ChaudharyVasu Punj, Han Yi, Yulan Suo and Preet M. Hittu Matta, Ramakrishnan Gopalakrishnan, 

B in a Negative Feedback MannerκFactor-K13 and Blocks K13-induced Nuclear Viral FLICE Inhibitory Protein (vFLIP)Sarcoma-associated Herpesvirus-encoded A20 Is Induced by KaposiSignal Transduction:

doi: 10.1074/jbc.M111.224048 originally published online April 29, 20112011, 286:21555-21564.J. Biol. Chem. 

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A20 Is Induced by Kaposi Sarcoma-associatedHerpesvirus-encoded Viral FLICE Inhibitory Protein (vFLIP)K13 and Blocks K13-induced Nuclear Factor-�B in a NegativeFeedback Manner*

Received for publication, January 22, 2011, and in revised form, April 17, 2011 Published, JBC Papers in Press, April 29, 2011, DOI 10.1074/jbc.M111.224048

Hittu Matta, Ramakrishnan Gopalakrishnan, Vasu Punj, Han Yi, Yulan Suo, and Preet M. Chaudhary1

From the Jane Ann Nohl Division of Hematology and Center for the Study of Blood Diseases, University of Southern California KeckSchool of Medicine, Los Angeles, California 90033

ExpressionofA20, a negative regulator of theNF-�Bpathway,is frequently lost in several subtypes of Hodgkin and non-Hodg-kin lymphoma. We report that A20 is expressed in Kaposi sar-coma-associated herpesvirus (KSHV)-infected primary effusionlymphoma cell lines, and its expression correlates closely withthe expression of KSHV-encoded viral FLICE inhibitory proteinK13. Ectopic expressionofK13 inducedA20expression throughNF-�B-mediated activation of A20 promoter. In turn, A20blocked K13-induced NF-�B activity and up-regulation of pro-inflammatory cytokines CCL20 and IL-8 in a negative feedbackfashion. Both the N-terminal deubiquitinating domain and theC-terminal zinc finger domain of A20 were involved in the inhi-bition of K13-induced NF-�B activity. Overexpression of A20blocked K13-induced I�B� phosphorylation, NF-�B nucleartranslocation, and cellular transformation. Consistent with theabove, K13-induced I�B� phosphorylation and NF-�B tran-scriptional activation were enhanced in A20-deficient cells.Finally, A20 was found to interact physically with K13. Takencollectively, these results demonstrate that K13 is a key deter-minant of A20 expression in KSHV-infected cells, and A20 is akey negative regulator of K13-induced NF-�B activity. A20might serve to control the inflammatory response to KSHVinfection and protect KSHV-infected cells from apoptosis.

The NF-�B2 pathway induces the expression of a number ofgenes involved in mediation of the inflammatory response, andattenuatingNF-�B signaling is essential formodulating inflam-matory reactions and maintaining homeostatic balance (1). Assuch, identification of genes that control NF-�B activationhas been the focus of intense research. A20 gene (also known astumor necrosis factor �-induced protein 3 or TNFAIP3) wasfirst identified 20 years ago as a gene that is rapidly induced inhuman umbilical vein endothelial cells upon treatment with

proinflammatory cytokines, such as TNF� and IL-1 (2). Subse-quent studies reported that A20 is also induced by other stim-uli, including phorbol esters, signaling via CD40 and TRAILreceptors, and activation of B cell surface receptor (3). In addi-tion, Epstein-Barr virus (EBV)-encoded latent membrane pro-tein 1 (LMP1) and human T cell lymphotropic virus 1 Tax pro-tein are known to induce A20 expression (4, 5). Overexpressionof A20 was shown to block NF-�B activation triggered by vari-ous stimuli, including TNF, IL-1, and lipopolysaccharide (LPS)(6, 7). Consistent with the above role, A20-deficient cells dem-onstrate prolongedNF-�B activation in response to TNF�, andmice deficient in A20 develop severe inflammation, showhypersensitivity to LPS and TNF�, and die early (8). Collec-tively, these studies have establishedA20 as a physiological reg-ulator of NF-�B activation.

