Regulation of Virus-triggered Signaling by OTUB1- and OTUB2-mediated Deubiquitination of TRAF3 and TRAF6*

Ubiquitination and deubiquitination have emerged as critical post-translational regulatory mechanisms for activation or attenuation of the virus-triggered type I interferon (IFN)2 induction pathways. In this study, we identified two deubiquitinating enzymes, OTUB1 and OTUB2, as negative regulators of virus-triggered type I IFN induction. Overexpression of OTUB1 and OTUB2 inhibited virus-induced activation of IRF3 and NF-κB, transcription of the IFNB1 gene as well as cellular antiviral response, whereas knockdown of OTUB1 and OTUB2 had opposite effects. Coimmunoprecipitations indicated OTUB1 and -2 interacted with TRAF3 and TRAF6, two E3 ubiquitin ligases required for virus-triggered IRF3 and NF-κB activation, respectively. Furthermore, we found that OTUB1 and OTUB2 mediated virus-triggered deubiquitination of TRAF3 and -6. These findings suggest that OTUB1 and OTUB2 negatively regulate virus-triggered type I IFN induction and cellular antiviral response by deubiquitinating TRAF3 and -6.

Viral infections triggered a series of signaling events that lead to induction of type I interferons (IFNs). Type I IFNs then activate the JAK-STAT signal transduction pathways, leading to transcriptional induction of a wide range of downstream antiviral genes and subsequent innate antiviral response (1)(2)(3)(4). Transcriptional induction of type I IFN genes requires the coordinate activation of multiple transcription factors and their cooperative assembly into transcriptional enhancer complexes in vivo. For example, the IFNB1 gene promoter contains conserved enhancer elements recognized by NF-B (B site) and phosphorylated IRF3 (ISRE site, also known as PRDIII or IRF-E). It has been shown that transcriptional activation of the IFNB1 gene requires coordinate and cooperative assembly of an enhanceosome that contains all of these transcription factors (2,5,6). The innate immune system has developed at least two types of pathogen recognition receptors for the recognition of viral RNAs (7).One is mediated by membrane-bound Toll-like receptors (TLRs) such as TLR3. Engagement of TLR3 by double-stranded RNA triggers TRIF-mediated signaling pathways, leading to IRF3 and NF-B activation (8). The second one involves the cytosolic RIG-I-like receptor family members RIG-I, MDA5, and Lgp2. Both RIG-I and MDA5 contain two CARD modules at their N terminus and a DexD/H-box RNA helicase domain at their C terminus (9,10). Upon viral infection, the RNA helicase domains of RIG-I and MDA5 serve as intracellular viral RNA receptors, whereas their CARD modules are associated with the downstream CARD-containing adapter protein VISA (also known as MAVS, IPS-1, and Cardif) (11)(12)(13)(14)(15). The essential roles of VISA in antiviral innate immune response were demonstrated by the observations that VISAdeficient mice failed to mount a proper IFN response to viral infections (14,16). Various studies have demonstrated that VISA plays a central role in assembling a complex that activates distinct signaling pathways leading to NF-B and IRF3 activation, respectively. VISA is associated with TRAF6 through its TRAF interaction motifs. It has been shown that TRAF6 is required for activation of IKK and subsequent NF-B (17,18). VISA is also associated with TRAF3, another member of the TRAF protein family (19,20). Gene knock-out studies have demonstrated that TRAF3 is essential in virus-triggered IRF3 activation and type I IFN induction (20,21).
