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.

Interestingly, several recent studies have demonstrated that overexpression of p115 or Sec16A in highly transfectable cell lines and depletion of Sec16 or p115 resulted in identical cellular outcomes (i.e. Golgi fragmentation (see Figure S4 and Figure 6 ) and delayed ER-to-Golgi transport), thereby suggesting that they are required in stoichiometric amounts [24, 25, 52] . Thus, when ectopically expressed in high amounts in 293T cells, p115 and Sec16A were expected to blunt TRAF3-dependent transcriptional activation. Indeed, transfection of increasing amounts of p115 or Sec16A efficiently blunted TRIF-, RIG-I-, and MAVS-induced IFNb promoter activation ( Figure S7A -C) as well as NF-kB promoter activation (data not shown). Importantly, adding increasing amounts of TRAF3 in this specific reporter gene assay dose-dependently reversed the inhibitory effect of p115 and Sec16A, once more substantiating the relationship that exist between Sec16A, p115 and TRAF3 ( Figure S7D ). Moreover, transfection of these plasmids also blunted TBK1-induced ISRE promoter activation ( Figure S7E ), but did not affect the transactivation response induced by the use of a constitutively active form of IRF-3 (IRF3-5D) ( Figure S7F) , suggesting that the ER-to-Golgi compartment plays upstream of IRF-3 in type I IFN signalling. To further confirm the implication of p115 and Sec16A in the type I IFN response, loss-of-function experiments were conducted next. As suspected, an RNAi approach targeting Sec16A and p115, which leads to Golgi fragmentation (see Figure S4 and Figure 6 ) significantly diminished Ifnb, ifit1 (ISG56), and oas1 mRNA induction following poly I:C and poly dA:dT transfection and SeV infection (Figure 9 ). To verify whether this approach affected IRF-3 activation and the induction of an IRF-3dependent antiviral protein [53] , we next verified the phosphorylation state of IRF-3 and the induction of ISG54 in HeLa cells expressing either shRNA duplexes targeting p115 and Sec16A or cells expressing a non-targeting (NT) shRNA duplex. The phosphorylation of IRF-3 and the expression of ISG54 were readily observed upon SeV infection, poly I:C and poly dA:dT transfection in HeLa cells expressing the NT shRNA duplex but was clearly reduced in cells expressing different shRNA duplexes targeting p115 ( Figure 10A ) and Sec16A ( Figure 10B ). Altogether, these data indicate that TRAF3 localization to the ER-to-Golgi vesicular pathway is necessary for a proper type I IFN response.