Kaposi sarcoma-associated herpesvirus (KSHV), also knownas human herpesvirus 8, is an oncogenic �2 herpesvirus that isthe etiological agent of Kaposi sarcoma (9) and has been asso-ciated with the pathogenesis of primary effusion lymphoma(PEL) and some aggressive forms of multicentric Castlemandisease (10).One of the KSHV proteins that is expressed in all cells

latently infected with KSHV is K13 (11). Based on its structuralhomology to the prodomain of caspase 8/FLICE (12), K13 wasoriginally classified as a viral FLICE inhibitory protein (vFLIP)(12). However, subsequent work showed that K13 is not aninhibitor of caspase 8/FLICE but is a potent activator of theNF-�B pathway and uses this pathway to promote cellular pro-liferation, survival, transformation, cytokine secretion, andKSHV latency (13–20). Ectopic expression of K13 in humanvascular endothelial cells transforms them into spindle cells,reminiscent of the spindle cells found in theKS lesions, which isaccompanied by increased expression of proinflammatorycytokines and chemokines known to be involved in the patho-genesis of KS lesions (18, 20). Because A20 is a key regulator ofinflammatory response, in this study, we have examined theeffect of K13 on A20 expression. Our results demonstrate thatA20 expression is up-regulated by K13, and in turn A20 blocksK13-induced NF-�B activity.

MATERIALS AND METHODS

Cell Lines and Reagents—293T, BC1, BC3, BCBL1 cells wereobtained from American Type Culture Collection (Manassas,VA), and APK1 cells were obtained from Dr. Jae Jung (Univer-

* This work was supported, in whole or in part, by National Institutes of HealthGrants CA85177, CA124621, and DE019811. This work was also supportedby the Leukemia and Lymphoma Society.

1 To whom correspondence should be addressed. Tel.: 323-865-3916; Fax:323-865-0060; E-mail: preet.chaudhary@med.usc.edu.

2 The abbreviations used are: NF-�B, nuclear factor �B; EBV, Epstein-Barr virus;ELIE, elongation inhibitory element; ER, estrogen receptor; HTLV, human Tcell lymphotropic virus; IKK, I�B kinase; KSHV, Kaposi sarcoma-associatedherpesvirus; LMP1, latent membrane protein 1; MEF, mouse embryonicfibroblast; 4OHT, 4-hydroxytamoxifen; PEL, primary effusion lymphoma;vFLIP, viral FLICE inhibitory protein; OTU, ovarian tumor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 24, pp. 21555–21564, June 17, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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sity of Southern California). 293T and BCBL1 cells expressingan empty vector and K13 have been described previously (21,22). A20�/� and A20�/� mouse embryo fibroblast (MEF) cellswere generously provided by Dr. Averil Ma (University of Cal-ifornia, San Francisco, CA). NF-�B inhibitors Bay-11-7082 andPS-1145 were purchased from Calbiochem, and arsenic triox-ide (As2O3) was from Sigma-Aldrich.Plasmids—Plasmids encoding K13, E8, Tax, phosphoryla-

tion-resistant mutants of I�B�, kinase-defective mutants ofIKK1/IKK2 and 4-hydroxytamoxifen (4OHT)-inducible K13-ERTAM have been described previously (14–15, 18, 23, 24). TheA20 WT-Luc, A20 mNF-�B-Luc, and A20 m1-Luc luciferasereporter constructs were kind gifts fromDr. Dikstein (TheWeiz-mann Institute of Science, Rehovot, Israel) (25).Thewild-type andmutantA20 constructs were kindly provided byDr. Lin Rongtuan(McGill University, Montreal, QC, Canada) (26). Recombinantretroviruses were generated and used to infect 293T, BCBL1, andMEF cells essentially as described previously (27).Luciferase Reporter—The 293T cells were transfected in a

24-well plate with various test plasmids (250 ng/well) alongwith the WT or mutant A20 luciferase reporter constructs (75ng/well) and a pRSV/LacZ (�-galactosidase) reporter construct(75 ng/well) using calcium phosphate as described previously(24). Cells were lysed 24–36 h later, and extracts were used forthemeasurement of firefly luciferase and �-galactosidase activ-ities, respectively. Luciferase activity was normalized relative tothe �-galactosidase activity to control for the difference in thetransfection efficiency.Western Blotting—Western blot analysis was performed

