UBL4A Augments Innate Immunity by Promoting the K63-Linked Ubiquitination of TRAF6

This information is current as Shu-Jie Peng, Ran-Ran Yao, Shuang-Shuang Yu, Hong-Yan of September 28, 2021. Chen, Xuewen Pang, Yu Zhang and Jun Zhang J Immunol 2019; 203:1943-1951; Prepublished online 26 August 2019; doi: 10.4049/jimmunol.1800750

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Supplementary http://www.jimmunol.org/content/suppl/2019/08/25/jimmunol.180075 Material 0.DCSupplemental http://www.jimmunol.org/ References This article cites 57 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/203/7/1943.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

UBL4A Augments Innate Immunity by Promoting the K63-Linked Ubiquitination of TRAF6

Shu-Jie Peng,1 Ran-Ran Yao,1 Shuang-Shuang Yu, Hong-Yan Chen, Xuewen Pang, Yu Zhang, and Jun Zhang

Human UBL4A/GdX, encoding an ubiquitin-like protein, was shown in this study to be upregulated by viral infection and IFN stimulation. Then the functions of UBL4A in antiviral immune response were characterized. Overexpression of UBL4A promoted RNA virus–induced ISRE or IFN-b or NF-kB activation, leading to enhanced type I IFN transcription and reduced virus replication. Consistently, knockdown of UBL4A resulted in reduced type I IFN transcription and enhanced virus replication. Additionally, overexpression of UBL4A promoted virus-induced phosphorylation of TBK1, IRF3, and IKKa/b. Knockdown of UBL4A inhibited virus-induced phosphorylation of TBK1, IRF3, and IKKa/b. Coimmunoprecipitation showed that UBL4A interacted with TRAF6, and this interaction was enhanced upon viral infection. Ubiquitination assays showed that UBL4A Downloaded from promoted the K63-linked ubiquitination of TRAF6. Therefore, we reveal a novel positive feedback regulation of UBL4A in innate immune response combating virus invasion by enhancing the K63-linked ubiquitination of TRAF6. The Journal of Immunology, 2019, 203: 1943–1951.

iral nucleic acids are sensed by pattern recognition re- promote the production of type I IFNs and proinﬂammatory

