The ubiquitin E3 ligase Huwe1 facilitates viral and self RNA sensing by RIG-I-like receptors

RIG-I-like receptors (RLRs) are cytoplasmic RNA sensors that promote type I and type III interferon (IFN) production in response to RNA ligands of viral or endogenous origin. The RLR pathway is tightly regulated by dynamic post-translational modifications, including ubiquitination. Huwe1 is a HECT domain-containing giant ubiquitin E3 ligase that has not been implicated in the RLR or IFN pathway. Here, we investigated whether Huwe1 is required for type I IFN induction downstream of RLRs. We demonstrate that loss of Huwe1 severely attenuates the expression of IFN-{beta}, IFN-{lambda}1 and IFN-stimulated genes (ISGs) in ADAR1-deficient human cells and primary murine bone-marrow derived macrophages, in which unedited self RNAs that serve as RLR ligands accumulate. In addition, depletion of Huwe1 reduces the induction of type I and III IFNs upon transfection with synthetic viral RNA mimetics or infection with a picornavirus. Using proteomics, we identified several putative Huwe1 substrates, which include key components of the RLR pathway (MAVS, TRAFs). We demonstrate that these substrates interact with Huwe1 and that Huwe1 is essential for the activity of TRAF5 in type I IFN induction. Collectively, our results put Huwe1 on the map as an important ubiquitin E3 ligase in the RLR pathway and provide new insights into ubiquitin-dependent regulation of cell-intrinsic antiviral immune pathways.