Accumulating evidence has revealed critical roles of posttranslational modification, particularly ubiquitination and deubiquitination, in RIG-I-like receptor-mediated IFN signaling. It has been shown that the E3 ubiquitin ligase TRIM25 catalyzes Lys 63 -linked ubiquitination of RIG-I, and this ubiquitination is essential for the interaction of RIG-I with VISA as well as for its ability to signal (22). The Riplet/REUL E3 ubiquitin ligase also targets RIG-I for ubiquitination, which positively regulates RIG-I-mediated signaling (23). In contrast, the E3 ubiquitin ligase RNF125 catalyzes Lys 48 -linked ubiquitination of RIG-I and negatively regulates RIG-I-mediated signaling (24). In addition, an OTU (ovarian tumor domain) containing deubiquitinating enzyme, CYLD, dissociates Lys 63 -linked ubiquitin moieties from RIG-I, and therefore negatively regulates RIG-Imediated signaling (25,26). The ubiquitination/deubiquitination systems also target other components in the virus-triggered pathways. For example, MITA and IRF3 are ubiquitinated by RNF5 and RBCK1, respectively, and subsequently degraded by proteasome-dependent processes (27,28). The E3 ubiquitin ligase Nrdp1 catalyzes the ubiquitination of TBK1, leading to its activation (29). A20, which contains an N-terminal OTU domain and a C-terminal E3 ligase domain, inhibits virus-triggered IFN signaling pathways through its E3 ligase activity toward an unidentified target (30 -32).
In this report, we found that two OTUB (Otubain) deubiquitinating enzyme family members, OTUB1 and OTUB2, deu-biquitinate TRAF3 and TRAF6, leading to the inhibition of virus-induced IFN-␤ expression and cellular antiviral responses. Our findings provide insights into the mechanisms on regulation of TRAF3 and TRAF6 mediated signaling by deubiquitination.
Expression Cloning-The expression clones on GFC transfection arrays were obtained from Origene. The clones were transfected together with the IFN-␤ luciferase reporter into 293 cells. Sixteen h after transfection, cells were infected with SeV or left uninfected for 8 h. The clones that inhibited SeV-triggered activation of the IFN-␤ promoter were isolated and validated by repeating the reporter assays.
Transfection and Reporter Assays-293 cells were seeded on 24-well dishes and transfected the following day by standard calcium phosphate precipitation. To normalize for transfection efficiency, 0.05 g of pRL-TK (Renilla luciferase) reporter plasmid was added to each transfection. Luciferase assays were performed using a dual-specific luciferase assay kit (Promega). Firefly luciferase activities were normalized based on Renilla luciferase activities. All reporter assays were repeated for at least three times.
Coimmunoprecipitation and Immunoblotting Analysis-For transient transfection and coimmunoprecipitation experiments, 293 cells (ϳ1 ϫ 10 6 ) were transfected for 24 h. The transfected cells were lysed in 1 ml of lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton, 1 mM EDTA, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). For each immunoprecipitation, 0.4 ml aliquot of lysate was incubated with 0.5 g of the indicated antibody or control IgG and 25 l of a 1:1 slurry of GammaBind G Plus-Sepharose (Amersham Biosciences) for 2 h. Sepharose beads were washed three times with 1 ml of lysis buffer containing 500 mM NaCl. The precipitates were analyzed by standard immunoblot procedures.
For endogenous coimmunoprecipitation experiments, 293 cells (5 ϫ 10 7 ) were infected with SeV for the indicated times or left uninfected. The coimmunoprecipitation and immunoblot experiments were performed as described above.
VSV Plaque Assay-293 cells grown in media containing 1% fetal bovine serum were incubated with VSV at a multiplicity of infection 0.1 for 1 h before replacement with the complete media containing 10% fetal bovine serum. Twenty-four h later, the supernatant was harvested and diluted to infect confluent BHK21 cells cultured on 24-well dishes. One hour postinfection, cell culture medium was removed, and 2% methylcellulose was overlaid. On day 3 postinfection, overlay was removed and cells were fixed with 0.5% glutaraldehyde for 30 min and stained with 1% crystal violet in 70% methanol for 15 min. Plaques were counted, averaged, and multiplied by the dilution factor to determine viral titer as log 10 (plaque-forming unit/ml).