essentially as described previously (28). Primary antibodiesused in these experiments were: p65 (Santa Cruz Biotechnol-ogy), NEMO (Santa Cruz Biotechnology), IKK�/� (Santa CruzBiotechnology), A20 (Cell Signaling), p-I�B� (Cell Signaling),total I�B� (Santa Cruz Biotechnology), tubulin (mouse mono-clonal; Sigma), and mouse monoclonal M2 FLAG (Sigma). Amouse monoclonal antibody against K13 (8F6) was raised inour laboratory. FLAG, control mouse IgG, and goat anti-mouseIgG beads were obtained from Sigma and used in co-immuno-precipitation studies as described previously (28).NF-�B DNA Binding Assay—The DNA binding activity of

the p65 subunit was measured in triplicate in the nuclearextracts using an enzyme-linked immunosorbent assay(ELISA)-based NF-�B DNA binding assay, as described previ-ously (21).ELISA—Human CCL20 and IL-8 were measured in the cell

culture supernatant using ELISA kits from R&D Systems(Minneapolis, MN) and BD Pharmingen, respectively. TheELISA was performed following the recommendations of themanufacturer.Soft Agar Assay—Soft agar assay was performed essentially as

described previously (14). Briefly, Rat1 K13 cells expressing anempty vector or A20 were overlaid as a single cell suspension of1000 cells in 1.0 ml of 0.4% Bacto-agar onto a 3.5-cm tissueculture dish containing a 0.6% agar base. All agar media weremade with Dulbecco’s modified Eagle’s medium supplementedwith 10% serum, penicillin/streptomycin. Triplicate plateswereprepared for each tested cell line and inspected for colony for-mation after incubation at 37 °C for 14 days.

RNA Interference (RNAi)—siRNA oligonucleotides againstA20 were purchased from Santa Cruz Biotechnology. p65siRNA has been described previously (18). siRNA oligonu-cleotides were transfected using Neon transfection system(Invitrogen) and following the recommendations of themanufacturer. Following transfection, cells were immedi-ately transferred to a 24-well plate containing prewarmedantibiotic-free medium and incubated at 37 °C. Cells werefirst examined for knock-down of A20 expression by West-ern blotting, and then the lysates were assayed for NF-�Bluciferase reporter activity.Statistical Analyses—A two-tailed paired Student’s t test was

used to test for differences between two groups. Differenceswith a p � 0.05 were considered as statistically significant. Allexperimentswere repeated aminimumof three timeswith trip-licate samples.

RESULTS

A20 Expression in PEL Cell Lines Correlates with Their Levelof Endogenous K13 Expression—A20 is a tumor suppressor, andrecent studies have demonstrated a loss of its expression inseveral subtypes of Hodgkin and non-Hodgkin lymphoma (3).To determine whether A20 expression is also lost in PEL, weusedWestern blotting to examine its expression in four PEL celllines, BC1, BC3, BCBL1, and APK1. The BC1 cells are duallyinfected with KSHV and EBV, whereas the BC3, BCBL1 andAPK1 cells are infected with KSHV only. As shown in Fig. 1A,we observed robust expression of A20 in BC1 and BC3 and lowlevel expression in BCBL1 and APK1 cell lines. Immunoblot-ting using a K13 monoclonal antibody revealed that expressionof A20 in the four PEL cell lines correlated closely with theirlevel of endogenous K13 expression, implying a causal associa-tion (Fig. 1A). To test this hypothesis further, we took advan-tage of BCBL1 cells that had been engineered to express FLAG-tagged K13 ectopically by retroviral-mediated gene transfer(21). Consistent with the notion that K13 up-regulates A20expression, the BCBL1-K13 cells demonstrated a markedincrease in A20 expression compared with the BCBL1-vectorcells (Fig. 1B). Induction of A20 expression by ectopic K13expression, however, was not limited to BCBL1 cells but wasalso observed in the 293T cell line (Fig. 1B). Taken collectively,the above results demonstrate that K13 is a key determinant ofA20 expression in PEL.

FIGURE 1. K13 up-regulates A20 expression in PEL cell lines. A, Westernblots showing expression of A20 and K13 in PEL cell lines. B, Western blotsshowing up-regulation of A20 in BCBL1 and 293 cells by ectopic K13 expres-sion. Stable pools of BCBL1 and 293 cells expressing an empty vector andFLAG-K13 were generated by retroviral gene transfer. Cell lysates from thesecells were used for Western blot analysis. Tubulin blot shows equal proteinloading (bottom panel).

