Project Summary: The intestine serves as a physical and immune barrier to protect against infection by enteric pathogens. The Cherry lab uses Drosophila as an in vivo model of human and mosquito enteric infection to understand the intestinal innate immune response and how it is influenced by the composition of the microbiota. Sindbis virus is a mosquito-borne virus that is transmitted to mosquito vectors orally during a blood meal. Our preliminary data suggest the Drosophila homologue of Stimulator of Interferon Genes (STING) is antiviral against Sindbis virus within the intestine. Mammalian STING is activated by binding cyclic dinucleotides produced endogenously by cyclic GMP-AMP synthase (cGAS) or exogenously by bacteria. Mammalian cGAS produces a cyclic dinucleotide after binding cytosolic DNA from bacteria or DNA viruses but is not activated by binding RNA. Our preliminary data shows that the closest cGAS ortholog is antiviral against enteric Sindbis infection. Sequence analysis of this Drosophila cGAS candidate reveals it lacks the DNA binding domain found in mammalian cGAS, suggesting that d-cGAS senses a non-DNA ligand, perhaps viral RNA. Metagenomic data reveals that Drosophila commensal bacteria encode CDN synthases indicating that the microbiota could provide an exogenous CDN pool to prime basal STING activity from subsequent viral infection. Indeed, our preliminary data suggests exogenous feeding of CDNs to flies lacking their microbiota protects against enteric Sindbis virus infection. Moreover, we found that STING may be antiviral through the activation of autophagy and inflammatory NF-kB activation. We hypothesize that both d-cGAS and the microbiome produce CDNs that activate dSTING to protect from RNA virus infection through infection-dependent autophagy within the intestine. To expand on this hypothesis in Aim 1 we propose to determine the role of d-cGAS and CDNs in STING-dependent antiviral defense. Additionally, in Aim 2 we propose to define the mechanism by which STING is antiviral in enterocytes. This work sheds light on evolution of the ancient cGAS- STING defense pathway and defines how additional microbiota-derived products, such as CDNs, may influence intestinal immunity.