ceptors (PRRs) and then induce the production of type I cytokines (24, 25). http://www.jimmunol.org/ V IFNs and proinflammatory cytokines (1, 2). For RNA TNF receptor–associated factor (TRAF) family members have viruses, TLR family member TLR3 senses dsRNA (3, 4), whereas been reported to be essential for the recruitment of the down- TLR7/8 senses ssRNA in the endosomes (5, 6). RIG-I–like re- stream kinases to the adaptor proteins. Among them, TRAF6 is a ceptors (RLRs) recognize viral dsRNA in the cytosol (7–9). For ubiquitin E3 ligase that attaches the K63-linked polyubiquitin DNA viruses, TLR9 senses unmethylated DNA in the endosomes chains on substrate proteins or itself (26). The autoubiquiti- (10). cGMP-AMP (cGAMP) synthase (cGAS) is the main iden- nation at Lys124 of TRAF6 is a key activation step for TRAF6 tified cytosolic viral DNA sensor (CDS) (2, 11). Besides, several (27). It is critical for the recruitment of IKKa/b/g and/or TBK other DNA sensors are reported, including AIM2, DAI, DDX41, complex to adaptor proteins (28, 29). By this, TRAF6 func- IFI16, RNA polymerase III, Lsm14A, etc. (12–17). Upon viral tions as an important signaling scaffold downstream of multiple by guest on September 28, 2021 invasion, viral nucleic acids are recognized by corresponding PRR such as TLRs, NLRs, RLRs, and CDSs, which eventually PRRs and then recruit the downstream adaptor proteins such as leads to the production of proinflammatory cytokines and type I MyD88, TRIF, MAVS, and STING (11, 18–20). Then, the acti- IFNs (19, 30–32). Therefore, the regulation at TRAF6 level vation signals transmitted by different sensors converge at may affect the activation of NF-kB signaling and multiple PRR the common kinase level IKKa/b/g and TBK1 (21–23). Then, signaling. phosphorylated by these kinases, the transcription factors NF-kB, Ubiquitin-like protein 4A (UBL4A)/X-linked gene in the IRF3, and IRF7 are activated and translocate into the nucleus and G6PD cluster at Xq28 (Gdx) was originally identified as a housekeeping gene that shares 43% identity to ubiquitin in the N terminus (33). However, the ubiquitin-related activity of UBL4A is not reported. UBL4A can form a complex with Bag6 and Department of Immunology, School of Basic Medical Sciences, NHC Key Laboratory Trc35 and localize to the endoplasmic reticulum (ER) to reg- of Medical Immunology, Ministry of Health (Peking University), Peking University Health Science Center, Beijing 100191, China ulate ER-associated degradation (34). UBL4A participates in the transport of tail-anchored proteins into the ER (35). UBL4A is 1These authors contributed equally to this work. also involved in DNA damage–induced cell death (36). UBL4A ORCIDs: 0000-0003-3074-3908 (S.-J.P.); 0000-0002-2733-0048 (R.-R.Y.). is a potential tumor suppressor by dephosphorylation of Stat3 Received for publication May 29, 2018. Accepted for publication July 24, 2019. via promoting the interaction between Tc45 and Stat3 (37). This work was supported by grants from the National Natural Science Foundation of For Akt activation, UBL4A mediates insulin-induced plasma- China (81873871 and 31470843) and the Nonprofit Central Research Institute Fund of Chinese Academy of Medical Sciences (2018PT31039). membraned translocation of Akt. UBL4A is also an Arp2/3 Address correspondence and reprint requests to Dr. Jun Zhang, Peking University binding partner and mediates actin branching (38). Until now, Health Science Center, 38 Xue Yuan Road, Hai Dian District, Beijing 100191, China. the functions of UBL4A in antiviral immune response are not E-mail address: [email protected] characterized. The online version of this article contains supplemental material. In the current study, we report that UBL4A is a novel IFN- Abbreviations used in this article: CDS, cytosolic viral DNA sensor; ER, endoplas- stimulated gene (ISG). Overexpression of UBL4A promoted mic reticulum; iBMDM, immortalized bone marrow–derived macrophage; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; NC, negative control; PRR, pattern virus-induced signaling, whereas knockdown of UBL4A had the recognition receptor; RLR, RIG-I–like receptor; SeV, Sendai virus; siRNA, small opposite effects and promoted viral replication. Further studies interfering RNA; TRAF, TNF receptor–associated factor; UBL4A, ubiquitin-like showed that UBL4A physically associated with TRAF6 and protein 4A; VSV, vesicular stomatitis virus. then augment the antiviral immune response by promoting the Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 K63-linked ubiquitination of TRAF6. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800750 1944 POSITIVE REGULATION OF VIRUS-INDUCED SIGNALING BY UBL4A

Materials and Methods Ab was added. Finally, ECL assay (ImageQuant LAS 500) was used to Cells, reagents, and plasmids detect the signal. HEK293, HEK293T, HeLa cells, and immortalized bone marrow–derived Coimmunoprecipitation macrophages (iBMDMs) were maintained in DMEM (Thermo Fisher HEK293T cells were seeded at a density of 2.0 3 105 cells/ml in a 10 cm Scientific, Waltham, MA) supplemented with 10% heat-inactivated FBS cell culture dish. After 24 h, the plasmids were transfected into cells. (CellMax) and penicillin (100 U/ml)/streptomycin (100 mg/ml). Lipofectamine The cells were harvested after another 24 h and then lysed for 1 h, and 3000 and Opti-MEM were purchased from Thermo Fisher Scientific. the supernatant was collected. Each sample was coincubated together Poly(I:C) and poly(dA:dT) were purchased from InvivoGen (San Diego, with the indicated Abs or mouse IgG for control overnight. The superna- CA). pENTER-Flag-His-UBL4A was purchased from Vigene Biosciences tant was coincubated together with protein A agarose beads for another (Rockville, MD). HA-tagged UBL4A was constructed by standard 2 h. Centrifugal pellets were washed with washing buffer. The results molecular biology techniques. Human IFN-a was from BioLegend, and were analyzed according to immunoblot analysis. For endogenous im- IFN-b was from PeproTech (Rocky Hill, NJ). munoprecipitation, HEK293 cells were treated with or without SeV infection for 24 h. Then, the cells were harvested, lysed, and immuno- Abs precipitated by IgG control or anti-UBL4A Ab. The blot was monitored Abs specific for p-TBK1, TBK1, p-IRF3, IRF3, p-IKKa/b, TRAF6, by anti-TRAF6 Ab. p-IkBa, and K63-linkage–specific polyubiquitin were purchased from Cell Signaling Technology (Danvers, MA). The TRAF6 Ab used for immu- In vivo ubiquitination assay noprecipitation was from Santa Cruz Biotechnology (Dallas, TX). UBL4A The HEK293T cells were transfected with the corresponding plasmids or Ab was from Proteintech (Rosemont, IL). Abs specific for GAPDH small interfering RNA (siRNA) oligonucleotides. Twenty-four hours (BioWorld Technology), anti-Flag (Medical & Biological Laboratories posttransfection, the cells were treated with MG132 (10 mM) (Sigma-

