Signaling by Stabilizing STAT1 Ubiquitin-Specific Protease 13

Ubiquitin-Specific Protease 13 Regulates IFN Signaling by Stabilizing STAT1 Hom-Ming Yeh, Chia-Yi Yu, Ho-Chun Yang, Shih-Han Ko, Ching-Len Liao and Yi-Ling Lin This information is current as of October 1, 2021. J Immunol 2013; 191:3328-3336; Prepublished online 12 August 2013; doi: 10.4049/jimmunol.1300225 http://www.jimmunol.org/content/191/6/3328 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2013/08/12/jimmunol.130022 Material 5.DC1 References This article cites 52 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/191/6/3328.full#ref-list-1 http://www.jimmunol.org/

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ubiquitin-Speciﬁc Protease 13 Regulates IFN Signaling by Stabilizing STAT1

Hom-Ming Yeh,*,† Chia-Yi Yu,† Ho-Chun Yang,†,‡ Shih-Han Ko,†,x Ching-Len Liao,x and Yi-Ling Lin*,†,x,{

The IFN immune system comprises type I, II, and III IFNs, signals through the JAK-STAT pathway, and plays central roles in host defense against viral infection. Posttranslational modifications such as ubiquitination regulate diverse molecules in the IFN pathway. To search for the deubiquitinating enzymes (DUBs) involved in the antiviral activity of IFN, we used RNA interference screening to identify a human DUB, ubiquitin-specific protease (USP) 13, whose expression modulates the antiviral activity of IFN-a against dengue virus serotype 2 (DEN-2). The signaling events and anti–DEN-2 activities of IFN-a and IFN-g were reduced in cells with USP13 knockdown but enhanced with USP13 overexpression. USP13 may regulate STAT1 protein because the protein level and stability of STAT1 were increased with USP13 overexpression. Furthermore, STAT1 ubiquitination was reduced in cells with Downloaded from USP13 overexpression and increased with USP13 knockdown regardless of with or without IFN-a treatment. Thus, USP13 positively regulates type I and type II IFN signaling by deubiquitinating and stabilizing STAT1 protein. Overall, to our knowledge, USP13 is the first DUB identified to modulate STAT1 and play a role in the antiviral activity of IFN against DEN-2 replication. The Journal of Immunology, 2013, 191: 3328–3336.

