CircRNA-encoded protein fine-tunes ROS homeostasis and engages conserved JAK-STAT antiviral defenses in Drosophila.

Multicellular organisms rely on multilayered immune systems to defend against pathogen invasion. In Drosophila melanogaster, the primary antiviral barrier is RNA interference (RNAi). However, many viruses encode suppressors that disable RNAi, forcing hosts to activate complementary defense strategies. One such strategy involves the circular RNA circZfh1, which encodes the protein CRAV. CRAV is essential for activating the JAK-STAT pathway and providing antiviral protection when RNAi is neutralized. However, the molecular mechanism linking CRAV expression to upd3 induction and JAK-STAT activation remains unclear. Here, we show that CRAV directly interacts with the Ca²(+)-binding domain of the NADPH oxidase Nox, enhancing its enzymatic activity. This interaction promotes the generation of moderate reactive oxygen species (ROS) that act as signaling intermediates rather than stress inducers. CRAV-induced ROS selectively activate the ASK1-p38 mitogen-activated protein kinase cascade, which in turn triggers JAK-STAT signaling and the expressions of antiviral cytokines and effectors. Loss of Nox or inhibition of ASK1-p38 abolishes CRAV-mediated protection, underscoring the necessity of finely tuned redox signaling. These findings reveal a direct mechanistic link between a circRNA-derived protein and conserved innate immune pathways, highlighting the pivotal role of controlled ROS signaling in antiviral defense. IMPORTANCE: Antiviral immunity depends on the balance between host defenses and viral countermeasures. In fruit flies, RNA interference (RNAi) represents the primary barrier to viral infection, but viruses often disable this pathway. We show that the circRNA-encoded protein CRAV provides a backup defense by directly binding the NADPH oxidase Nox to generate moderate reactive oxygen species (ROS). Unlike damaging oxidative stress, these ROS serve as signaling cues that activate p38 and JAK-STAT pathways, which in turn drive antiviral cytokine production. This study uncovers how a circRNA-derived protein engages conserved redox-sensitive immune signaling, illustrating an adaptive strategy that ensures protection when RNAi is compromised. The results provide fundamental insights into the evolutionary diversification of circRNA-encoded proteins and broaden our understanding of how finely tuned ROS signaling contributes to innate antiviral immunity.