The transcription factors IRF3 and NF-κB are crucial in innate immune signalling in response to many viral and bacterial pathogens. However, mechanisms leading to their activation remain incompletely understood. Canonical RLR signalling and detection of viral RNA is dependent upon the receptors RIG-I, MDA5 and TLR3. Alternatively, the DExD-Box RNA helicases DDX1-DDX21-DHX36 activate IRF3/NF-κB in a TRIF-dependent manner independent of RIG-I, MDA5 or TLR3. Here we describe DDX50, which shares 55.6% amino acid identity with DDX21, as a component of the dsRNA sensing machinery and signalling pathway. Deletion of DDX50 in mouse and human cells impaired activation of the IFNβ promoter, IRF3-dependent endogenous gene expression and cytokine/chemokine production in response to cytoplasmic dsRNA (polyIC transfection), and infection by RNA and DNA viruses. Mechanistically, DDX50 co-immunoprecipitated with TRIF and DDX1, promoting complex formation upon stimulation. Furthermore, whilst MAVs/TBK1 induced signalling is intact in Ddx50 KO cells, TRIF-dependent signalling was impaired suggesting DDX50 drives TRIF-dependent Ifnβ transcription. Importantly, loss of DDX50 resulted in increased replication and dissemination of vaccinia virus, herpes simplex virus and Zika virus highlighting its important role as a viral restriction factor.

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