Project Summary: During infection by arthropod borne flaviviruses such as Dengue (DV) and Zika (ZV), infected cells accumulate non-coding RNAs termed Subgenomic Flaviviral RNAs (sfRNAs) that consist of the viral genomic RNA’s 3 untranslated region (UTR). sfRNAs are generated when the host 5 to 3 exoribonuclease Xrn1 encounters exoribonuclease-resistant RNA (xrRNAs) structures in the genome’s 3 UTR that halt its progress. Analysis of flaviviral genomes has revealed that the majority of mosquito-borne flaviviruses have multiple xrRNAs “in tandem”. It was hypothesized that these structures were functionally redundant and working independently to generate sfRNA. However, data in our lab has revealed that within certain flaviviruses the structural integrity of one xrRNA is sensed by the other, affecting its function. We hypothesize that this “coupling” or “coordination” is due to tandem xrRNAs interacting through intervening sequences and/or structures. To test this hypothesis, we developed a surrogate reporter system that allows us to test the functional effects of mutations in the intervening sequence of the Dengue virus tandem xrRNAs. Specifically, our assay reports on changes in the patterns of produced sfRNAs, which have been well characterized for the wild type sequences. For aim 1 we will use this system to explore the mechanism of this coupling between xrRNAs in multiple flaviviruses. In addition, this research will also include uncovering the mechanism in which sfRNAs interfere with the mosquito immune response. Specifically, it has been shown that sfRNAs are capable of interacting with the mammalian Dicer protein, reducing the amount of small interfering RNA (siRNA) formed in vitro. Since mosquitos rely on RNA interference (RNAi) as their primary defense against viral infection, and mammalian and mosquito Dicer proteins are well conserved, we hypothesize that sfRNAs dampen the RNAi response in mosquitos by interacting with the Dicer-2 (Dcr2) protein of the mosquito RNAi pathway. Under aim 2 we will utilize a series of in vitro biochemical assays to test how this interaction between sfRNA and Dcr2 is taking place and elucidate on how sfRNAs work to dampen the immune response of the mosquito vector. Uncovering the dynamics of these host pathogen interactions could lead to new strategies for attenuating sfRNA production in mosquitos to curb the spread of flaviviruses.