he cytokines IFNs play important roles in antiviral, anti- STAT1 and activates IFN-stimulated genes by binding to IFN-g proliferative, and antitumor activities. There are three activation sequence (GAS) (4, 5). http://www.jimmunol.org/ T IFN families: type I IFNs (including IFN-b and diverse The IFN pathway is tightly regulated by several posttranslation- species of IFN-a) are produced by numerous cell types, and type al mechanisms such as phosphorylation, acetylation, and ubiq- II (IFN-g) and type III (IFN-l) IFNs are largely restricted to uitination (6–8). Ubiquitin, a 76-aa polypeptide, can be covalently immune and epithelial cells, respectively (1, 2). After binding to attached to cellular proteins by an enzymatic cascade involving IFN receptors, IFNs primarily signal through receptor-associated three classes of enzymes, E1, E2, and E3 (9). Ubiquitin forms an JAK kinases, which leads to activation of STAT proteins (3). With isopeptide linkage with substrate protein via the C-terminal glycine type I IFN stimulation, phosphorylated STAT1 and STAT2 di- residue of ubiquitin and the lysine residue on the substrate protein. merize and associate with IFN regulatory factor 9 to form IFN- Ubiquitin has seven lysine residues (K6, K11, K27, K29, K33, K48, by guest on October 1, 2021 stimulated gene factor 3, which then binds to specific DNA and K63); each can form an isopeptide bond with the C-terminal sequences named IFN-stimulated response elements (ISREs). glycine residue of the following ubiquitin. Monoubiquitination Type II IFN triggers the homodimer formation of phosphorylated refers to only one ubiquitin conjugating to the lysine of the substrate protein. However, ubiquitins are often conjugated to substrates by forming a polyubiquitin chain, such as K48-linked polyubiquitination, which marks proteins for proteasome degradation, and K63-linked *Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan; †Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan; polyubiquitination, which can regulate signal transduction and DNA ‡Graduate Institute of Microbiology, National Taiwan University College of Medi- repair processes (10). cine, Taipei 10051, Taiwan; xDepartment of Microbiology and Immunology, National { Several components of the IFN signaling pathway are modified and Defense Medical Center, Taipei 11490, Taiwan; and Genomic Research Center, Academia Sinica, Taipei 11529, Taiwan controlled by ubiquitination. For example, a cullin-based E3 ligase Received for publication January 22, 2013. Accepted for publication July 14, 2013. facilitates the ubiquitination and degradation of type I IFN receptor This work was supported by National Science Council, Taiwan Grants NSC100- IFNAR1 (11), and suppressor of cytokine signaling 1, a component 2923-B-001-002-MY3 and NSC101-2321-B-001-028-MY3 and by Academia Sinica, of an E3 ligase, promotes JAK2 polyubiquitination and degradation Taiwan. (12, 13). STAT1 could also be regulated by ubiquitination, and IFN-g Address correspondence and reprint requests to Dr. Yi-Ling Lin, Institute of Biomedical triggers STAT1 phosphorylation and ubiquitin-mediated protein Sciences, Academia Sinica, No. 128, Section 2, Academy Road, Taipei 115, Taiwan. E-mail address: [email protected] degradation (14). A nuclear E3 ligase called STAT-interacting LIM (SLIM) protein (also known as PDLIM2) causes STAT1 and STAT4 The online version of this article contains supplemental material. ubiquitination and degradation (15, 16). Another E3 ligase, Smad Abbreviations used in this article: DEN, dengue virus; DEN-2, dengue virus serotype 2; DUB, deubiquitinating enzyme; GAS, IFN-g activation sequence; GAS-Luc, IFN-g ubiquitination regulating factor 1 (Smurf1), was recently found to activation sequence–luciferase; HA, hemagglutinin; HA-Ub, hemagglutinin-tagged catalyze K48 polyubiquitination and proteasomal degradation of ubiquitin; IB, immunoblotting; IP, immunoprecipitation; ISRE, IFN-stimulated re- STAT1 (17). Overexpression of SLIM or Smurf1 attenuates IFN-g– sponse element; ISRE-Luc, IFN-stimulated response element–driven luciferase; MOI, multiplicity of infection; OTU, ovarian tumor; RNAi, RNA interference; shLuc, mediated STAT1 activation, whereas knockdown of SLIM or luciferase-targeted short hairpin RNA; shRNA, short hairpin RNA; shUSP13, Smurf1 enhances IFN-g–mediated STAT1 activation and the anti- ubiquitin-specific protease 13–targeted short hairpin RNA; SLIM, STAT-interacting LIM; Smurf1, Smad ubiquitination regulating factor 1; TRAF, TNFR-associated viral immune response (15, 17). factor; UBA, ubiquitin-associated; USP, ubiquitin-specific protease; USP13-res, RNA Ubiquitination is a reversible process and the human genome interference–resistant ubiquitin-specific protease 13; WT, wild-type; ZnF-UBP, zinc contains ∼100 deubiquitinating enzymes (DUBs) (18, 19). Similar finger ubiquitin-specific protease. to phosphorylation and dephosphorylation, deubiquitination pro- Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 vides an important layer of regulation for ubiquitination by re- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300225 The Journal of Immunology 3329 moving ubiquitin from their substrates. Several DUBs negatively of DEN infection as compared with the luciferase-targeted shRNA (shLuc) regulate immune responses. CYLD specifically removes K63-linked control and the clones have a live cell number not ,40% of the shLuc polyubiquitin chains from TNFR-associated factors (TRAFs), thus control. leading to IkB kinase inhibition in the NF-kB activation pathway Generation of recombinant lentivirus (20). A20 is a dual-function enzyme with DUB activity, which The lentiviral vectors expressing shRNAs targeting USP13 (G9, 59-GCA- preferentially cleaves K63-linked polyubiquitin chains, and E3 li- GATAAAGAAGTTCACTTT-39 [TRCN0000007248] and B10, 59-CGAT- gase activity, which builds K48-linked polyubiquitin chains (21, 22). TTAAATAGCGACGATTA-39 [TRCN0000007251]) were from the Taiwan Thus, A20 inhibits NF-kB activation upstream of IkBkinaseby National RNAi Core Facility. Lentivirus was prepared by cotransfection of USP13-targeted shRNA (shUSP13)/pLKO.1-puromycin with helper plas- removing K63-linked polyubiquitin chains and promoting K48- mids, pCMVDR8.91 and pMD.G, into HEK293T cells with use of Lip- linked polyubiquitination and degradation of receptor-interacting ofectamine 2000 (Invitrogen). The culture supernatants were harvested 24, protein 1 (23–25). Additionally, ovarian tumor (OTU) B1 and 36, and 48 h after transfection and further concentrated by centrifuging at OTUB2 inhibit virus-triggered IFN-b expression by deubiquiti- 20,000 3 g for 3 h at 4˚C. nating TRAF3 and TRAF6 (26). Furthermore, ubiquitin-specific Plasmid construction protease (USP) 17 (also called DUB3) positively regulates virus- The human USP13 cDNA with or without hemagglutinin (HA) tag was induced type I IFN signaling through interaction and deubiquiti- PCR amplified from Flag-HA-USP13 (Addgene, plasmid 22568) with the nation of RIG-I and MDA5 (27). However, research into deubi- primer pair sequences for HA-USP13, 59-CCCAAGCTTATGTACCCA- quitination lags behind that of ubiquitination, and not many DUBs TACGATGTTCCA-39 and 59-AACGGGCCCTTAGCTTGGTATCCT- with a role in cellular immunity have been revealed. GC-39, and USP13, 59-TAAAAGCTTATGCAGCGCCGGGGCGCCCT- 39 and 59-AATGGGCCCGAGCTTGGTATCCTGCGGTAAA-39 (the To gain more insight into the role of cellular DUBs in host defense,