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K13 Activates A20 Promoter Activity—To determine themechanism of A20 up-regulation by K13, we examined theeffect of ectopic K13 expression on the transcriptional activityof a luciferase reporter construct (A20-Luc) driven by the A20promoter region (25). We transfected the A20-Luc reporterplasmid into 293T cells along with an empty vector or anexpression plasmid encoding K13 and measured the luciferaseactivity in the cell lysates 28–30 h after transfection. As shownin Fig. 2A, transient expression of K13 resulted in a �8-fold

increase in A20 luciferase reporter activity compared withtransfection with an empty vector, suggesting that K13 up-reg-ulates A20 expression at the transcriptional level.The NF-�B Sites at �54 and �66 in the A20 Promoter Are

Essential for K13-mediated Activation—The A20 promoterregion contains two NF-�B sites residing at �54 and �66nucleotides from the putative transcription start site whichhave been shown to play a key role in response to TNF� stim-ulation (25, 29). In addition, the A20 promoter has a negative

FIGURE 2. K13 activates A20 promote through NF-�B pathway. A and B, the 293T cells were transfected with a control vector and a vector encodingFLAG-K13 (250 ng/ml) along with either A20-Luc or A20 mNF-�B-Luc or A20 m1-Luc reporter construct (75 ng/well) and a pRSV/LacZ (�-galactosidase)reporter construct (75 ng/well), and the reporter assay was performed as described under “Materials and Methods.” The values shown are averages(mean � S.E.) of one representative experiment of three in which each transfection was performed in duplicate. Expression of the K13 was confirmed byWestern blot analysis with an antibody against the FLAG epitope tag. C, K13-ERTAM induces A20 promoter activity upon treatment with 4OHT. Theexperiment was performed essentially as described in A. The values shown are averages (mean � S.E.) of one representative experiment ofthree in whicheach transfection was performed in duplicate. D, wild-type K13 but not vFLIP E8- or NF-�B-defective mutants of K13 induces A20 promoter activity. 293Tcells were transfected with the indicated FLAG-tagged expression constructs along with a A20 promoter luciferase construct. The experiment wasperformed as described for A. The values shown are averages (mean � S.E.) of one representative experiment of three in which each transfection wasperformed in duplicate. Lower panel shows the expression of the transfected proteins as dermined by Western blotting with a FLAG antibody. E, dom-inant negative mutants of I�B� (I�B��N and I�B�S32A/S36A), IKK1 and IKK2 block K13-induced A20 promoter activity. The 293T cells were transfectedeither with an empty vector or K13, along with an A20 luciferase reporter construct and a reporter construct, as described in A. The amount of inhibitorplasmids (500 ng/well) was five times the amount of vector or K13 (100 ng/well) plasmid, and the total amount of transfected DNA was kept constantby adding empty vector. The values shown are averages (mean � S.E.) of one representative experiment of three in which each transfection wasperformed in duplicate. Expression of the K13 was confirmed by Western blot analysis with the FLAG antibody. Asterisks indicate significance at levelsof p � 0.05. F, pharmacologic inhibitors of NF-�B block K13-induced A20 promoter activation. The 293T cells were transfected with an empty vector ora vector encoding K13 along with A20-Luc and pRSV/LacZ reporter constructs. Approximately 3 h after transfection, cells were treated with dimethylsulfoxide (vehicle) or the indicated compounds for 18 h before cell lysis and measurement of reporter activities. Asterisks indicate significance at levelsof p � 0.05 compared with vehicle-treated cells.

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upstream promoter element called ELIE (Elongation InhibitoryElement) (30). To examine the involvement of theseNF-�B andELIE sites in K13-induced A20 promoter activation, we trans-fected 293T cells with either a luciferase reporter constructcontaining the wild-type A20 promoter (A20-WT-Luc) orreporter constructs bearing mutations in the NF-�B (A20-mNF-�B-Luc) and ELIE sites (A20-m1-Luc), respectively. Asshown in Fig. 2B, K13-induced A20 promoter activity wasmarkedly attenuated in the A20-mNF-�B-Luc construct. Onthe other hand, mutation of the ELIE site had no effect on K13-induced A20 promoter activation. The role of the NF-�B sitesin K13-induced A20 promoter activation was confirmed by anindependent experiment involving expression of a K13-ERTAM

fusion protein in which K13 activity is controlled at the post-translational level by fusion with a mutated estrogen receptor(18). The mutated estrogen receptor (ERTAM) in this constructdoes not bind to the physiological ligand estrogen but bindswith very high affinity to the synthetic ligand 4OHT and regu-lates the activity of K13 in a 4OHT-dependent fashion (18).Consistent with the previous results, 4OHT treatment stronglyactivated the luciferase activity in cells transfected with K13-ERTAM and A20-WT-Luc construct but not in those trans-fected with K13-ERTAM and A20-mNF-�B-Luc (Fig. 2C). Col-lectively, these results demonstrate the importance of theNF-�B sites in K13-mediated A20 gene expression.K13 Stimulates A20 Promoter Activity via NF-�B Activation—