Company, Nagoya, Japan), anti-GFP (Sungene Biotech, Tianjin, China), Aldrich) for 8h before harvest. The cells were first lysed in 100 mlof Downloaded from anti-ubiquitin pAb (PTM Biolabs, Chicago, IL), anti-HA (California coimmunoprecipitation lysis buffer containing 1% SDS for 2 h and then Bioscience), and anti-His (Origene, Rockville, MD) were from indicated boiled for 5 min. The supernatant was diluted with lysis buffer to SDS companies. Mouse control IgG and rabbit control IgG were from Sigma- concentration of 0.1%, sonicated for 20 s, and then centrifuged, and the Aldrich. HRP-conjugated goat anti-mouse or -rabbit IgG was from Beijing supernatant was recovered. The supernatant was incubated with the appro- Biodragon Immunotechnologies Company (China). priate Abs overnight and then incubated with protein A agarose beads for 3–4 h. Centrifugal pellets were washed six to nine times with washing buffer. Luciferase assays The ubiquitin-modified proteins were analyzed with anti-ubiquitin Ab. 3 5 http://www.jimmunol.org/ HEK293T cells were seeded into 24-well plates (1.0 10 cells per well). Knockdown of UBL4A in human cells A total of 24 hours later, expressing vectors were transfected with reporter plasmids such as 100 ng of ISRE or IFN-b or NF-kB luciferase reporter The siRNA oligonucleotides targeting human UBL4A (37) (sequence, plasmid, along with 1 ng of pRL-SV40 by polyethylenimine transfection. 59-GCUCAACCUAGUGGUCAAA-39 [1#] and 59-GGAACAGCUACA- If necessary, the empty control plasmid was cotransfected to ensure the GAGGGAU-39 [2#]) were synthesized by GenePharma Company. The se- same amount of total DNA in each transfection. A total of 24 hours later, quence of negative control (NC) is 59-UUCUCCGAACGUGUCACGU-39. cells were infected with Sendai virus (SeV), HSV-1, or transfected by The HEK293 cells were transfected with UBL4A-siRNA 1# or 2# or an poly(I:C) or poly(dA:dT) for 24 h. Luciferase reporter assays were mea- NC using PerMute Reagent (SignaGen Laboratories, Rockville, MD) for sured with the Dual-Luciferase Reporter Assay System (Promega) fol- 18–24 h, then cells were infected by SeV or VSV-GFP or transfected with lowing the manufacturer’s instructions. Renilla luciferase reporter plasmid virus mimics for the indicated hours, and then the cells were harvested for was added to each transfection to normalize the transfection efficiency. PCR, real-time PCR, or immunoblot assays. by guest on September 28, 2021 RT-PCR and quantitative real-time PCR Isolation of primary murine peritoneal macrophages and Cells treated with diverse stimuli were harvested in TRIzol reagent (Thermo RNA interference Fisher Scientific). The first-strand cDNA was synthesized using a reverse The isolation of murine peritoneal macrophages was as described (39). transcription system (Promega). Gene expression was detected to measure Briefly, 1 ml of 4.0% (w/v) thioglycollate medium (Sigma-Aldrich) was mRNA abundance of the tested genes with the Bio-Rad real-time PCR injected i.p. into 6–8-wk-old mice. Three days later, peritoneal macro- system with Power SYBR Green PCR Master mix (Bio-Rad, Hercules, CA). phages were harvested by rinsing peritoneal lavage with PBS. Collected Data shown were the relative expression of the tested mRNA normalized macrophages were resuspended in RPMI 1640 containing 10% FBS in to that of GAPDH or b-actin. Gene-specific primer sequences used were 12-well plates (3 3 105/ml) for further experiments. For RNA interference as follows: IFNB1,59-ACTGCCTCAAGGACAGGATG-39 (forward) and assays, all siRNAs (GenePharma) were transfected by INTERFERin 59-GGCCTTCAGGTAATGCAGAA-39 (reverse); ISG15,59-GGTGGAC- (Polyplus-transfection) at 50 nM according to the manufacturer’s AAATGCGACGAAC-39 (forward) and 59-ATGCTGGTGGAGGCCCTT- instructions. To determine efficiency of protein knockdown, at 48 h post- AG-39 (reverse); ISG56,59-GCCATTTTCTTTGCTTCCCCTA-39 (forward) transfection, cells were harvested, lysed, and immunoblotted with anti- and 59-TGCCCTTTTGTAGCCTCCTTG-39 (reverse); GAPDH,59-A- mouse UBL4A Ab (Proteintech). The sequences of the siRNAs used were CCCACTCCTCCACCTTTGA-39 (forward) and 59-CTGTTGCTG- as follows: NC, 59-UUCUCCGAACGUGUCACGU-39;mouseUbl4a 1#, TAGCCAAATTCGT-39 (reverse); vesicular stomatitis virus (VSV), 59-GGAACAACUACAGAGGGAU-39;mouseUbl4a 2#, 59-AGCUCAAC- 59-ACGGCGTACTTCCAGATGG-39 (forward) and 59-CTCGGTTCAAGA- CUAGUUGUUAA-39 (37). TCCAGGT-39 (reverse). Mouse Ifnb1 (forward): 59-GGCAGTGTAACTCTT- CTGCAT-39; mouse Ifnb1 (reverse): 59-CAGCTCCAAGAAAGGACGAAC-39; Statistical analyses mouse b-actin (forward): 59-AGAGGGAAATCGTGCGTGAC-39, mouse b-actin (reverse): 59-CAATAGTGATGACCTGGCCGT-39.MouseUbl4a Data were shown as mean 6 SD. Statistical analyses were performed using (forward): 59-GCAACGTCTGCTGTTCAAGG-39,mouseUbl4a (reverse): GraphPad Prism 5.0. Statistical significance was determined by the un- 59-CTGCTGGCATCTGCTACACT-39. paired two-tailed Student t test: *p , 0.05, **p , 0.01, and ***p , 0.001. Immunoblot analysis Results HEK293 or HEK293T cells were transfected for 18–36 h, then the cells UBL4A is a putative virus-induced ISG were infected with SeV or VSV-GFP for the indicated time points or left In an attempt to identify virus-induced genes, we found that UBL4A untreated. Cells were lysed in lysis buffer with proteinase inhibitor (Roche) and PMSF. The protein concentration was quantified with a BCA quanti- is upregulated upon RNA viruses (SeV and VSV-GFP) infection fication kit (Promega) before the immunoblot was performed. For detec- both in HEK293T (Fig. 1A, 1B) and HeLa cells (Fig. 1C, 1D). In tion of p-TBK1, p-IRF3, TBK1, IRF3, UBL4A, and GAPDH, 20 mgof addition, IFN-a and IFN-b induce the expression of UBL4A in proteins were loaded for the immunoblot analysis. For detection of HEK293T cells (Fig. 1E, 1F). Similar results were also observed p-IKKa/b, p-IkBa,30mg of proteins were loaded for immunoblot analysis. The Abs were diluted in 5% (w/v) fat-free milk or 5% BSA in TBST, in primary murine peritoneal macrophages and iBMDM upon IFN and then the membrane was incubated at 4˚C with gentle shaking over- stimulation (data not shown). Taken together, UBL4A may be a night. After at least three rounds of washing, HRP-conjugated secondary putative novel ISG. The Journal of Immunology 1945