restriction enzyme recognition sequences are in italics and USP13 sequences Downloaded from we performed RNA interference (RNAi) screening to search for are in bold). The HA-USP13 and USP13 cDNAs were subcloned into DUBs involved in the antiviral activity of type I IFN against dengue pCR3.1 and pcDNA3.1, respectively. To generate HA-USP13/pTY for virus (DEN) infection. DEN belongs to the Flavivirus genus of the lentivirus production, HA-USP13 was PCR amplified and subcloned into the family Flaviviridae and has emerged as the most important vector- lentiviral vector pTY-EF1a. To generate RNAi-resistant USP13 (USP13- res), we introduced two point mutations for USP13-shRNA targeting borne viral disease in tropical and subtropical areas (28). DEN has sequences by the single-primer mutagenesis method (32) with the primer four serotypes, and people infected with DEN may develop mild sequences for shUSP13-G9, 59-CTGAATACTTGGTAGTGCAGATCAA- dengue fever or severe dengue hemorrhagic fever and dengue shock GAAATTCACTTTTGGTCTTGACTGGG-39, and shUSP13-B10, 59- http://www.jimmunol.org/ syndrome. The magnitude of viral replication is thought to be one of ATCTAGATACTGATGACGATTTGAATAGTGACGATTATGAATATG- AAGATGAA-39 (mutations are underlined). We also used the single-primer the determining factors of DEN pathogenesis. DEN replication is mutagenesis method to generate the mutated USP13 (C345A and M664/ sensitive to IFN in cell-based assays and in experimental animals 739E) with the primer sequences for C345A, 59-AACCTGGGCAAC- (29, 30). Thus, understanding the antiviral actions of IFN against AGCGCATATCTCAGCTCTGTC-39,forM664E,59-GTGATGCAGC- DEN may shed light on the mechanisms of basic IFN signaling and TGGCCGAGGAGGGTTTCCCGCTGGAAGCA-39,andforM739E,59- also of DEN pathogenesis. By using a set of lentiviruses targeting GAAATCGTAGCTATCATCACCTCCGAGGGATTTCAGCGAAATCA- GGCT-39, respectively (the mutated codons are underlined). 84 human DUBs, we identified a DUB named ubiquitin-specific To construct the C-terminal Flag-tagged STAT1 expression plasmid protease 13 (USP13) whose expression affected the anti-DEN ac- (STAT1-Flag), we PCR amplified the human STAT1 cDNA fragment from by guest on October 1, 2021 tivity of type I and type II IFNs. Furthermore, ubiquitination and STAT1-DsRed2 plasmid (33) with the primer sequences 59-CTCAGA- protein degradation of STAT1 were reduced in cells with USP13 TCTATGTCTCAGTGGTACGAACT-39 and 59-TAAGGATCCCTAC- TGTGTTCATCATACTGT-39 (the restriction enzyme recognition se- overexpression. Thus, USP13, a cellular DUB, may deubiquitinate quences are in italics and the STAT1 sequences are in bold), and then the STAT1 and play a role in host defense against viral infection. cDNA was cloned into the pFlag/pCR3.1 vector. The HA-tagged ubiquitin- expressing plasmid HA-Ub (34), provided by Dr. Hsiu-Ming Shih (Insti- Materials and Methods tute of Biomedical Sciences, Academia Sinica, Taiwan), was further mutated to HA-Ub-GG/AA with the primer sequence 59-GTGTTGAGACTT- Virus and cell lines CGTGCTGCTTAAGTCGAGCATGCATCT-39 (mutations are underlined DEN serotype 2 (DEN-2) PL046 strain (31) was propagated in mosquito and vectors are in italics) or mutated to HA-Ub-K48/63R with the primer C6/36 cells with RPMI 1640 medium supplemented with 5% FBS (Life sequences for K48R, 59-GACTGATCTTTGCTGGCAGGCAGCTGGAA- Technologies) and 2 mM L-glutamine (Life Technologies). Human lung GATGGACG-39, and K63R, 59-ACTTTGTCTGACTACAATATTCAAA- carcinoma A549 and human embryonic kidney (HEK293T) cells were GGGAGTCTACTCTTCATCTTGTG-39 (mutations are underlined), cultured in 10% FBS and 2 mM L-glutamine containing F-12 and DMEM respectively. medium (both Life Technologies), respectively. Baby hamster kidney Western blot assay fibroblasts (BHK-21) were cultured in RPMI 1640 medium containing 5% FBS and 2 mM L-glutamine. For viral infection, cells were absorbed with Cells were lysed in SDS sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% SDS, virus at the indicated multiplicity of infection (MOI) for 2 h at 37˚C. The 10% glycerol) containing protease and phosphatase inhibitors (Roche). unbound virus was removed by gently washing with HBSS (HyClone), and EquivalentamountsofproteinswereseparatedbySDS-PAGEandtransferred then cells were cultured at 37˚C. At the indicated times postinfection, the to nitrocellulose membranes (Hybond-C Super; Amersham Biosciences), culture supernatants were collected and sequentially diluted for plaque- which were blocked with 5% skim milk in PBS with 0.1% Tween 20 and forming assays on BHK-21 cells as previously described (31). thenreactedwiththeindicatedAbs.Theanti-USP13Ab(HPA004827)was from Sigma-Aldrich, and anti-HA (3724), anti-pSTAT1 (Tyr701) (9171), DUB screening anti-STAT1 (9172), anti-pSTAT2 (Tyr690) (4441), anti-STAT2 (4594), anti- 705 The human deubiquitinating enzyme set (C6-2-5, v1.0; Taiwan National pSTAT3 (Tyr ) (9145), and anti-STAT3 (9132) Abs were from Cell Sig- RNAi Core Facility) contains the individual vesicular stomatitis virus-G– naling Technology. pseudotyped lentivirus targeting 84 human DUBs with at least five short Immunoprecipitation-immunoblotting analysis hairpin RNAs (shRNAs) for each DUB (Supplemental Table I). A549 cells seeded in 96-well plates were transduced with lentivirus expressing shRNA HEK293T cells were cotransfected with the indicated plasmids (e.g., HA- targeting the individual human DUBs. Cells were selected with puromycin USP13 and STAT1-Flag) and lysed with protein lysis buffer (50 mM Tris- (10 mg/ml) for 3 d, treated with 1000 U/ml IFN-a-2a (Roferon-A) for 16 h, HCl [ph 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing then infected with DEN-2 (MOI of 1) for 2 d. The degree of DEN infection protease and phosphatase inhibitors mixture. Cell lysates were precleared was detected by immunofluorescence assay with anti-dengue NS3 Ab by mouse IgG-agarose (A0919; Sigma-Aldrich), then immunoprecipitated and quantified by use of Cellomics ArrayScan HT image reader (Thermo with anti-HA affinity gel (E6779; Sigma-Aldrich) or anti-Flag M2 affinity Scientific). We selected the candidate gene USP13 for further study gel (A2220; Sigma-Aldrich) overnight at 4˚C. The proteins were eluted by based on the criteria that at least two clones show a .2-fold induction sample buffer with 2-ME and analyzed by Western blot analysis with anti- 3330 USP13 DEUBIQUITINATES STAT1

