Terminal loop sequences in viral double-stranded RNAs modulate RIG-I signaling

Detection of foreign RNAs is a crucial activation step for innate immunity pathways in response to viral infections. Retinoic acid-inducible gene I (RIG-I) is a cytoplasmic RNA sensor that triggers type I and III interferon (IFN) expression and activates the antiviral response during RNA virus infections. The activating ligand for RIG-I has been shown to be 5'-triphosphated, blunt-ended, double-stranded (ds)RNA, but questions remain on the impact of other RNA motifs on RIG-I activation. Here we show that immune-activating copy-back viral genomes (cbVGs) contain RNA stem-loops away from the 5' end of the RNA that enhance RIG-I signaling and IFN expression. Importantly, the sequence of the terminal loops of the activating motifs impacts the strength of IFN expression. Additionally, we show that synthetic versions of these cbVG-derived stem-loops trigger innate immune responses in mice, demonstrating their potential as immunostimulants in vivo.