Fish and Shellfish Immunology 84 (2019) 857–864 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article A20 (tnfaip3) is a negative feedback regulator of RIG-I-Mediated IFN induction in teleost T Emilie Mérour, Raphaël Jami, Annie Lamoureux, Julie Bernard, Michel Brémont, ∗ Stéphane Biacchesi VIM, INRA, Université Paris-Saclay, 78350, Jouy-en-Josas, France ARTICLE INFO ABSTRACT Keywords: Interferon production is tightly regulated in order to prevent excessive immune responses. The RIG-I signaling A20 pathway, which is one of the major pathways inducing the production of interferon, is therefore finely regulated TNFAIP3 through the participation of different molecules such as A20 (TNFAIP3). A20 is a negative key regulatory factor RIG-I of the immune response. Although A20 has been identified and actively studied in mammals, nothing is known Interferon about its putative function in lower vertebrates. In this study, we sought to define the involvement of fish A20 Teleost orthologs in the regulation of RIG-I signaling. We showed that A20 completely blocked the activation of IFN and ISG promoters mediated by RIG-I. Furthermore, A20 expression in fish cells was sufficient to reverse the antiviral state induced by the expression of a constitutively active form of RIG-I, thus allowing the efficient replication of a fish rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We brought evidence that A20 interrupted RIG-I signaling at the level of TBK1 kinase, a critical point of convergence for many different pathways that activates important transcription factors involved in the expression of many cytokines. Finally, we showed that A20 expression was directly induced by the RIG-I pathway demonstrating that fish A20 acts as a negative feedback regulator of this key pathway for the establishment of an antiviral state. 1. Introduction cytokines. Both TANK-binding kinase 1 (TBK1) and inhibitor-κB kinase ε (IKKε) protein kinases phosphorylate IRF3/IRF7, although TBK1 The innate immune response against virus infection is characterized seems to be the main kinase involved in IRF3/IRF7 activation [3–5]. by the induction of a rapid non-specific antiviral state in order to block Given the critical role of the RIG-I-mediated IFN induction pathway virus replication and spread. This primary immune response involves a and to avoid extensive tissue damage upon resolution of infection, ne- large panel of different pattern recognition receptors (PRRs), including gative regulatory loops are essential to maintain the immune homeo- TLRs and RIG-I-like receptors (RLRs), able to detect distinct viral mo- static balance and to ensure the proper termination of the antiviral lecular patterns, such as nucleic acids and viral proteins, collectively response [6,7]. Among a long list of molecules, the NF-κB dependent known as pathogen-associated molecular patterns (PAMPs). PRR acti- gene, A20, also known as tumor necrosis factor alpha-induced protein 3 vation triggers multiple signaling cascades that lead to the induction of or TNFAIP3 [8], has emerged as a key player in the termination of type I interferons (IFNs) which establish the antiviral state and stimu- tumor necrosis factor (TNF)-induced apoptosis [9], the negative feed- late the adaptive immune response [1]. Among the PRRs, RLRs play a back regulation of inflammation mediated by NF-κB[10,11], and the key role in sensing viral nucleic acids in the cell cytosol and are es- negative control of the IFN pathway by preventing prolonged IRF3 sential in the early induction of type I IFN [2]. Upon viral RNA detec- activation and IFN overexpression following viral infection [12,13]. tion, RIG-I-like receptors reconform and interact with the mitochon- A20 is a cytoplasmic protein of 90 kDa that is expressed in most cell drial activator of virus signaling (MAVS) protein, also known as IPS-1, types and with dual catalytic activity. A20 is an ubiquitin-editing pro- VISA or Cardif. This interaction induces the recruitment of numerous tein with a deubiquitinase activity mediated by its ovarian tumor (OTU) adaptor proteins and kinases to activate IRF3/IRF7 and NF-κB tran- domain at the N-terminal and a C-terminal domain characterized by a scription factors which then translocate from the cytosol to the nucleus seven zinc finger (ZF) structure functioning as an E3 ubiquitin ligase. and induce the expression of the type I IFNs and inflammatory A20 catalyzes the K48-linked ubiquitylation of target proteins through ∗ Corresponding author. E-mail address: [email protected] (S. Biacchesi). https://doi.org/10.1016/j.fsi.2018.10.082 Received 30 August 2018; Received in revised form 1 October 2018; Accepted 29 October 2018 Available online 30 October 2018 1050-4648/ © 2018 Elsevier Ltd. All rights reserved. E. Mérour et al. Fish and Shellfish Immunology 84 (2019) 857–864 its C-terminal ZF domain, a signal for proteasomal degradation. In salmon TO cells were extracted using RNeasy kit (QIAGEN) according parallel, A20 removes K63-linked ubiquitin chains, which function as to the manufacturer's instructions. The