STAT1, anti-pSTAT1 (Tyr 701), and anti-USP13 Abs, respectively. To that was provided by the Taiwan National RNAi Core Facility. determine the interaction of endogenous STAT1 and USP13, lysates of A549 cells transduced with these DUB-targeting lentiviruses were HEK293T cells with or without IFN-a treatment were immunoprecipitated selected with puromycin for 3 d and treated with IFN-a over- with anti-USP13 Ab (HPA004827; Sigma-Aldrich), anti-STAT1 Ab (9172; Cell Signaling Technology), or anti-pSTAT1 (Tyr701) Ab (9171; Cell Sig- night. Cells were then infected with DEN-2 for 2 d (Fig. 1A). naling Technology). The immune complexes bound to Protein A Mag The levels of viral replication were determined by immunofluo- Sepharose Xtra (28-9670-56; GE Healthcare Life Sciences) were washed rescence assay with Ab against DEN-2 NS3 protein and measured three times with protein lysis buffer plus 1% Nonidet P-40. The immuno- by a Cellomics ArrayScan HT image reader. IFN-a greatly re- precipitated proteins were then subjected to Western blot analysis with the indicated Abs. duced DEN-2 viral NS3 protein expression in control cells trans- To detect STAT1 ubiquitination, HEK293T cells were cotransfected with duced with lentivirus expressing shLuc (Fig. 1Ba,1Bb), but its STAT1-Flag plus the wild-type (WT) HA-Ub or mutated HA-Ub-K48R, anti–DEN-2 activity was repressed in cells transduced with len- -K63R, -K48/63R, and -GG/AA in the presence of 2 mM MG-132 tivirus expressing shUSP13 (Fig. 1Bc). Thus, USP13 was picked (474791; Calbiochem) for 16 h. To assess the role of USP13 in STAT1 as a candidate gene that may be involved in IFN-a–mediated ubiquitination, USP13 or LacZ-V5 control was cotransfected with STAT1- Flag plus WT or mutated HA-Ub. Cell lysates were immunoprecipitated antiviral responses. with mouse anti-Flag M2 Ab (F3165; Sigma-Aldrich) overnight. The im- Knockdown of USP13 hampers the anti–DEN-2 effect of IFN-a mune complex bound to Protein G Mag Sepharose beads (28-9670-70; GE Healthcare Life Sciences) was washed five times with RIPA lysis buffer To verify the screening data, we established two stable USP13- (0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 10 knockdown A549 cell lines by transduction with lentiviruses mM Tris-HCl [pH 7.5], and 5 mM EDTA) at 4˚C for 30 min. The immunoprecipitated proteins were separated by SDS-PAGE and detected by expressing shRNAs targeting two different regions of USP13, Western blot analysis. shUSP13-G9 and shUSP13-B10. DEN-2 NS3 protein expres- Downloaded from Reporter assay sion measured by Western blotting and viral progeny production measured by plaque-forming assay were higher in both USP13- The type I IFN reporter (pISRE-driven luciferase [pISRE-Luc], 0.5 mg) or knockdown cell lines with IFN-a treatment as compared with type II IFN reporter (pGAS-driven luciferase [pGAS-Luc], 0.5 mg) plus the shLuc control cells (Fig. 2A). We then rescued the USP13 pro- internal control pRL-TK (0.1 mg) were cotransfected to shUSP13-A549 or shLacZ control A549 cells for 16 h. The reporters were also cotransfected tein expression by overexpressing USP13-res generated by in- with HA-USP13 or GFP control (1 mg) to A549 cells for 16 h. Before troducing two wobble mutations into the targeting sequences harvesting for dual luciferase assay (Promega), cells were treated with of each shUSP13 (Fig. 2B). The enhanced DEN-2 replication http://www.jimmunol.org/ 1000 U/ml IFN-a or IFN-g (CYT-402; ProSpec) for 6 h. Firefly luciferase in IFN-a–treated USP13-knockdown cells was significantly lost activity was normalized to that of Renilla luciferase. in cells with USP13-res but not GFP overexpression (Fig. 2B). Immunofluorescence assay Furthermore, USP13 did not affect the replication of DEN-2 Cells were fixed with 4% paraformaldehyde in PBS and then permeabilized per se, because the levels of DEN-2 replication in cells without with 0.5% Triton X-100. Cells were blocked with skim milk in PBS with IFN-a treatment were not significantly different in cells with or 0.1% Tween 20 and then incubated with mouse anti-DEN-2 NS3 Ab (31) without USP13 knockdown (Fig. 2C). Thus, the enhanced DEN-2 and Alexa Fluor 488–conjugated secondary Ab (Invitrogen). DAPI was used replication in IFN-a–treated USP13-knockdown cells was likely to stain nuclei. DEN-2 NS3 content was measured by use of the Cellomics ArrayScan HT image reader. due to the effect of USP13 on the IFN-a–mediated signaling event. by guest on October 1, 2021 RNA extraction and quantitative RT-PCR Total cellular RNA was extracted by use of the RNeasy Total RNA kit (Qiagen), and cDNA was reverse transcribed from 1 mg RNA by the Ther- moScript RT-PCR system (Invitrogen). PCR quantification involved use of Power SYBR Green PCR Master Mix (Applied Biosystems) with primer sequences for STAT1 (59-GCCGCCCAATGCTTGCTTGG-39 and 59- AAATTCAGCCGCCAGACTGC-39) and b-actin (59-TCCTGTGGCATC- CACGAAACT-39 and 59-GAAGCATTTGCGGTGGACGAT-39). The relative expression of the target gene was normalized to that of actin by the second derivative maximum method (Roche). Melting curves were used to validate the specificities of products. Ex vivo DUB assay HEK293T cells were transfected with HA-GFP, HA-USP13, or HA-USP13- M664/739E (1.5 mg) or with STAT1-Flag (1 mg) plus HA-Ub (1 mg) in the presence of MG-132 (2 mM) for overnight. The cell lysates were immunoprecipitated with anti-HA affinity gel for HA-tagged control GFP, WTand mutated USP13, or with anti-Flag M2 affinity gel for STAT1-Flag, respectively. The immune complex was washed four times with protein lysis buffer plus 1% Nonidet P-40. STAT1-Flag proteins were eluted with elution buffer (100 mM glycine-HCl [pH 3.0]) and then immediately neutralized with 1 M Tris-HCl (pH 8.5). The purified STAT1-Flag was then incubated with HA- GFP, HA-USP13, or HA-USP13-M664/739E immune complex in 150 ml FIGURE 1. RNAi screening of USP13 identified as an IFN-a regulator. (A) Overview of RNAi screening to identify human DUBs involved in deubiquitination buffer (50 mM Tris-HCl [pH 8.0], 5 mM MgCl2, and 1 mM DTT) at 37˚C for 2 h. The deubiquitination reaction was stopped by adding antiviral activity of IFN-a against DEN-2. A549 cells transduced with SDS sample buffer containing 2-ME and then the supernatant was collected lentiviruses expressing shRNAs targeting cellular DUBs were selected for Western blot analysis with anti-ubiquitin Ab (U0508; Sigma-Aldrich). with puromycin (10 mg/ml) for 3 d and treated with IFN-a (1000 U/ml) overnight (o/n). Cells were then infected with DEN-2 (MOI of 1) for 2 d Results and stained with Ab against DEN-2 NS3 for the Cellomics ArrayScan Screening of human RNAi library for cellular DUBs involved in HT image reader. (B) Representative immunofluorescence images from 3 a anti–DEN-2 activity of IFN-a screening (20 objective). Control A549 cells with shLuc without ( )or with (b) IFN-a treatment were stained with anti-DEN NS3 Ab (green) and To identify the cellular DUBs involved in the IFN-mediated antiviral DAPI for cell nuclei (blue). (c) The extent of DEN-2 infection in IFN-a– effect, we screened a set of lentiviruses targeting 84 human DUBs treated shUSP13-lentivirus–transduced A549 cells. The Journal of Immunology 3331

