in human primary bronchia...