Key Role of Lys63-Linked Polyubiquitination in Viral Activation of IRF3

Viral nucleic acids exposed during invasion and proliferation are detected by mammalian cells through receptors belonging to pattern-recognition receptors family (PRRs). Among PRRs, RIG-I-like receptors (RLRs), including RIG-I, MDA5 and LGP2, are responsible for sensing intracellular viral RNAs. MAVS, a mitochondria-localized transmembrane protein, transduces signaling from RIG-I and MDA5 to activate downstream transcription factors IRF3 and NF-kB, which contribute to the induction of IFNb. Despite growing list of components revealed in RIG-I/MAVS/IRF3 pathway, molecular mechanism by which MAVS activates IRF3 upon viral infection has remained largely unclear. In current study, employing a cell-free system together with conventional fractionation procedures, Ubc5 was identified as a specific ubiquitin-conjugating enzyme (E2) involved in IRF3 activation. Taking advantages of inducible-RNAi strategy, catalytically active Ubc5 was shown to be essential for viral activation of IRF3. Furthermore, evidences were obtained indicating that Lys63-linked polyubiquitination played a key role in MAVS-mediated IRF3 activation both in vitro and in vivo. Finally, NEMO was demonstrated to function as a ubiquitin-chain adaptor recruiting and activating TBK1, the kinase for IRF3 phosphorylation. Those results offered insights into the mechanism underlying IRF3 activation mediated by K63-linked polyubiquitination.