FIGURE 2. USP13 knockdown reduced the anti–DEN-2 activity of IFN-a.(A) A549 cells transduced with lentiviruses expressing shRNAs targeting 2 regions of USP13 (shUSP13-G9 and shUSP13-B10) and shLuc control cells were pretreated with IFN-a (1000 U/ml) for 16 h and then infected with DEN-2 (MOI of 1) for 2 d. Western blot analysis of protein levels of DEN-2 NS3, USP13, and actin is shown. The culture supernatants were collected for plaque-forming assay of viral progeny production. Quantitative data are means 6 SD virus titers (PFUs/ml) from two independent experiments analyzed by a two- tailed Student t test. (B) USP13-knockdown cells were rescued with lentivirus expressing the USP13-res or GFP control and analyzed as in (A). (C) shLuc control and shUSP13-A549 cells were Downloaded from infected with DEN-2 (MOI of 1) for 2 d without IFN-a treatment and analyzed as in (A). http://www.jimmunol.org/

USP13 positively regulates type I and type II IFN signaling USP13 prolonged the half-life of STAT1 protein (Fig. 5C) and in- pathways creased STAT1 expression. To address whether USP13 modulates the IFN-a–triggered JAK- USP13 physically interacts with STAT1 STAT signaling pathway, we measured IFN-a activation levels by using pISRE-Luc. The basal and IFN-a–triggered ISRE-Luc ac- To determine how USP13 modulates STAT1 protein, we tested tivities were lower in cells with USP13 knockdown (Fig. 3A), whether USP13 physically interacts with STAT1. By immunoprecipitation (IP)-immunoblotting (IB) assay, anti-Flag Ab brought whereas overexpression of USP13 enhanced the ISRE-Luc activ- by guest on October 1, 2021 ities (Fig. 3B), which suggests that USP13 positively regulates the down not only Flag-tagged STAT1 but also USP13 with or without JAK-STAT signaling pathway. To identify the molecules targeted IFN-a treatment (Fig. 6A, lanes 5 and 6). Furthermore, anti-HA by USP13, we detected the expression and activation of STAT Ab coprecipitated HA-tagged USP13 with STAT1 but barely with proteins. Interestingly, levels of total STAT1 and pSTAT1 but not phosphorylated STAT1 (Fig. 6B, lanes 5 and 6). We further tested much of STAT2 and STAT3 were reduced in cells with USP13 whether the endogenous STAT1 and USP13 proteins interact with knockdown (Fig. 3C). Furthermore, the levels of total STAT1 each other. Similar to the data of overexpressed proteins, anti-STAT1 and pSTAT1 protein were recovered in the USP13-rescued but not anti-pSTAT1 Ab coprecipitated STAT1 with USP13 in cell shUSP13 cells (Fig. 3C). Because the STAT1 transcript levels lysates with or without IFN-a treatment (Fig. 6C). Furthermore, were not signiﬁcantly different in cells with altered USP13 ex- anti-USP13 Ab brought down USP13 with STAT1 but little with pression (Fig. 3D), the effect of USP13 on STAT1 likely occurred phosphorylated STAT1 (Fig. 6D). Thus, USP13 interacts with at the protein but not RNA level. STAT1 but not much with activated STAT1. STAT1 is involved in both type I and type II IFN pathways, so USP13 decreases the ubiquitination of STAT1 we tested whether USP13 also plays a role in type II IFN signaling. In measuring the GAS-driven reporter activity (pGAS-Luc), USP13 To address whether USP13 affects the ubiquitination of STAT1, we ﬁrst knockdown reduced and USP13 overexpression augmented the determined the ubiquitination pattern of STAT1 in cells cotransfected reporter activity of IFN-g–triggered GAS-Luc (Fig. 4A, 4B). with plasmids expressing STAT1-Flag and HA-Ub. By IP-IB assay Furthermore, the anti–DEN-2 activity of IFN-g was also reduced with anti-Flag and anti-HA Abs, smeared protein reactivity with with UPS13 knockdown and rescued by USP13 overexpression a molecular mass larger than the expected size of STAT1 protein in shUSP13 cells (Fig. 4C, 4D). Thus, USP13 may target STAT1 indicated polyubiquitination and/or multiple monoubiquitination of protein in both type I and type II IFN signaling events. STAT1 (Fig. 7A, lane 1). To determine whether the two lysine residues most commonly used in polyubiquitination, K48 and K63, USP13 stabilizes STAT1 protein level contributes to STAT1 polyubiquitination, we cotransfected cells with To address how USP13 regulates STAT1 protein levels, we traced the STAT1-Flag and HA-tagged ubiquitin with K48R, K63R, or K48/63R protein stability of STAT1 in cells treated with the protein synthesis mutation. The levels of STAT1 ubiquitination were slightly reduced inhibitor cycloheximide with and without USP13 overexpression. with K48R and K48/63R mutants, but not much with the K63R Endogenous STAT1 protein levels were higher in HEK293T cells mutant, as compared with WT ubiquitin (Fig. 7A, lanes 3–5), sug- transfected with USP13-expressing plasmid than in control GFP- gesting that K48- but not K63-linkage polyubiquitination is involved. transfected cells (Fig. 5A) at all of the time points tested. The trans- We further used the ubiquitin-GG/AA mutant, which cannot be fected STAT1-Flag protein levels were also increased in cells with conjugated to substrates because its C-terminal diglycine motif has USP13 overexpression (Fig. 5B). Thus, ectopic overexpression of been changed to alanine-alanine (9, 35). The GG/AA mutant ubiq- 3332 USP13 DEUBIQUITINATES STAT1

