UC San Diego

As a first line of defense, epithelial cells can detect pathogen associated molecular patterns (PAMPs) from an infecting virus and respond by producing type I and type III interferons. The timing and magnitude of the interferon response can greatly influence the course of disease. Here, we identified viral and host factors that modulate the interferon response to influenza A virus.A known viral contributor is the influenza protein NS1, a potent interferon signaling antagonist. To explore additional factors that may influence the interferon response, we first looked for viral features that modulate interferon induction in the absence of NS1, which allowed us to identify stages of the viral life cycle that contribute to or suppress the generation of immunostimulatory ligands. We found that de novo flu RNA is essential for interferon induction in the absence of NS1, and nuclear export of viral genomes contributes to this response. Defective viral genomes enhance the interferon response but only in conditions supporting replication. To identify host factors that modulate interferon induction within influenza-infected cells, we developed CRISPR with Transcriptional Readout (CRITR-Seq). CRITR-Seq is a method linking CRISPR guide sequence to activity at a promoter of interest. Employing this method, we find that depletion of the Negative Elongation Factor complex increases both flu transcription and an interferon response. We find that the process of flu transcription is a key contributor to the interferon response, both in the presence and absence of viral replication. Therefore, our findings validate CRITR-seq as a tool for genome-wide screens for phenotypes of gene expression and identify NELF and the host transcription machinery as a rate-limiting interface with the flu life cycle. Together, these studies highlight innate immune ligand concentration as a primary factor shaping the probability of the interferon response to influenza virus, and demonstrate that de novo viral RNA is both critical and rate-limiting for interferon induction. Our finding that flu transcription can contribute to interferon-induction by a replication-incompetent virus adds complexity to our understanding of the processes that generate PAMPs during flu infection and opens new questions about the identity of the relevant influenza immunostimulatory ligand(s).