AbstractThe 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 inDdx50KO cells, TRIF-dependent signalling was impaired suggesting DDX50 drives TRIF-dependentIfnβtranscription. 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.Author summaryThe detection of viral RNA or DNA by host RNA or DNA sensors and the subsequent antiviral immune response are crucial for the outcome of infection and host survival in response to a multitude of viral pathogens. Detection of viral RNA or DNA culminates in the upregulation of inflammatory cytokines, chemokines and pathogen restriction factors that augment the host innate immune response, restrict viral replication and clear infection. The canonical RNA sensor RIG-I is a member of the large family of DExD/H-box helicases, however the biological role of many DExD/H-box helicases remain unknown. In this report, we describe the DExD-Box helicase DDX50 as a new component of the RNA sensing machinery. In response to DNA and RNA virus infection, DDX50 functions to enhance activation of the transcription factor IRF3, which enhances antiviral signalling. The biological importance of DDX50 is illustrated by its ability to restrict the establishment of viral infection and to diminish the yields of vaccinia virus, herpes simplex virus and Zika virus. These findings increase knowledge of the poorly characterised host protein DDX50 and add another factor to the intricate network of proteins involved in regulating antiviral signalling in response to infection.