FIGURE 1. Viral infection and type I IFNs upregulate UBL4A expression. (A and B) Expression of UBL4A protein in HEK293T cells infected by SeV or VSV-GFP for indicated time periods. (C and D) Expression of UBL4A protein in HeLa cells infected by SeV or VSV-GFP for indicated time periods. GAPDH was used as loading control. (E and F) HEK293T cells were stimulated with IFN-a or IFN-b for indicated time periods. Downloaded from

UBL4A augments virus-induced ISRE, IFN-b, and To further examine whether UBL4A is also involved in DNA NF-kB activation virus–induced signaling, the cells were also infected with HSV-1 http://www.jimmunol.org/ Previously, accumulating evidence showed that ISGs may be or transfected with DNA virus mimics poly(dA:dT). Consistent important innate immunity regulators (40). Then we tested the with the data observed upon RNA virus infection, overexpressed UBL4A-augmented DNA virus (Supplemental Fig. 1A) or poly(dA:dT) possibility that UBL4A might regulate antiviral innate immunity. (Supplemental Fig. 1B) induced ISRE, IFN-b, and/or NF-kB ac- To achieve this goal, dual-luciferase reporter assays were ﬁrst tivation. Therefore, UBL4A augments both RNA- and DNA virus– used. UBL4A was overexpressed in HEK293T with ISRE or induced type I IFN signaling. IFN-b or NF-kB reporter plasmid. Then the cells were treated UBL4A promotes type I IFN signaling and inhibits with or without virus infection. As shown in Fig. 2A, overexpressed virus replication

