Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.

Induction of an antiviral innate immune response relies on pattern recognition receptors, including retinoic acid-inducible gene 1-like receptors (RLR), to detect invading pathogens, resulting in the activation of multiple latent transcription factors, including interferon regulatory factor 3 (IRF3). Upon sensing of viral RNA and DNA, IRF3 is phosphorylated and recruits coactivators to induce type I interferons (IFNs) and selected sets of IRF3-regulated IFN-stimulated genes (ISGs) such as those for ISG54 (Ifit2), ISG56 (Ifit1), and viperin (Rsad2). Here, we used wild-type, glycogen synthase kinase 3␣ knockout (GSK-3␣ ؊/؊ ), GSK-3␤ ؊/؊ , and GSK-3␣/␤ double-knockout (DKO) embryonic stem (ES) cells, as well as GSK-3␤ ؊/؊ mouse embryonic fibroblast cells in which GSK-3␣ was knocked down to demonstrate that both isoforms of GSK-3, GSK-3␣ and GSK-3␤, are required for this antiviral immune response. Moreover, the use of two selective small-molecule GSK-3 inhibitors (CHIR99021 and BIO-acetoxime) or ES cells reconstituted with the catalytically inactive versions of GSK-3 isoforms showed that GSK-3 activity is required for optimal induction of antiviral innate immunity. Mechanistically, GSK-3 isoform activation following Sendai virus infection results in phosphorylation of ␤-catenin at S33/S37/T41, promoting IRF3 DNA binding and activation of IRF3-regulated ISGs. This study identifies the role of a GSK-3/␤-catenin axis in antiviral innate immunity. Induction of an antiviral innate immune response relies on pattern recognition receptors, including those belonging to the retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLR), Tolllike receptor (TLR), and recently characterized DNA sensor families, to detect and respond to invading pathogens, resulting in the production of type I interferons (IFNs) and proinflammatory cytokines (1, 2). The expression of these cytokines is the result of the activation of signaling pathways that culminate in the activation of a number of latent transcription factors, including IFN regulatory factor 3 (IRF3) (3). C-terminal phosphorylation of IRF3 by the IB kinase (IKK)-related kinases TANK-binding kinase 1 (TBK1) and IKKi (4, 5) results in its dimerization and interaction with the transcriptional coactivators CREB-binding protein (CBP)/p300, which are required for the DNA binding activity of IRF3 to induce type I IFNs and selected sets of IRF3-regulated IFN-stimulated genes (ISGs) such as those for ISG54 (Ifit2), ISG56 (Ifit1), and viperin (Rsad2) (6). Recently, ␤-catenin has also been reported to act as a coactivator of IFN-␤ transcription allowing the recruitment of the acetyltransferase CBP/p300 to IRF3 (7-9). IRF3 and its coactivators are subject to positive or negative regulation by posttranslational modifications, protein phosphorylation being the most common (9-11).Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that is expressed ubiquitously in most cell types. In mammals, two distinct genes encode GSK-3, generating two related proteins, GSK-3␣ and GSK-3␤....

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are the main cytosolic sensors of single-stranded RNA viruses, including paramyxoviruses, and are required to initiate a quick and robust innate antiviral response. Despite different ligand-binding properties, the consensus view is that RIG-I and MDA5 trigger common signal(s) to activate interferon regulatory factor 3 (IRF-3) and NF-κB, and downstream antiviral and proinflammatory cytokine expression. Here, we performed a thorough analysis of the temporal involvement of RIG-I and MDA5 in the regulation of IRF-3 during respiratory syncytial virus (RSV) infection. Based on specific RNA interference-mediated knockdown of RIG-I and MDA5 in A549 cells, we confirmed that RIG-I is critical for the initiation of IRF-3 phosphorylation, dimerization and downstream gene expression. On the other hand, our experiments yielded the first evidence that knockdown of MDA5 leads to early ubiquitination and proteasomal degradation of active IRF-3. Conversely, ectopic expression of MDA5 prolonged RIG-I-induced IRF-3 activation. Altogether, we provide novel mechanistic insight into the temporal involvement of RIG-I and MDA5 in the innate antiviral response. While RIG-I is essential for initial IRF-3 activation, engagement of induced MDA5 is essential to prevent early degradation of IRF-3, thereby sustaining IRF-3-dependent antiviral gene expression. MDA5 plays a similar role during Sendai virus infection suggesting that this model is not restricted to RSV amongst paramyxoviruses.

Activation of NF-B transcription factors by locally produced angiotensin II (Ang II) is proposed to be involved in chronic inflammatory reactions leading to atherosclerosis development. However, a clear understanding of the signaling cascades coupling the Ang II AT1 receptors to the activation of NF-B transcription factors is still lacking. Using primary cultured aortic vascular smooth muscle cells, we show that activation of the IKK complex and NF-B transcription factors by Ang II is regulated by phosphorylation of the catalytic subunit IKK␤ on serine residues 177 and 181 in the activation T-loop. The use of pharmacological inhibitors against conventional protein kinases C (PKCs), mitogen-activated/extracellular signal-regulated kinase (MEK) 1/2, ribosomal S6 kinase (RSK), and silencing RNA technology targeting PKC␣, IKK␤ subunit, tumor growth factor ␤-activating kinase-1 (TAK1), the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor-6 (TRAF6), and RSK isoforms, demonstrates the requirement of two distinct signaling pathway for the phosphorylation of IKK␤ and the activation of the IKK complex by Ang II. Rapid phosphorylation of IKK␤ requires a second messenger-dependent pathway composed of PKC␣-TRAF6-TAK1, whereas sustained phosphorylation and activation of IKK␤ requires the MEK1/2-ERK1/2-RSK pathway. Importantly, simultaneously targeting components of these two pathways completely blunts the phosphorylation of IKK␤ and the proinflammatory effect of the octapeptide. This is the first report demonstrating activation of TAK1 by the AT1R. We propose a model whereby TRAF6-TAK1 and ERK-RSK intracellular pathways independently and sequentially converge to the T-loop phosphorylation for full activation of IKK␤, which is an essential step in the proinflammatory activity of Ang II.

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