The IB kinase-related kinases, TBK1 and IKKi, were recently shown to be responsible for the C-terminal phosphorylation of IRF-3. However, the identity of the phosphoacceptor site(s) targeted by these two kinases remains unclear. Using a biological assay based on the IRF-3-mediated production of antiviral cytokines, we demonstrate here that all Ser/Thr clusters of IRF-3 are required for its optimal transactivation capacity. In vitro kinase assays using full-length His-IRF-3 as a substrate combined with mass spectrometry analysis revealed that serine 402 and serine 396 are directly targeted by TBK1. Analysis of Ser/Thr-to-Ala mutants revealed that the S396A mutation, located in cluster II, abolished IRF-3 homodimerization, CBP association, and nuclear accumulation. However, production of antiviral cytokines was still present in IRF-3 S396A-expressing cells. Interestingly, mutation of serine 339, which is involved in IRF-3 stability, also abrogated CBP association and dimerization without affecting gene transactivation as long as serine 396 remained available for phosphorylation. Complementation of IRF-3-knockout mouse embryonic fibroblasts also revealed a compensatory mechanism of serine 339 and serine 396 in the ability of IRF-3 to induce expression of the interferon-stimulated genes ISG56 and ISG54. These data lead us to reconsider the current model of IRF-3 activation. We propose that conventional biochemical assays used to measure IRF-3 activation are not sensitive enough to detect the small fraction of IRF-3 needed to elicit a biological response. Importantly, our study establishes a molecular link between the role of serine 339 in IRF-3 homodimerization, CBP association, and its destabilization.

Collectively, our data demonstrate that the proinflammatory activity of Ang II is independent of the classical pathway leading to IB␣ phosphorylation and degradation but clearly depends on the recruitment of an IKK complex signaling cascade leading to phosphorylation of p65 on serine 536.

(18-20, 50, 53, 59). The mitochondrial adapter MAVS/ IPS-1/Cardif/VISA is directly downstream of the helicases and acts as a pivotal point in the cascade leading to activation of the transcription factors IRF-3 and -7 and NF-B, which synergistically regulate IFN-␤ gene expression (21,30,45,49,58). Secreted IFN-␣/␤ binds to its cognate IFNAR receptor in neighboring cells and initiates a second wave of IFN response, mediated by a complex known as ISGF3, which is composed of STAT-1, STAT-2, and IRF-9 transcription factors (27,39,54). During the second wave, IFN production is amplified with the expression of multiple IFN-␣ subtypes and hundreds of interferon-stimulated genes (ISGs), including recently identified IB kinase ε (IKKε)-specific genes such as ADAR-1, IFIT3, and OAS1 (51).The MAVS adapter contains an amino-terminal caspase activation and recruitment domain (CARD) that interacts with the CARDs of RIG-I/Mda5 and a carboxy-terminal transmembrane (TM) domain that anchors MAVS to the outer mitochondrial membrane (21,30,45,58). The essential role of MAVS in antiviral signaling was demonstrated by the failure of MAVS-deficient mice to mount a proper IFN response to poly(I ⅐ C) stimulation and by their severely compromised immune defense against virus infection (24, 49). Interestingly, MAVS expression alone, in the absence of virus infection, is sufficient to trigger the IRF and NF-B pathways leading to IFN production. Engagement of MAVS by active RIG-I/Mda5 leads to dimerization (3) and formation of a mitochondrial platform where multiple signaling molecules converge to mediate activation of the classical IKK complex and/or the IKK-related kinases 21,30,31,42,43,45,58,60,61).TBK-1 and IKKε activate the IRF pathway by direct phosphorylation of IRF-3 and IRF-7 in their C-terminal regulatory region (9,26,29,34,46,52). Analysis of knockout mice demonstrated that the ubiquitously expressed TBK-1 is the major mediator of IRF-3/-7 phosphorylation and initiator of the antiviral response (15,35,38). Disruption of IKKε expression on the other hand had a minimal effect on the activation of IRF-3/-7 and was considered dispensable for the induction of the IFN response (15,35,38). However, following viral infection IKKε, but not TBK-1, phosphorylates STAT-1 on serine 708 and increases expression of genes such as ADAR-1, IFIT3, and OAS1 (51). Importantly, this observation suggests that differences at the level of substrate specificity may exist for the two kinases (28, 51). Mechanistic differences in the activation of TBK-1 and IKKε may also be expected, based on differences in cytoplasmic localization of the two kinases as well as the observation that IKKε is directly recruited to the mitochondrial network via MAVS following virus infection, whereas TBK-1 remains largely cytoplasmic (16, 23, 25, 30).

Activation of the innate arm of the immune system following pathogen infection relies on the recruitment of latent transcription factors involved in the induction of a subset of genes responsible for viral clearance. One of these transcription factors, IFN regulatory factor 3 (IRF-3), is targeted for proteosomal degradation following virus infection. However, the molecular mechanisms involved in this process are still unknown. In this study, we show that polyubiquitination of IRF-3 increases in response to Sendai virus infection. Using an E1 temperature-sensitive cell line, we demonstrate that polyubiquitination is required for the observed degradation of IRF-3. Inactivation of NEDD8-activating E1 enzyme also results in stabilization of IRF-3 suggesting the NEDDylation also plays a role in IRF-3 degradation following Sendai virus infection. In agreement with this observation, IRF-3 is recruited to Cullin1 following virus infection and overexpression of a dominant-negative mutant of Cullin1 significantly inhibits the degradation of IRF-3 observed in infected cells. We also asked whether the C-terminal cluster of phosphoacceptor sites of IRF-3 could serve as a destabilization signal and we therefore measured the half-life of C-terminal phosphomimetic IRF-3 mutants. Interestingly, we found them to be short-lived in contrast to wild-type IRF-3. In addition, no degradation of IRF-3 was observed in TBK1−/− mouse embryonic fibroblasts. All together, these data demonstrate that virus infection stimulates a host cell signaling pathway that modulates the expression level of IRF-3 through its C-terminal phosphorylation by the IκB kinase-related kinases followed by its polyubiquitination, which is mediated in part by a Cullin-based ubiquitin ligase.

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