Subcellular Fractionation-The cell fractionation experiments were performed as described previously (36). In brief, 293 cells (6 ϫ 10 7 ) infected with SeV or left uninfected for various time points were washed with phosphate-buffered saline and lysed by douncing 40 times in 5 ml homogenization buffer (10 mM Tris-HCl, pH 7.4, 2 mM MgCl 2 , 10 mM KCl, 250 mM sucrose). The homogenate was centrifuged twice at 500 g for 10 min. The supernatant was centrifuged at 5,000 ϫ g for 10 min to precipitate mitochondria. The supernatant was further centrifuged at 50,000 ϫ g for 60 min to generate cytosol. The mitochondria fraction was lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Nonidet P-40, protease inhibitor mixture) for 20 min followed by immunoprecipitation or immunoblotting analysis.

Identification of OTUB1 and OTUB2 as Two Negative Regulators of Virus-triggered Signaling Pathways-Virus-triggered
IFN induction is delicately regulated to avoid excessive immune response. Because ubiquitination of several key components of the virus-triggered IFN pathways is important for their activation, we reasoned that these components are also regulated by certain deubiquitinating enzymes. To identify such enzymes, we screened a protease cDNA array containing 352 clones for proteases that inhibit SeV-induced activation of the IFN-␤ promoter in reporter assays. These experiments identified OTUB1 as an inhibitor of SeV-induced activation of the IFN-␤ promoter (Fig. 1A).
OTUB1 is a member of the OTU domain-containing Otubain family of cysteine proteases (33). The OTU domain confers the deubiquitinase activity of this family. During the course of this work, it was reported that the Otubain family member DUBA/OTUD5 deubiquitinates TRAF3 and negatively regulates TLR3-and RIG-I/MDA5-mediated IFN induction (34). Very recently, it was reported that two Otubain family members, OTUB2 and OTUD5/DUBA, are among 283 genes identified in a RNA interference screen for human genes asso-  ciated with West Nile virus infection (35). We reasoned that these proteins could act to inhibit virus-triggered IFN induction and cellular antiviral response and therefore were required for efficient West Nile virus infection. In reporter assays, OTUB2, like OTUB1 and DUBA, inhibited SeV-induced activation of the IFN-␤ promoter in a dose-dependent manner (Fig.  1A). OTUB1 and OTUB2 could also inhibit expression of endogenous IFNB1 and RANTES genes, and their effects were accumulative in these experiments (Fig. 1B&C), suggesting OTUB1 and OTUB2 have redundant functions in inhibiting virus-triggered signaling. Consistently, OTUB1 and OTUB2 also inhibited SeV-triggered ISRE and NF-B activation (Fig. 1,  D and E), as well as cytoplasmic poly(I⅐C)-and B-DNA-induced activation of the IFN-␤ promoter (Fig. 1F). The effects of OTUB1 and OTUB2 on virus-induced IFN signaling are not cell type-specific because OTUB1 and OTUB2 also inhibited SeV-induced IFN-␤ promoter in A549 cells (Fig. 1G). TRIF is an essential adapter for TLR3-and TLR4-mediated induction of type I IFNs. In reporter assays, OTUB1 and OTUB2 also inhibited TRIF-mediated activation of the IFN-␤ promoter (Fig. 1H). In similar experiments, OTUB1 and OTUB2 did not inhibit IFN-␥-induced activation of the IRF1 promoter (Fig.  1I). These results suggest that OTUB1 and OTUB2 specifically inhibit virus-and TLR3/4-triggered signaling.