UBL4A augmented SeV-induced ISRE, IFN-b,andNF-kB activa- by guest on September 28, 2021 To support the results above, we also detected the transcription of tion. In addition, UBL4A also augment RNA virus mimics, poly(I:C)– downstream genes induced by type I IFN signaling such as IFNB1, induced ISRE, IFN-b,andNF-kB activation (Fig. 2B). Notably, the ISG15, and ISG56. By monitoring the expression of these genes enhanced effects on ISRE, IFN-b,andNF-kBactivationbyUBL4A by RT-PCR and real-time PCR, it was found that overexpression were dose dependent (Fig. 2C, 2D). of UBL4A promoted virus-induced transcription of IFNB1, ISG15,

FIGURE 2. UBL4A positively regulates RNA-induced antiviral signaling. (A) Luciferase activity in HEK293T cells overexpressed with control vector (1 mg) or UBL4A plasmid (1 mg) together with ISRE/IFN-b/NF-kB luciferase reporter (100 ng) and pRL-SV40 (1 ng) for 24 h, and then the cells were infected with SeVor transfected with poly(I:C) (B) for another 24 h. (C) Luciferase activity in HEK293T cells overexpressed with control vector (1 mg) and UBL4A plasmid in an increasing dose (100/300/500 ng) together with ISRE/IFN-b/NF-kB luciferase reporter (100 ng) and pRL-SV40 (1 ng) for 24 h, and then the cells were infected with SeV for another 24 h. (D) Immunoblot analysis of Flag-UBL4A protein using the lysates of Luciferase activity. GAPDH served as loading control. Results were presented as mean 6 SD of at least two technical replicates. *p , 0.05, **p , 0.01. 1946 POSITIVE REGULATION OF VIRUS-INDUCED SIGNALING BY UBL4A

FIGURE 3. UBL4A positively regulates SeV- mediated type I IFN signaling and inhibits VSV replication. (A–D) Overexpression of UBL4A promoted SeV-induced transcription of IFNB1, ISG15, and ISG56. HEK293T cells were transfected with UBL4A expressing plasmid or control vector for 24 h. HEK293T were infected by SeV for the indicated hours or left untreated. The indicated mRNAs were detected by PCR (A) and quantiﬁed by real-time PCR (B–D). (E–H)Over-

expression of UBL4A attenuates VSV replication. Downloaded from HEK293T cells were transfected with UBL4A expressing plasmid or control vector for 24 h. HEK293T were harvested after being stimulated with VSV-GFP for the indicated hours. And then the PCR (E) and real-time PCR (F) and immunoblot analysis (G) were performed to detect the

replication of VSV-GFP. Moreover, 24 h after http://www.jimmunol.org/ VSV-GFP stimulation, the ﬂuorescence intensity was observed under a ﬂuorescence microscope (H). *p , 0.05, **p , 0.01. by guest on September 28, 2021

