The Mechanisms Of CIAP1/2 Mediated Regulation Of IFN-β Induction

Innate immune response is the front line of host defence over pathogen infection. After viral infection, the widely expressed pattern recognition receptors (PRRs) in host cells recognize pathogen-associated molecular patterns (PAMPs) and activate innate immune response, which lead to induction of type I interferons (IFNs) and proinflammatory cytokines. Type I IFNs initiate the JAK-STAT signal cascades and trigger transcription of thousands of downstream genes, which collaborately inhibit virus replication and induce apoptosis of infected cells. The transcriptional activation of type I IFNs demands the cooperative assembly of multiple transcription factors. For example, NF-kB and IRF3 are the most important transcription factors for the transcriptional induction of IFN-β.Recently, it has been reported that TRAF2/3 and cIAP1/2 form a cytoplasmic complex. Upon stimulation by BAFF and CD40L, the complex is recruited to the respective receptor, where cIAP1/2 mediate K48-linked ubiquitination and degradation of TRAF3, which is critical for activation of downstream MEKK and MAPK kinase cascades. Becasuse of the close relationship between TRAF3 and cIAP1/2, we speculated cIAPl and cIAP2 may be involved in the virus-triggered innate immune signaling pathways.Here we show that two E3 ligases cIAP1 and cIAP2 are critical in vius-triggered IFN-βinduction and cellular antiviral response. Kncokdown of cIAP1 and cIAP2 markedly inhibited virus-triggered activation of IRF3, NF-κB and IFN-βreporter. Interestingly, knockdown of cIAP1 and cIAP2 also attenuated cytoplasmic poly(I:C)-and B-DNA-triggered IFN-βpromoter activation. Consistently, knockdown of either cIAP1 or cIAP2 impaired SeV-triggered endogenous IRF3 dimerazation and transcription of endogenous IFN-βand downstream genes, while simultaneously knockdown of both cIAP1 and cIAP2 had accumulative effects. In plaque assay, knockdown of cIAP1 and cIAP2 enhanced VSV replication and abolished poly(I:C)-induced cellular antiviral response. Endogenous coimmunoprecipitation experiments indicated that viral infection or cytoplasmic poly(I:C) induction caused recruitment of cIAP1/2 to TRAF3/6 and VISA located at the mitochondria. Using linkage-specific ubiquitin, we found that SeV infection induced both K48-and K63-linked ubiquitination of endogenous TRAF3/6. Overexpression of both cIAP1 and cIAP2 mediated K48- and K63-linked ubiquitination of TRAF3/6, whereas E3 ligase inactive mutants of cIAP1 and cIAP2 could not. Furthermore, virus-triggered endogenous TRAF3 ubiquitination was abrogated by both RNAi-and Smac Mimetic-mediated knockdown of cIAP1/2.In this report, we found that cIAP1/2 caused TRAF3/6 ubiquitination at the mitochondria following viral infection and this is essential for virus-triggered IFN-βinduction. These findings provide insights into the mechanisms on how TRAF3/6 are positively regulated in the innate immune response. A recent study suggests that cIAP1/2 ubiquitinate RIP2, which is required for innate immune responses mediated by NOD1 and NOD2, which sense intracellular bacteria infection. In light of this and our study, it is possible that cIAP1 and cIAP2 are generally involved in innate immune responses mediated by a variety of PRRs.