ABSTRACT Epithelial cells are exposed to a wide range of environmental pathogens due to their location at body surfaces. Aside from functioning as a physical barrier, epithelial cells also coordinate immune defenses through receptor-mediated signaling. However, we do not fully understand the mechanisms that shape pathogen recognition and immune signaling in epithelial cells. The overall goal of this proposal is to elucidate the molecular mechanisms that regulate the outcome of viral infection in epithelial cells. There is a critical need for an in vivo animal model that facilitates mechanistic studies of epithelial immunity. Infection with Orsay virus–a naturally occurring intestinal pathogen of the nematode Caenorhabditis elegans–provides an innovative approach to study cellular interactions between a virus and its natural host in the context of a whole-animal model. An obligate, intracellular pathogen, Orsay virus invades C. elegans intestinal cells and induces the activation of the Intracellular Pathogen Response (IPR), a transcriptional defense program that confers pathogen resistance (Sowa et al. 2020). This antiviral transcriptional response requires DRH-1, a homolog of mammalian RIG-I-like receptors (RLRs). RLRs are intracellular pattern-recognition receptors (PRRs) that detect viral RNA to initiate an antiviral immune response. Notably, DRH-1/RLR is one of the few PRRs conserved between C. elegans and humans. Despite the similarity between RLRs and DRH-1, C. elegans lacks sequence-based homologs to the downstream signaling components of the RLR pathway–including MAVS, IRF3, NF-κB and interferon. It is unclear which host determinants are involved in DRH-1-mediated activation of the IPR and where DRH-1 is required to induce the IPR. The proposed studies integrate molecular tools and a natural infection model to address the central hypothesis that DRH-1 signals through a non-canonical RLR signaling pathway and functions in intestinal cells to induce the IPR. The central hypothesis will be tested through two specific aims. Aim 1. Determine how DRH-1 signals to activate the Intracellular Pathogen Response. Aim 2. Define the tissue specificity and subcellular localization of DRH-1 during Orsay virus infection. The expected outcome of this project is a mechanistic understanding of how C. elegans DRH-1 coordinates host-virus interactions at the epithelial barrier. This work will highlight either the evolutionary conservation or rewiring of RLR signaling in epithelial cells. Collectively, the proposed studies will reveal a novel form of antiviral immunity in C. elegans. More broadly, the proposed studies will have positive translational impact by elucidating a novel antiviral pathway that may provide insight into the regulation of innate immune responses during viral infection in humans.