Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

van Zuylen, Wendy J.; Doyon, Priscilla; Clément, Jean-François; Khan, Kashif Aziz; D'Ambrosio, Lisa M.; Dô, Florence; St-Amant-Verret, Myriam; Wissanji, Tasheen; Emery, Gregory; Gingras, Anne-Claude; Meloche, Sylvain; Servant, Marc J.

The results presented above suggest a role for the ER-to-Golgi compartment in TRAF3-dependent innate immune response. To investigate whether Sec16A or p115 play a role in the type I IFN response, we overexpressed both proteins in Hec1B cells and assessed NF-kB and IRF-3 transcription factor activation using reporter gene assays. Without any stimulation, overexpression of either protein did not significantly activate the IFNb promoter. However, following viral infection, the response was increased in cells overexpressing Sec16A or p115 ( Figure 8A ). Overexpression of Sec16A and p115 also increased the activation of the ISG56 promoter (IRF3-dependent promoter) ( Figure 8B ) and the NF-kBdependent promoter ( Figure 8C ) following SeV infection. Moreover, we observed a synergistic effect on IFNb promoter activity when Sec16A or p115 were co-expressed with MAVS ( Figure 8D ), TBK1 ( Figure 8E ) and, interestingly, the TLR3 essential effector TRIF ( Figure 8F ). Similar results were also obtained for the ISG56 promoter and the NF-kB-dependent promoter ( Figure S6 ). To further substantiate that the positive transcriptional effect of Sec16A and p115 is dependent on TRAF3, TRAF3-knockout MEF cells were transfected with p115 and Sec16A in the presence or absence of TRAF3 and used in the IFNb promoter reporter assay. As suspected, the enhanced promoter activation, induced by ectopically expressed p115 and Sec16A, was entirely dependent on the presence of TRAF3 ( Figure 8G ). Thus, when expressed in relatively low amounts in Hec1B and MEF cells (not shown), p115 and Sec16A positively participate in a TRAF3-dependent type I IFN response, probably reflecting the ability of a subpopulation of cytoplasmic TRAF3 to further associate with the ER-to-Golgi components under these conditions of mild ectopic expression.