FIGURE 3. USP13 is involved in the IFN-a signaling pathway and controls the protein but not mRNA level of STAT1. (A) shLacZ control and shUSP13- A549 cells were cotransfected with pISRE-Luc (0.5 mg) and pRL-TK (0.1 mg) for 16 h and then stimulated with IFN-a (1000 U/ml) for 6 h and harvested for dual- luciferase assay. Firefly luciferase activity was normalized to that of Renilla luciferase. Quantitative data are means 6 SD (n = 3/group) and analyzed by a two- tailed Student t test. (B) A549 cells were cotransfected with the reporter plasmids (0.5 mg pISRE-Luc and 0.1 mg pRL-TK) plus GFP control or USP13 expression plasmid (1 mg) for 16 h and then treated with IFN-a Downloaded from (1000 U/ml) for 6 h. Cells were analyzed as in (A). (C) Western blot analysis of total and pSTAT proteins in IFN-a–treated cells with USP13 knockdown and rescue. (D) Quantitative RT-PCR analysis of the relative mRNA level of STAT1 with USP13 knockdown and rescue and normalized to that of actin (n = 2/group). Data are means 6 SD and analyzed by a two-tailed http://www.jimmunol.org/ Student t test. by guest on October 1, 2021 uitin, with which neither polyubiquitination nor monoubiquitination importance of the cysteine catalytic motif and UBA domain of can occur, greatly reduced STAT1 ubiquitination (Fig. 7A, lane 2). USP13 on its function. We then performed an ex vivo DUB exper- Previously, both K48-linkage polyubiquitination (17) and mono- iment to directly demonstrate that USP13 is able to deubiquitinate ubiquitination (36) have been reported for STAT1 protein. Our results STAT1. The ubiquitination level of STAT1 protein was reduced by thus suggest that polyubiquitination through K48 and the other five incubation with USP13, but not with M664/739E-mutated USP13, less used lysines (K6, K11, K27, K29, and K33) as well as mono- when compared with the GFP control (Fig. 8B). Thus, our results ubiquitination may occur on STAT1 protein. suggest that USP13 can deubiquitinate STAT1 and increase the We then determined whether USP13 affects STAT1 ubiquitination. stability of STAT1 protein. Interestingly, STAT1 ubiquitination was greatly reduced in cells with USP13 overexpression as compared with unrelated LacZ-V5 over- Discussion expression (Fig. 7B). We further tested whether the ubiquitination of By RNAi screening, we identified USP13 as a positive regulator of endogenous STAT1 can be downregulated by endogenous USP13. the IFN signaling pathway. STAT1 is involved in both type I and type Similar to IFN-g treatment (14), the overall STAT1 and pSTAT1 II IFN signaling and is targeted by the ubiquitin–proteasome system ubiquitination was increased in cells treated with IFN-a (Fig. 7C). (14). As outlined in Fig. 9, USP13 can reduce the ubiquitination Furthermore, stronger STAT1/pSTAT1 ubiquitination signal was levels of STAT1, stabilize its protein level, and result in a stronger noted in cells with USP13 knockdown when compared with the IFN antiviral response. Thus, to our knowledge, USP13 is the first control LacZ knockdown with or without IFN-a treatment (Fig. 7C). cellular DUB involved in deubiquitinating and stabilizing STAT1 Thus, USP13 is able to enhance the ubiquitination of both the non- protein in both type I and type II IFN signaling. To address whether phosphorylated and phosphorylated STAT1 at basal level as well as IFN and/or viral infection can modulate USP13 expression to reg- at the stimulated state. ulate STAT1 and the IFN system, we determined the protein ex- To demonstrate the ability of USP13 to deubiquitinate STAT1, we pression level of endogenous USP13 by Western blotting. Similar determined the sequences required for USP13 to stabilize STAT1 USP13 protein levels were noted in mock- and DEN-infected cells protein. Both the cysteine catalytic motif (37) and ubiquitin-asso- with or without IFN-a treatment (Supplemental Fig. 1), indicating ciated (UBA) domain (38) have been implicated in USP13 function, that USP13 protein expression is not modulated by type I IFN or so we constructed two USP13 mutants, C345A with mutated cys- DEN infection. Recently, accumulating evidence suggests that the teine catalytic motif and M664/739E with mutated UBA domain. activity of DUBs can be reversibly inhibited by oxidation mediated The ability of USP13 to increase STAT1 protein level was reduced by reactive oxygen species (39). Thus, whether the activity of by either C345A or M664/739E mutation (Fig. 8A), confirming the USP13 could be blocked by reactive oxygen species in DEN- The Journal of Immunology 3333