RNA was used to generate full- docking sites for protein-protein interactions, from its target proteins length cDNAs using the SMART RACE cDNA amplification kit (BD through its N-terminal OTU domain. The removal of K63-linked ubi- Clontech) with universal primers provided by the manufacturer. PCR quitin chains inactivates the signaling function of the target proteins amplifications were performed using the Advantage 2 PCR kit (BD and facilitates its K48-linked ubiquitylation and degradation [11]. Clontech) by following the instructions recommended by the manu- A20 has been demonstrated as an important negative regulator of facturer for the PCR reaction mixture and the amplification conditions RIG-I signaling [14]. In response to viral infection, RIG-I-like receptors and with gene-specific primers (Table S1A) designed from the ESTs (RIG-I/MDA5) recognize viral RNA and trigger the downstream cascade sequences found in GenBank. The overall nucleotide identities of the through the mitochondrial adaptor MAVS and the E3 ubiquitin ligase sequences between zebrafish and fathead minnow and zebrafish and TRAF3. TRAF3 mediates K63-linked polyubiquitinations of some TBK1 salmon were 83% and 73%, respectively. RT-PCR products were pur- lysine residues, a post-translational modification required for TBK1 ified with QIAquick PCR purification kit (QIAGEN), cloned into the activation by trans-autophosphorylation [15]. As a critical kinase in- eukaryotic expression vectors pcDNA1.1/Amp (Invitrogen), modified to volved in IFN expression, the activity of TBK1 must be tightly regulated fused an HA tag at the 5′ end of the gene of interest, and fully se- [7]. The A20 regulatory complex, including tax1-binding protein 1 quenced. The Neighbor-joining (NJ) phylogenetic tree of A20 was cal- (TAX1BP1) and A20 binding inhibitor of NF-κB 1 (ABIN1) in associa- culated by MEGA6 software [22] based on a multiple alignment (using tion with A20, antagonizes K63-linked polyubiquitination of TBK1 by ClustalW) of full-length A20 amino-acid sequences from fish and other disrupting the interaction between TBK1 and TRAF3 [16,17]. However, vertebrates (protein accession numbers Table S2) and a 1000-boostrap it is still unclear whether the inhibitory effect on IRF3/IRF7 is direct, was performed. through A20 binding to TBK1 and decrease of TBK1 kinase activity, and/or indirect through A20 recruitment to the adaptor molecule 2.3. Transfection, fluorescence microscopy and luciferase activity assay TAX1BP1, where it cooperates with ABIN1 to inhibit TBK1 activation [14]. EPC cells were plated into 6-well plates at a density of 5 × 106 cells The IFN system is remarkably conserved in vertebrates. In parti- per well 24 h prior to transfection by electroporation (Amaxa cular, teleost fish possess functional RLR pathway whose induction by Biosystems; Lonza) as previously described [23]. At 24 or 48 h post- viral pathogens leads to the induction of type I interferon. Several transfection, cells were fixed or lysed for further experiments. Plasmids groups showed that fish have orthologs of human RLRs, including RIG- constructs used in the present study were previously described (RIG-I I, MDA5 and LGP2, as well as several downstream signaling molecules, Nter-eGFP, MAVS, eGFP-MAVS, TBK1, 3xFlag-TBK1, PPM1Bb and such as MAVS, TBK1/IKKε and IRF3/IRF7 [18,19]. To date, almost IRF3ΔCter) [23–25]. nothing is known about the fine regulation of the RIG-I-mediated IFN For immunofluorescence microscopy, the cell monolayers were expression [20]. In the present study, we sought to define the in- fixed with a mixture of alcohol and acetone [1:1 (v/v)] or with 80% volvement of fish A20 orthologs in the regulation of RIG-I signaling. methanol, after mitochondria staining using 500 nM of MitoTracker Activation of IFN or ISG promoters by RIG-I was completely inhibited DeepRed FM (Invitrogen) at −20 °C for 15 min. Antigen detection was by A20 expression in fish cells. We showed that A20 interrupted RIG-I performed by incubation with mouse anti-Flag M2 (1/200) or rabbit signaling at the level of TBK1 by physically interacting and co-loca- anti-HA mAbs (1/50; Sigma) diluted in 1x PBS containing 0.05% Tween lizing with this important kinase. Furthermore, fish cells expressing the 20 for 45 min at room temperature. Cells were then washed three times, active form of RIG-I induced an antiviral state that totally blocked re- incubated with Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor plication of a Piscine Novirhabdovirus, the viral hemorrhagic septicemia 594-conjugated anti-rabbit immunoglobulins (Invitrogen) for 45 min at virus (VHSV), an effect that was reversed by co-expression of A20. Fi- room temperature and washed again. Cell monolayers were then vi- nally, we showed that A20 was up-regulated by the RIG-I pathway itself sualized directly with a UV-light microscope (Carl Zeiss) after mounting demonstrating that fish A20 acts as a negative feedback regulator of of the coverslips using Pro-Long Gold antifade reagent with 4’, 6-dia- RIG-I-mediated induction of the antiviral state.