Mammalian orthoreovirus (MRV) genomic RNA is detected by host cells to induce an innate immune response. Detection of MRV RNA also contributes to the induction of cell death. Because viral genomic RNA remains contained within particles throughout infection, how the RNA becomes exposed and accumulates to the level that induces a host response remains unknown. In this proposal, we will uncover how viral RNA becomes detectable by the host cell. In Aim 1 of this proposal, we will build on the data that specific endosomal uptake pathways, endosomal protease activity, and endosomal maturation are required for exposure of viral genomic RNA from incoming particles. We will identify how these endosomal properties influence genomic RNA exposure. We will also determine how viral determinants control exposure and transport of viral genomic dsRNA for detection by cytoplasmic sensors. In Aim 2, we will determine how the assembly of the viral µ1 capsid protein on progeny cores blocks excessive accumulation of viral RNA. We will perform biochemical and virological experiments to identify structure-function relationships in µ1 that mediate these effects. We will also define the structural basis of µ1-mediated shut down of viral RNA transcription by solving the structure of a MRV assembly intermediates. In Aim 3 of the proposal, we will follow up on our preliminary data indicating that cellular RNA transporter SIDT2 controls MRV RNA levels. We will define whether the RNautophagy function of SIDT2 governs its antiviral effect and determine whether viral RNA turnover is influenced by RNautophagy. Finally, we will define how RNAs are specifically recognized by the RNautophagy machinery. Completion of this work will reveal viral and cellular factors that control the detection of MRV and other related dsRNA viruses by host cells.