and ISG56 in HEK293T cells (Fig. 3A–D). Besides, overexpression Listeria monocytogenes–induced expression of Ifnb1 (Supplemental of UBL4A also enhanced DNA virus mimic poly(dA:dT)–induced Fig. 2B). These data showed that knockdown of UBL4A suppresses transcription of IFNB1 in HEK293T cells (Supplemental Fig. 1C). both virus- and bacteria-induced type I IFN signaling. In con- Then the viral replication was examined upon VSV-GFP infection, sistent with attenuated type I IFN signaling, VSV replication was which includes a GFP tag and facilitates the observation of virus enhanced in HEK293 cells with UBL4A knockdown (Fig. 4E). infection by ﬂuorescent microscopy or by anti-GFP Ab. By RT-PCR Therefore, UBL4A is indeed a positive regulator in innate immune and real-time PCR, it was shown that overexpression of UBL4A response. It may act as an extensive positive regulator in multiple inhibited VSV replication (Fig. 3E, 3F). Additionally, ﬂuorescence innate immune signaling such as RLR-, CDS-, or TLR-mediated microscopy and Western blot showed similar results (Fig. 3G, 3H). signaling. These data showed that overexpression of UBL4A promoted virus- UBL4A promotes TBK1 and NF-kB activation induced signaling and suppressed virus replication. Then we tried to answer how UBL4A exerts its functions in an- Knockdown of UBL4A suppresses virus-induced type I tiviral signaling. The data above show that UBL4A may play a role IFN signaling in RLR-, CDS-, and TLR-induced signaling. It raised the possi- To examine the functions of endogenous UBL4A in antiviral bility that UBL4A acts at the common signaling pathway down- signaling, we knocked down the endogenous expression of UBL4A stream of multiple PRR signaling. As we know, downstream of the in HEK293 cells; both siRNA oligonucleotides against UBL4A adaptor protein MAVS (RNA virus) or STING (DNA virus), had the knockdown effects (Fig. 4A). As shown in Fig. 4B, 4C, the kinase TBK1 or IKKa/b/g are shared in both signal pathways knockdown of UBL4A resulted in reduced transcription of IFNB1, (41–43). Then we monitored whether UBL4A can have an effect ISG15, and ISG56. We further knocked down Ubl4a in primary on the phosphorylation of TBK1 and IKK kinases. As shown murine peritoneal macrophages and iBMDM. It was shown that in Fig. 5A, UBL4A augmented SeV-induced TBK1 and IRF3 knockdown of mouse Ubl4a in primary peritoneal macrophages phosphorylation in HEK293T cells. Moreover, it also promoted led to decreased expression of Ifnb1 upon SeV infection (Fig. 4D). SeV-induced IKKa/b and IkBa phosphorylation in HEK293T cells Knockdown of mouse Ubl4a expression in iBMDM also inhibited (Fig. 5B). In addition, UBL4A enhanced the phosphorylation virus-induced transcription of Ifnb1 (Supplemental Fig. 2A). Inter- of TBK1, IRF3, IKKa/b,andIKBa stimulated by poly(dA:dT) estingly, knockdown of mouse Ubl4a in iBMDM also suppressed (Supplemental Fig. 1D, 1E). The Journal of Immunology 1947 Downloaded from http://www.jimmunol.org/

FIGURE 4. Knockdown of UBL4A inhibits SeV-induced type I IFN signaling and potentiates VSV replication. (A) HEK293 cells were transfected with NC or UBL4A siRNAs. The expression level of UBL4A protein was analyzed by immunoblot using indicated Abs. (B and C) Knockdown of UBL4A inhibits SeV-induced transcription of IFNB1, ISG15,andISG56. HEK293 cells were transfected with UBL4A siRNAs or NC siRNA for 36 h. HEK293 cells were stimulated with SeV for the indicated hours or left unstimulated. The indicated mRNAs were detected by PCR (B)and quantiﬁed by real-time PCR (C). (D) Murine peritoneal macrophages were transfected by INTERFERin with mouse Ubl4a #1 siRNA or control by guest on September 28, 2021 siRNA. A total of 36 hours after transfection, the cells were infected with SeV for indicated hours, and the transcription of Ifnb1 was detected. (E) Knockdown of UBL4A potentiates VSV replication. HEK293 cells were transfected with UBL4A siRNAs or control siRNA for 36 h. HEK293 were harvested after being stimulated with VSV-GFP for the indicated hours. Then the immunoblot analysis was performed to detect the replication of VSV-GFP. *p , 0.05, **p , 0.01, ***p , 0.001.