To determine the roles of endogenous OTUB1 and OTUB2, we investigated the effects of knockdown of OTUB1 and OTUB2 on virus-triggered IFN signaling. Reporter assays indicated that knockdown of OTUB1 and OTUB2 potentiated SeV-induced activation of the IFN-␤ promoter (Fig. 2, A and  B). In these experiments, knockdown of DUBA had a weaker potentiation effect than knockdown of OTUB1 or OTUB2 (Fig. 2B). Knockdown of OTUB1 and OTUB2 also potentiated SeV-induced expression of endogenous IFNB1 and RANTES genes (Fig. 2, C and D). (The OTUB1-1 OTUB1-RNAi and OTUB2-1 OTUB2-RNAi plasmids were used for all the following experiments.) Consistently, knockdown of OTUB1 and OTUB2 also potentiated SeV-triggered ISRE and NF-B activation (Fig. 2, E and  F).Similar results were obtained in A549 cells (Fig. 2G). Knockdown of OTUB1 and OTUB2 also potentiated TRIF-mediated activation of the IFN-␤ promoter (Fig. 2H).  were transfected with an IFN-␤ promoter reporter (0.05 g) and the indicated expression plasmids (0.1 g each) for 20 h before luciferase assays were performed. *, p Ͻ 0.05, n ϭ 3; **, p Ͻ 0.01, n ϭ 3. B, OTUB1 and OTUB2 interacts with TRAF3, TRAF6, and VISA but not RIG-I. 293 cells (2 ϫ 10 6 ) were transfected with the indicated plasmids. Immunoprecipitation (IP) and Western blot analysis were performed with the indicated antibodies (Ab). C, virus-induced recruitment of OTUB1 and OTUB2 to the VISA-associated complex at the mitochondria. 293 cells (6 ϫ 10 7 ) were infected with SeV for the indicated times or left uninfected. The mitochondria were isolated by cell fractionation. The mitochondrial lysates were immunoprecipitated and analyzed by Western blots with the indicated antibodies. NS, nonspecific; AIF, apoptosis-inducing factor, serving as a mitochondrial control protein; Rel. Lucif. Act, relative luciferase activity; F, FLAG.
Taken together, these results suggest that OTUB1 and OTUB2 are two negative regulators of viruses, as well as TLR3/4-triggered signaling pathways.
OTUB1 and OTUB2 Negatively Regulate Cellular Antiviral Response-Because OTUB1 and OTUB2 negatively regulate virus-triggered IFN signaling, we further confirmed their roles in cellular antiviral response. We determined the effects of knockdown of these proteins on viral replication. In plaque assays, knockdown of OTUB1 and OTUB2 inhibited VSV replication and enhanced the inhibitory effect triggered by cytoplasmic poly(I⅐C) (Fig. 3). These results suggest OTUB1 and OTUB2 negatively regulate cellular antiviral response.
OTUB1 and OTUB2 Inhibit VISA-but Not TBK1-mediated Signaling-Various components are involved in virus-induced signaling. To determine at which levels that OTUB1 and OTUB2 regulate virus-induced signaling, we performed reporter assays. As shown in Fig. 4A, OTUB1 and OTUB2 inhibited RIG-I-N, MDA5-and VISA-mediated but not TBK1and IRF3-mediated activation of the IFN-␤ promoter, suggesting that OTUB1 and OTUB2 target a step downstream of VISA but upstream of TBK1.
OTUB1 and OTUB2 Are Associated with TRAF3 and TRAF6 and Recruited to the VISA-associated Complex-Because TRAF3 and TRAF6 functions downstream of VISA in virus-triggered IFN-␤ induction, we hypothesized that TRAF3 and TRAF6 are the targets of OTUB1 and OTUB2. Coimmunoprecipitation experiments indicated that OTUB1 and OTUB2 interacted with TRAF3 and TRAF6 (Fig. 4B). In the same experiments, OTUB1 also interacted with VISA but not RIG-I (Fig. 4B). Moreover, endogenous coimmunoprecipitation experiments indicated that OTUB1 and OTUB2 are both recruited to the VISA-associated complex after SeV infection, though the dynamics was different among the proteins (Fig. 4C). These results suggest that components of the VISA-associated complex are potential targets of OTUB1 and OTUB2.
