A TRAF3-NIK module differentially regulates DNA vs RNA pathways in innate immune signaling.

Detection of viral genomes by the innate immune system elicits an antiviral gene program mediated by type I interferons (IFNs). While viral RNA and DNA species induce IFN via separate pathways, the mechanisms by which these pathways are differentially modulated are unknown. Here we show that the positive regulator of IFN in the RNA pathway, TRAF3, has an inhibitory function in the DNA pathway. Loss of TRAF3 coincides with increased expression of the alternative NF-κB-inducing molecule, NIK, which interacts with the DNA pathway adaptor, STING, to enhance IFN induction. Cells lacking NIK display defective IFN activation in the DNA pathway due to impaired STING signaling, and NIK-deficient mice are more susceptible to DNA virus infection. Mechanistically, NIK operates independently from alternative NF-κB signaling components and instead requires autophosphorylation and oligomerization to activate STING. Thus a previously undescribed pathway for NIK exists in activating IFN in the DNA pathway

Ubiquitin Dependent Regulation of Innate Antiviral Signaling

Induction of type I interferons by the transcription factors IRF3 and IRF7 is essential in the initiation of antiviral innate immunity. Activation of IRF3/7 requires C-terminal phosphorylation by the upstream kinases TBK1/IKKi, where IRF3/7 phosphorylation promotes dimerization, and subsequent nuclear translocation to the IFN-beta promoter. Recent studies have described the ubiquitin-editing enzyme A20 as a negative regulator of IRF3 signaling by associating with TBK1/IKKi, however the regulatory mechanism of A20 inhibition remains unclear. Here we describe the adaptor protein, TAX1BP1, as a key regulator of A20 function in terminating signaling to IRF3. Murine embryonic fibroblasts (MEFs) deficient in TAX1BP1 displayed increased amounts of IFN-beta production upon viral challenge compared to WT MEFs. TAX1BP1 inhibited virus-mediated activation of IRF3 at the level of TBK1/IKKi. TAX1BP1 and A20 blocked antiviral signaling by disrupting K63-linked polyubiquitination of TBK1/IKKi independently of the A20 deubiquitination (DUB) domain. Furthermore, TAX1BP1 was required for A20 effector function as A20 was defective for the targeting and inactivation of TBK1 and IKKi in Tax1bp1–/– MEFs. Additionally, we found the E3 ubiquitin ligase TRAF3 to play a critical role in promoting TBK1/IKKi ubiquitination. Collectively, our results demonstrate TBK1/IKKi to be novel substrates for A20 and further identifies a novel mechanism whereby A20 and TAX1BP1 restrict antiviral signaling by disrupting a TRAF3/TBK1/IKKi signaling complex. Several viruses utilize a number of strategies to evade the host innate immune response by inhibiting the production of type I interferons. The Human T-cell leukemia virus type 1 (HTLV-1) has been shown to block interferon signaling, however the mechanism of inhibition is poorly understood. We show here that the HTLV-1 encoded protein, Tax plays a critical role in blunting the activation of type I interferons. Tax expression rendered MEFs hyper-permissive in supporting virus replication. Correspondingly, Tax blocked the production of IFN-beta. Interestingly, Tax did not require NEMO interaction to inhibit antiviral signaling to IRF3/7. Instead, Tax targeted RIP1 and further blocked IRF7 K63-linked polyubiquitination. Altogether, we show that Tax inhibits IFN activation by disrupting the ubiquitin dependent activation of IRF7 mediated by RIP1

The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signalling

The RING domain protein RNF11 is overexpressed in breast cancers and promotes tumour growth factor-beta (TGF-β) signalling. RNF11 has been proposed to regulate TGF-β signalling by interacting with HECT- and SCF-type E3 ligases; however, the role of RNF11 in other signalling pathways is poorly understood. Here, we demonstrate a novel function of RNF11 as a negative regulator of NF-κB and jun N-terminal kinase (JNK) signalling pathways. Knockdown of RNF11 with siRNA resulted in persistent tumour necrosis factor (TNF)- and lipopolysaccharide (LPS)-mediated NF-κB and JNK signalling. RNF11 interacted with the NF-κB inhibitor A20 and its regulatory protein TAX1BP1 in a stimulus-dependent manner. RNF11 negatively regulated RIP1 and TRAF6 ubiquitination upon stimulation with TNF and LPS, respectively. Furthermore, RNF11 was required for A20 to interact with and inactivate RIP1 to inhibit TNF-mediated NF-κB activation. Our studies reveal that RNF11, together with TAX1BP1 and Itch, is an essential component of an A20 ubiquitin-editing protein complex that ensures transient activation of inflammatory signalling pathways

TAX1BP1 Restrains Virus-Induced Apoptosis by Facilitating Itch-Mediated Degradation of the Mitochondrial Adaptor MAVS

The host response to RNA virus infection consists of an intrinsic innate immune response and the induction of apoptosis as mechanisms to restrict viral replication. The mitochondrial adaptor molecule MAVS plays critical roles in coordinating both virus-induced type I interferon production and apoptosis; however, the regulation of MAVS-mediated apoptosis is poorly understood. Here, we show that the adaptor protein TAX1BP1 functions as a negative regulator of virus-induced apoptosis. TAX1BP1-deficient cells are highly sensitive to apoptosis in response to infection with the RNA viruses vesicular stomatitis virus and Sendai virus and to transfection with poly(I·C). TAX1BP1 undergoes degradation during RNA virus infection, and loss of TAX1BP1 is associated with apoptotic cell death. TAX1BP1 deficiency augments virus-induced activation of proapoptotic c-Jun N-terminal kinase (JNK) signaling. Virus infection promotes the mitochondrial localization of TAX1BP1 and concomitant interaction with the mitochondrial adaptor MAVS. TAX1BP1 recruits the E3 ligase Itch to MAVS to trigger its ubiquitination and degradation, and loss of TAX1BP1 or Itch results in increased MAVS protein expression. Together, these results indicate that TAX1BP1 functions as an adaptor molecule for Itch to target MAVS during RNA virus infection and thus restrict virus-induced apoptosis

Comprehensive Mutagenesis of Herpes Simplex Virus 1 Genome Identifies UL42 as an Inhibitor of Type I Interferon Induction.

In spite of several decades of research focused on understanding the biology of human herpes simplex virus 1 (HSV-1), no tool has been developed to study its genome in a high-throughput fashion. Here, we describe the creation of a transposon insertion mutant library of the HSV-1 genome. Using this tool, we aimed to identify novel viral regulators of type I interferon (IFN-I). HSV-1 evades the host immune system by encoding viral proteins that inhibit the type I interferon response. Applying differential selective pressure, we identified the three strongest viral IFN-I regulators in HSV-1. We report that the viral polymerase processivity factor UL42 interacts with the host transcription factor IFN regulatory factor 3 (IRF-3), inhibiting its phosphorylation and downstream beta interferon (IFN-β) gene transcription. This study represents a proof of concept for the use of high-throughput screening of the HSV-1 genome in investigating viral biology and offers new targets both for antiviral therapy and for oncolytic vector design.IMPORTANCE This work is the first to report the use of a high-throughput mutagenesis method to study the genome of HSV-1. We report three novel viral proteins potentially involved in regulating the host type I interferon response. We describe a novel mechanism by which the viral protein UL42 is able to suppress the production of beta interferon. The tool we introduce in this study can be used to study the HSV-1 genome in great detail to better understand viral gene functions