Besides, during knockdown of UBL4A in HEK293 cells, the degradation of TRAF6 (45, 46). The K63-linked ubiquitination of phosphorylation of TBK1, IRF3, IKKa/b,andIkBa was examined. TRAF6 is associated with activation of NF-kB signaling and As shown in Fig. 5C, 5D, knockdown of UBL4A inhibited virus- diverse PRR signaling (27, 28). Then we monitored whether induced phosphorylation of TBK1, IRF3, IKKa/b,andIkBa. UBL4A affected the ubiquitination of TRAF6. It was found that Furthermore, knockdown of mouse Ubl4a expression in primary UBL4A promoted the K63-linked ubiquitination of TRAF6 but murine peritoneal macrophages resulted in reduced phosphorylation not K48-linked ubiquitination (Fig. 6D). Moreover, we detected of TBK1, IRF3, IKKa/b,andIkBa (Fig. 5E, 5F). Knockdown of the K63-linked ubiquitination of TRAF6 when we knocked down mouse Ubl4a expression in iBMDM showed similar results the expression of endogenous UBL4A expression. As shown in (Supplemental Fig. 2C, 2D). These data suggested that UBL4A acts Fig. 6E, knockdown of UBL4A led to reduced K63-linked ubiq- upstream or at the level of kinases TBK1 and IKKa/b. uitination of TRAF6. These data together suggest that UBL4A is a TRAF6 binding partner and promotes the K63-linked ubiq- UBL4A physically interacts with TRAF6 and augments the uitination of TRAF6. K63-linked ubiquitination of TRAF6 As demonstrated above, UBL4A acts as the common signaling Discussion molecules in both DNA and RNA virus signaling pathways. Then TRAF6 is an important adaptor protein in diverse signaling we tested whether UBL4A can interact with TBK1, IKK, or IKKa/b. pathways. It can mediate the activation of NF-kB, MAPK, It was found that UBL4A cannot interact with TBK1, IKK, or PI3K, and IFN regulatory factor pathways (47). Downstream of IKKa/b (data not shown). TRAF family members are always TLRs, TRAF6 is involved in both MyD88-dependent and shared by multiple signaling pathways (44). We also tested whether TRIF-dependent NF-kB activation (19, 30). Besides TLRs, it UBL4A can interact with TRAF family members. As shown in wasalsoreportedtobeinvolvedinRLR-andCDS-mediated Fig. 6A, coimmunoprecipitation assays showed that UBL4A can spe- signaling pathways. In DNA virus–induced signaling, dsDNA- ciﬁcally interact with TRAF6. Overexpression and endogenous im- mediated NF-kB activation needs the formation of STING- munoprecipitation conﬁrmed the interaction and demonstrated that this TRAF6-TBK1 axis (32). In RLR signaling, the adaptor protein association was enhanced upon viral infection (Fig. 6B, 6C). Then it MAVS inducing NF-kB activation requires the recruitment of was asked how UBL4A affects the functions of TRAF6. It was well IKK a/b/g complextoMAVSbyTRAF6(29,31).TRAF6 known that K48-linked ubiquitination of TRAF6 is linked with the also possesses a ubiquitin E3 ligase activity (47). TRAF6 can 1948 POSITIVE REGULATION OF VIRUS-INDUCED SIGNALING BY UBL4A Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. UBL4A promotes virus-induced phosphorylation of TBK1 and IKKa/b.(A and B) HEK293T cells were transfected with UBL4A expressing plasmid or control vector for 24 h and were harvested after being treated with SeV for the indicated hours. Immunoblot was performed to examine the phosphorylated state of TBK1, IRF3, IKKa/b, and IkBa.(C and D) HEK293 cells were transfected with NC or UBL4A-siRNAs for 36 h, and then cells were infected and harvested for the indicated hours. Phosphorylation of TBK1, IRF3, IKKa/b,andIkBa was detected by immunoblot with corresponding Abs. GAPDH is performed as the loading control. Data are representative of at least three independent experiments with similar results. (E and F) Murine peritoneal macrophages were transfected by INTERFERin with mouse Ubl4a #1 siRNA or NC siRNA. A total of 36 hours after transfection, the cells were infected with SeV for indicated hours, and the phosphorylated state of TBK1, IRF3 (E), IKKa/b,andIkBa (F)were detected by immunoblotting. undergo K48-linked and K63-linked ubiquitination. TRAF6 promotes the degradation of TRAF6 (45). Inositol polyphosphate promotes K63-linked autopolyubiquitination (27, 28). This is multikinase (IPMK) enhances TLR-induced inﬂammation by re- essential for the downstream signaling transduction. Therefore, moving the K48-linked ubiquitination of TRAF6 and prevents it the regulation at TRAF6 level may affect the strength of diverse from degradation (46). NLRP11 attenuates type I IFN signaling PRR signaling. by promoting the degradation of TRAF6 in an MAVS-dependent Deubiquitinases (CYLD, A20, MYSM1) remove the K63-linked manner (52). TRAF6 promotes the ubiquitination of GSK3b, and polyubiquitination of TRAF6 and inhibit the innate immune re- it is essential for poly(I:C)–induced cytokine production (53). sponse (48–50). RNA sensor LGP2 negatively regulates innate TRIM12c binds with TRAF6 and reciprocally enhances their immune response by inhibiting TRAF ubiquitin ligase activity ubiquitination, resulting in cooperative activation of type I IFN (including TRAF6) (51). K48-linkded ubiquitination of TRAF6 signaling (54). The Journal of Immunology 1949