FIGURE 4. USP13 enhances the signaling and anti– DEN-2 activity of IFN-g.(A) shLacZ control and shUSP13-A549 cells were cotransfected with pGAS- Luc (0.5 mg) and pRL-TK (0.1 mg)for16handthen treated with IFN-g (1000 U/ml) for 6 h. Cells were analyzed as in Fig. 3A. (B) A549 cells were cotransfected with the reporter plasmids (0.5 mgpGAS-Luc and 0.1 mg pRL-TK) plus GFP control or USP13 expression plasmid (1 mg) for 16 h and then treated with IFN-g (1000 U/ml) for 6 h. Cells were analyzed as in (A). (C) Western blot analysis of DEN-2 NS3, USP13, and actin protein levels in cells pretreated with IFN-g (1000 U/ml) overnight and infected with DEN-2 (MOI Downloaded from of 1) for 2 d. (D) Plaque-forming assay of viral titration in culture supernatants. Data are means 6 SD virus titers (PFUs/ml) from two independent experiments analyzed by a two-tailed Student t test. http://www.jimmunol.org/

infected cells that then results in less STAT1 protein to beneﬁt viral might have speciﬁc substrates regulating distinct cellular processes

replication remains elusive. (18, 41). USPs are Cys-dependent proteases containing a catalytic by guest on October 1, 2021 The human genome encodes ∼95 DUBs categorized into five core domain including an N-terminal Cys-box and a C-terminal major families, including USP, ubiquitin C-terminal hydrolase, His-box, which comprise the catalytic Cys and His residues, re- OTU domain containing protease, Machado–Josephin disease spectively. USPs also contain other functional ubiquitin-binding protease, and JAB1/MPN/Mov34 metalloenzyme (18, 40). USP domains, such as the zinc finger ubiquitin-specific protease (ZnF- represents the largest family of DUBs, with .50 members that UBP) domain, the ubiquitin-interacting motif, and the UBA domain

FIGURE 5. STAT1 protein levels are increased by USP13. Western blot analysis of protein levels in HEK293T cells transfected with (A) HA-Ub plus GFP or USP13 plasmid or (B) HA-Ub and STAT1-Flag plus GFP or USP13 overnight and then treated with cycloheximide (CHX, 50 mg/ml) for the indicated times. (C) The protein band density was quantiﬁed with use of ImageJ and the level of STAT1 was normalized to that of actin. The protein levels of STAT1 relative to that at time 0 of each group were calculated and are shown. 3334 USP13 DEUBIQUITINATES STAT1 Downloaded from

FIGURE 6. USP13 mainly interacts with the nonactivated form of STAT1. IP-IB analysis with control IgG, anti-Flag afﬁnity gel, or anti-HA afﬁnity gel (A, B) in HEK293T cells cotransfected with plasmids expressing HA-USP13 and STAT1-Flag for 18 h and then treated with or without IFN-a (1000 U/ml) for 30 min. IP-IB analysis of protein levels of USP13, STAT1, and pSTAT1 in the immune complex and in total cell lysates is shown. (C and D) The interaction of endogenous USP13 and STAT1 was investigated by IP-IB analysis. Immunoprecipitation of HEK293 cells with or without IFN-a treatment was carried out with control IgG, anti-STAT1, anti-pSTAT1, or anti-USP13 Abs as indicated. The immunoprecipitated proteins were then analyzed by http://www.jimmunol.org/ Western blot with anti-USP13, anti-STAT1, and anti-pSTAT1 Abs, respectively.

ﬂanking or inserting in the core domain (40, 41). The UBA and shares a similar fold with that of USP5, but it cannot bind ubiquitin, ubiquitin-interacting motif domains can facilitate substrates bind- and USP13 does not function similar to USP5 in recycling ubiquitin ing onto enzymes, and the ZnF-UBP domain might stimulate the (44). As compared with USP5, USP13 shows weaker deubiquiti- hydrolysis reaction or regulate other biological functions (42, 43). nation activity, probably through its tandem UBA domains binding USP13, also called isopeptidase T3, shares ∼80% sequence with ubiquitin (44). similarity with USP5. Both proteins have four ubiquitin-binding A couple of proteins involved in diverse cellular functions have sites, including two UBA domains, a ZnF-UBP domain, and a cat- been reported as targets of USP13. For example, USP13 appears to be by guest on October 1, 2021 alytic domain (44). The ZnF-UBP domain of USP5 recognizes and responsible for the protein stability of microphthalmia-associated recycles the unanchored polyubiquitination chains to keep the free transcription factor, which is essential for melanocyte develop- ubiquitin pool stable (45, 46). The ZnF-UBP domain of USP13 ment (37). USP13 can reduce the ubiquitination and degradation of

FIGURE 7. STAT1 ubiquitination is reduced by USP13. (A) IP-IB and IB analysis with the indicated Abs in HEK293T cells cotransfected with STAT1-Flag plus the WTor mutated GG/AA, K48R, K63R, or K48/63R HA-Ub in the presence of MG-132 (2 mM) for 16 h. (B) IP-IB and IB analysis with the indicated Abs in HEK293T cells transfected with STAT1-Flag plus USP13 or LacZ-V5 as well as HA-Ub or HA-Ub-GG/AAwith MG-132 (2 mM) for 16 h. (C) IP-IB and IB analysis with the indicated Abs in HEK293T cells with control LacZ knockdown or USP13 knockdown with or without IFN-a treatment (1000 U/ml, 30 min) . The Journal of Immunology 3335