OTUB1 and OTUB2 Deubiquitinate TRAF3 and TRAF6-Because OTUB1 and OTUB2 are deubiqutinating enzymes, we determined whether OTUB1 and OTUB2 could remove ubiquitin moieties from components of the VISA-associated complex. As shown in Fig. 5A, overexpression of OTUB1 and OTUB2 caused deubiquitination of TRAF3 and TRAF6, but not RIG-I or VISA. The combination of OTUB1 and OTUB2 further enhanced the deubiquitination effects (Fig. 5A). Conversely, knockdown of OTUB1 and OTUB2 potentiated SeV-induced ubiquitination of TRAF3 and TRAF6 (Fig. 5B). These results suggest that OTUB1 and OTUB2 specifically deubiquitinate TRAF3 and TRAF6 after viral infection.

DISCUSSION
Virus-triggered induction of type I IFNs is crucial for the early innate antiviral response as well as late stage adaptive immunity. This process is delicately regulated in a spatio-temporal manner by various molecules and distinct mechanisms. Ubiquitination and deubiquitination have emerged as critical post-translational regulatory mechanisms for activation or attenuation of the virus-triggered IFN response pathways. Previous studies have demonstrated a critical role for the members of the TRAF protein family in the virus-triggered induction of type I IFNs. However, how TRAFs are regulated in virus-triggered IFN induction pathways is not understood. In this report, we demonstrated that TRAF3 and TRAF6 were negatively regulated through deubiquitination by OTUB1 and OTUB2 in the virus-triggered IFN induction pathways.
Because several components of the virus-triggered IFN induction pathways are ubiquitinated and their ubiquitinations are essential for their abilities to signal, we attempted to identify deubiquitination enzymes that negatively regulate the pathways. These efforts led to the identification of OTUB1 and OTUB2, two members of the Otubain deubiquitinating enzyme family. We found that overexpression of either OTUB1 or OTUB2 inhibited SeV-triggered activation of ISRE and NF-B as well as the IFN-␤ promoter. Moreover, knockdown of either OTUB1 or OTUB2 potentiated SeV-triggered activation of ISRE and NF-B as well as the IFN-␤ promoter. Previously, it has been demonstrated that DUBA/OTUD5, also a member of the Otubain family, deubiquitinates TRAF3 and negatively regulates TLR3-and RIG-I/MDA5mediated IFN induction (34). In our experiments, knockdown of DUBA had a relative minor potentiation effect on virus-triggered signaling in comparison to either OTUB1 or OTUB2. These studies suggest that OTUB1, OTUB2, and DUBA have redundant roles in inhibition of virus-triggered signaling. In the course of this work, one study reported that two Otubain family members, OTUB2 and OTUD5/DUBA, were among 283 genes identified in a RNA interference screen for human genes associated with the West Nile virus infection (35). In light of this observation and our studies, we propose that OTUB1, OTUB2, and DUBA promote viral infection by inhibiting virus-triggered induction of type I IFNs and cellular antiviral response.
Several observations suggest that OTUB1 and OTUB2 mediate the inhibition of the virus-triggered signaling through deubiquitination of TRAF3 and TRAF6. Firstly, OTUB1 and OTUB2 inhibited upstream RIG-I-, MDA5-, and VISA-mediated but not downstream TBK1-mediated activation of the IFN-␤ promoter. Second, OTUB1 and OTUB2 were recruited to the VISA complex after viral infection. Third, overexpression of OTUB1 and OTUB2 abolished ubiquitination of TRAF3 and TRAF6 but not that of upstream component RIG-I and VISA, whereas knockdown of OTUB1 and OTUB2 increased SeV-induced ubiquitination of TRAF3 and TRAF6. These results suggest that OTUB1 and OTUB2 function to deubiquitinate TRAF3 and TRAF6, two E3 ubiquitin ligases required for virus-triggered IRF3 and NF-B activation pathways, respectively.
Regulation of the virus-triggered IFN pathways and OTUB1 and OTUB2 are further supported by their abilities to regulate viral replication. In plaque assays, knockdown of OTUB1 and OTUB2 had opposite effects. In conclusion, our findings revealed the regulatory mechanism of TRAF3 and TRAF6 in virus-triggered IFN induction pathways and further underscored the importance of ubiquitination and deubiquitination in precise control of cellular antiviral response.