FIGURE 6. UBL4A interacts with TRAF6 and promotes the K63-linked ubiquitination of TRAF6. (A) HEK293T cells were transfected with His-Flag-UBL4A and Flag-TRAFs, respectively (left), or HA-UBL4A and Flag-TRAF6 (right). The cell lysates were immunoprecipitated with anti-His (left) or anti-Flag (right) and analyzed with immunoblot by indicated Abs. (B) HEK293T cells were transfected with His- UBL4A and Flag-TRAF6 for 36 h and infected with SeV for the indicated hours. Immunoprecipitation was performed with anti-His and analyzed by immunoblot. (C)HEK293cellswere left infected or uninfected with SeV for 24 h. Then the cells were harvested for further endogenous immunoprecipitation. The cell lysate was pulled down by anti-UBL4A Ab and detected by anti-TRAF6 Ab. (D) HEK293T cells were transfected with the indicated plasmids. Twenty-four hours

posttransfection, the cells were treated with MG132 for 8 h Downloaded from before harvest. Then the cells were harvested in lysis buffer. The supernatant was incubated with the appropriate Abs overnight. The ubiquitin-modiﬁed proteins were analyzed with anti-Ub Ab. Lysine 48(K48)–linked polyubiquitination (right) or K63-linked polyubiquitination (left) were both detected. (E) HEK293 cells were transfected with NC or

UBL4A siRNA#1, then were infected by SeV for 12 h. Before http://www.jimmunol.org/ harvest, the cells were treated with MG132 for 8 h. Then the cells were harvested in lysis buffer and immunoprecipitated with anti-TRAF6 Ab. The immunoblot was performed with anti-Ub-K63 Ab. by guest on September 28, 2021

TRAF6 is also used by viral components to mediate viral evasion. Notably, in this study, we found that the positive regulation of EBV-encoded BPLF1 deubiquitinates TRAF6 to inhibit NF-kB UBL4A is not restricted to RNA virus infection. It also applies activation (55). HSV Us3 removes TRAF6 polyubiquitination and to DNA virus and bacteria L. monocytogenes infection. These inhibits TLR2 signaling (56). Severe acute respiratory syndrome data suggest that UBL4A is an extensive positive regulator in coronavirus papain-like protease removes the K63-linked poly- innate immune response. ubiquitination of TRAF3 and TRAF6 and inhibits the TLR7 sig- Our data indicate that UBL4A may act at RLR-, CDS-, or naling pathway (48). Therefore, the regulation at TRAF6 level is TLR-mediated signaling. It tells us UBL4A may play a role at the very critical for innate immune response. common signaling pathway downstream of multiple PRR signal- UBL4A is a protein sharing high homology with ubiquitin (33). ing. We first tested the effects of UBL4A on the common kinases However, it has no ubiquitin activity. The functions of UBL4A or downstream transcription factors and found that UBL4A pro- include ER-associated degradation (34), potential tumor suppres- moted virus-induced phosphorylation of TBK1, IRF3, IKKa/b,or sive activity (37), mediating DNA damage–induced cell death (36) IkBa. Knockdown of UBL4A inhibited the phosphorylation of and cell migration (57), etc. Its participation in the immune system these molecules. These demonstrated again that UBL4A posi- is poorly understood. tively regulates antiviral signaling and acts at the shared signaling In an attempt to identify novel virus-induced proteins, unex- pathways downstream of multiple PRR signaling. Then we asked pectedly, we found that the ubiquitous expressed gene UBL4A how UBL4A plays the positive role in antiviral immune response. was induced by virus infection and IFN stimulation. The up- We coimmunoprecipitated UBL4A with the common kinases such regulated expression of UBL4A is slight, not very significant. The as TBK1, IKKa/b. It showed no interaction between them. Then possible reason for that is the ubiquitously high basal expres- we tested the important bridge molecule TRAF family members sion of UBL4A in cells. The upregulation of UBL4A upon viral and found that UBL4A was specifically associated with TRAF6. and IFN stimulation raised the possibility that UBL4A may Endogenous immunoprecipitation demonstrated again the inter- be involved in antiviral immune response. To test this possibility, action. Notably, this interaction is enhanced by viral infection. we first overexpressed UBL4A in HEK293T cells and by dual- Then we tested how UBL4A affects the functions of TRAF6. luciferase activity to test whether UBL4A had an effect on As mentioned above, the K63-linked ubiquitination of TRAF6 virus-induced ISRE, IFN-b, and NF-kB activation. We indeed is essential for the activation of downstream signal transduction. found that UBL4A positively regulates RNA virus–induced sig- Unexpectedly, the ubiquitinationassaysshowedthatUBL4A naling. Consistent with this, the downstream genes were upregu- specifically promoted the K63-linked ubiquitination of TRAF6. lated by UBL4A overexpression. Viral replication was inhibited. Therefore, this study is the first report, to our knowledge, that links Knockdown of UBL4A expression exerts the opposite effects. UBL4A with the ubiquitinated modification of associated proteins. 1950 POSITIVE REGULATION OF VIRUS-INDUCED SIGNALING BY UBL4A

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