mids expressing STAT1 and ubiquitin (Fig. 7). Furthermore, ubiquitin with K48R but not K63R mutation could be conjugated to STAT1 protein to a level lower than that of WT ubiquitin but higher than that of the GG/AA mutant. These results suggest that STAT1 might be monoubiquitinated as previously reported (36) and/or polyubiquitinated with K48 linkage as well as linkage through other lysines such as K6, K11, K27, K29, and/or K33. However, regardless of the types of ubiquitin linkage, STAT1 ubiquitination was greatly reduced in cells with USP13 overexpression. USP13 has been reported to elicit cleavage of both K48- and K63-linked polyubiquitin chains (44, 47), and USP5 can hydrolyze the K63-, K48-, K11-, and K29-linked and linear polyubiquitination chains (50, 51). Thus, USP13 might possess broad DUB activity against several types of ubiquitin linkages and warrants further study to reveal its enzymatic activity. The incidence of DEN infection has grown around the world, and according to the World Health Organization, dengue now threatens .40% of the world’s population. Despite the clinical signiﬁcance of the infection, no speciﬁc treatment or vaccine is available for DEN infection. DEN infection may cause a wide spectrum of illnesses Downloaded from ranging from mild dengue fever to severe dengue hemorrhagic fever and dengue shock syndrome. The pathogenesis mechanisms for DEN-related diseases are not clear, but high viral replication might be a risk factor for the severe form of DEN infection. DEN replication was found sensitive to type I and type II IFNs in cell-based assays and in infected animals (29, 30). Different DEN strains may http://www.jimmunol.org/

FIGURE 8. USP13 deubiquitinates STAT1. (A) Western blot analysis of protein levels in HEK293T cells cotransfected with STAT1-Flag plus WT USP13, USP13-C345A, USP13-M664/739E, or GFP control for 20 h. (B) STAT1 proteins were puriﬁed from HEK293T cells cotransfected with STAT1-Flag and HA-Ub in the presence of MG-132 (2 mM) by anti-Flag

beads and then were incubated with HA-GFP, HA-USP13, or HA-USP13 by guest on October 1, 2021 (M664/739E) mutant proteins immunoprecipitated by anti-HA afﬁnity gel in vitro at 37˚C for 2 h with deubiquitination buffer. The products of deubiquitination reaction were analyzed by immunoblotting with anti-ubiquitin Ab.

Sky2, the F-box adaptor of the E3 ubiquitin ligase SCFSkp2 (47). It can bind to and stabilize another E3 ligase, the RING finger E3 ubiquitin ligase Siah2, even though it reduces the ubiquitin ligase activity of Siah2 (38). USP13 interacts with and deubiquitinates Beclin 1, a tumor suppressor and autophagy inducer (48). Further- more, interaction with Beclin 1 can enhance the DUB activity of USP13. USP10, known as a DUB of p53 (49), is another substrate of USP13. USP10 also interacts with USP13 and increases its DUB activity (48). Additionally, USP13 interacts with some components of P97/VCP complex, a key chaperone in endoplasmic reticulum– associated degradation (19) and increases the protein level of CD3d, an endoplasmic reticulum–associated degradation substrate (44). In this study, we add STAT1 to the growing list of USP13 substrates by demonstrating that USP13 can remove STAT1 ubiquitination and increase STAT1 protein stability. Because STAT1 is involved in type I, II, and III IFN signaling, USP13 thus also regulates the innate immunity and host defense mechanism. FIGURE 9. USP13 reduces STAT1 protein ubiquitination and enhances Two E3 ligases, SLIM and Smurf1, have been reported to con- the signaling and antiviral activity of type I and type II IFNs. (A) STAT1 is tribute to STAT1 ubiquitination (15–17). SLIM, a nuclear E3 ligase, an essential molecule in the signaling cascades of type I and type II IFNs, can interact with phosphorylated STAT4 and promote the ubiq- which leads to gene activation mediated by promoters containing ISRE and GAS, respectively. STAT1 proteins are ubiquitinated and targeted for pro- uitination and degradation of STAT4 protein (15). SLIM also protein degradation. USP13 can reduce the STAT1 ubiquitination and increase motes the ubiquitination of STAT1 and inhibits STAT1-mediated its protein level. (B) USP13 regulates the outcomes of IFN antiviral activity gene expression (15). Smurf1, a HECT-type E3 ligase, catalyzes by controlling the protein levels of STAT1. Increased USP13 expression K48-linked polyubiquitination of STAT1 and promotes STAT1 may stabilize STAT1 proteins, enhance the IFN response, and inhibit virus degradation, independent of STAT1 phosphorylation (17). We also replication. In contrast, reduced USP13 level may reduce the STAT1 protein detected STAT1 polyubiquitination in cells transfected with plas- level, weaken the IFN response, and increase virus replication. 3336 USP13 DEUBIQUITINATES STAT1 regulate the IFN pathway differently (52), but the determining fac- uitin ligase domains of A20 downregulate NF-kB signalling. Nature 430: 694– 699. tors controlling the levels of DEN replication in infected patients are 24. Shembade, N., N. S. Harhaj, D. J. Liebl, and E. W. Harhaj. 2007. Essential role largely unclear. Therefore, more understanding of the antiviral for TAX1BP1 in the termination of TNF-a-, IL-1- and LPS-mediated NF-kBand mechanism, such as the cellular proteins involved in IFN antiviral JNK signaling. EMBO J. 26: 3910–3922. 25. Iha, H., J. M. Peloponese, L. Verstrepen, G. Zapart, F. Ikeda, C. D. Smith, activity against DEN replication, will shed light on the overall in- M. F. Starost, V. Yedavalli, K. Heyninck, I. Dikic, et al. 2008. Inflammatory terplay between DEN and host immunity. Our study revealed cardiac valvulitis in TAX1BP1-deficient mice through selective NF-kB activa- a cellular protein USP13 that participates in the antiviral action of tion. EMBO J. 27: 629–641. 26. Li, S., H. Zheng, A. P. Mao, B. Zhong, Y. Li, Y. Liu, Y. Gao, Y. Ran, P. Tien, and type I and II IFN against DEN infection and paves the way to a new H. B. Shu. 2010. Regulation of virus-triggered signaling by OTUB1- and direction in research into IFN signaling. 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