DZIP3 is a viral RNA co-sensor for RIG-I that promotes type-I interferon responses.

RNA-binding E3 ubiquitin ligases have the ability to link RNA regulation to the protein ubiquitination machinery, thereby influencing diverse biological processes. Here, we identify the RNA-binding E3 ubiquitin ligase DZIP3 as an innate immune sensor that couples viral RNA recognition to downstream immune signaling. Upon viral infection, binding of viral RNA to the DZIP3 RNA-binding motif induces conformational changes that promote K63-linked auto-ubiquitination, oligomerization, and stabilization of DZIP3. Activated DZIP3 associates with RIG-I and MAVS at the mitochondrial membrane, promoting K63-linked ubiquitination of RIG-I and robust activation of the type-I interferon response. Consequently, DZIP3 depletion impairs host antiviral defense, whereas DZIP3 restricts infection by a broad range of RNA viruses. Our findings establish DZIP3 as an innate immune co-sensor that integrates viral RNA detection with ubiquitin-mediated signaling, revealing a regulatory mechanism of RIG-I-MAVS-mediated antiviral immunity.