PROJECT SUMMARY Work from many laboratories has established that DNA sensors – both membrane-localized Toll-like receptors (e.g. TLR9) and intracellular DNA sensors (e.g. cGAS) – sense herpesvirus infection. In striking contrast, our knowledge about the physiologic relevance of cytoplasmic RNA sensors of the RIG-I-like receptor (RLR) family in the detection of herpesviruses, such as herpes simplex virus type 1 (HSV-1), is rudimentary. The proposed study builds on a recent discovery by the Gack laboratory that RIG-I plays a crucial role in the detection and restriction of HSV-1 infection, where RIG-I and intracellular DNA sensors act in a temporal manner. Furthermore, we identified that during HSV-1 infection the host-derived 5S ribosomal RNA pseudogene transcript 141 (RNA5SP141) activates RIG-I and elicits cytokine-mediated antiviral innate immune defense. In collaborative work we recently identified that ablated RNA5SP141 gene expression is associated with severe HSV-1 encephalitis in humans. Furthermore, we discovered that RNA5SP141 is exported from infected cells via exosomes and taken up by uninfected cells, where it triggered RIG-I signaling. Using a coordinated series of molecular, biochemical, and cell biological approaches combined with CRISPR-gene editing techniques, we will define the role of RNA5SP141 gene expression in the antiviral innate immune response to HSV-1 infection and in HSV-1-associated disease (AIM 1). Furthermore, we will elucidate the molecular mechanism(s) underlying RNA5SP141 exosomal loading (AIM 2), and also determine the physiologic relevance of exosomal delivery of RNA5SP141 in host immunity to HSV-1 infection (AIM 3). Our studies will provide a molecular understanding of innate immune detection of herpesvirus infection, which may provide the foundation for new therapeutic approaches or guide the design of novel adjuvants.