To confirm the role of the NF-�B pathway in K13-induced A20promoter activation, we used three previously described pointmutants of K13, K13-58AAA (E58A/C59A/L60A), K13-65AAA (R65A/R66A/D67A), and K13-67AAA (D67A/L68A/L69A), which are defective in NF-�B activation (14) Consistentwith their loss of NF-�B activating ability, these mutants failedto activate the A20-Luc construct (Fig. 2D). Furthermore, noA20 promoter activation was observed upon co-expression ofthe vFLIP E8 from the equine herpesvirus 2, which resemblesK13 in structure but lacks the ability to activate the NF-�Bpathway (24, 31) (Fig. 2D). On the other hand, co-expression ofhuman T cell leukemia-encoded Tax protein, which resemblesK13 in activating the NF-�B pathway, led to A20 promoteractivation (Fig. 2D). Collectively, these results indicate thatK13-induced NF-�B activity is likely involved in A20 promoteractivation.Abrogation of K13-induced A20 Promoter Activation by

Genetic and Pharmacological Inhibitors of the NF-�B Pathway—K13 activates the NF-�B pathway by activating a �700-kDaIKK complex consisting of IKK1/IKK�, IKK2/IKK�, andNEMO/IKK�which phosphorylates I�B�, targeting it for ubiq-uitination and subsequent degradation via the proteasome(28). We examined the effect of dominant negative mutants ofI�B�, IKK1/IKK�, and IKK2/IKK� on K13-induced A20 pro-moter activity. As shown in Fig. 2E, K13-inducedA20 promoteractivity was completely blocked by a phosphorylation-resistantmutant of I�B� in which the two critical serine residues havebeen mutated to alanine (I�B� S32A/S36A), and a deletionmutant of I�B� lacking the N-terminal 36 amino acids(I�B��N). Similarly, kinase-dead mutants of IKK1 and IKK2effectively blocked K13-induced A20 promoter activity (Fig.2E).

We next examined whether K13-induced A20 promoteractivity could be antagonized by pharmacologic inhibitors ofthe NF-�B pathway. We have previously reported that Bay-11-7082, a specific inhibitor of the NF-�B, can block K13-inducedNF-�B activity and spindle cell transformation of human vas-cular endothelial cells (18). Consistent with these results, treat-ment with Bay-11-7082 also blocked K13-induced A20 pro-moter activity in 293T cells (Fig. 2F). Because K13 activates theNF-�B pathway by activating the IKK complex (24, 28), weexamined the ability of a specific inhibitor of this complex,PS1145 (32), to block K13-induced A20 promoter activity. Asshown in Fig. 2F, this inhibitor effectively blocked K13-trans-activated A20 promoter activity. Arsenic trioxide, anotherknown inhibitor of K13-induced NF-�B activation (33), alsoblocked K13-induced A20 promoter activity (Fig. 2F). Takencollectively, these results confirm the importance of the IKKcomplex and the NF-�B pathway in K13-induced A20 pro-moter activation.Inhibition of K13-induced NF-�B Activation by Overexpres-

sion of A20—A20 is a key player in the negative feedback regu-lation of NF-�B signaling in response to multiple stimuli,including TNF� and IL-1� (34). In contrast, NF-�B activationby human T cell lymphotropic virus 1 Tax protein, whichresembles K13 in activating NF-�B by directly binding toNEMO, has been reported to be resistant to A20 (7). Therefore,we next examined the effect of A20 on K13- and Tax-inducedNF-�B activity. For this purpose, we transfected 293T cells withanNF-�Bdriven luciferase-based reporter construct alongwithK13 and Tax in the presence or absence of A20 and �24 h aftertransfection examined the induction NF-�B promoter activityby a luciferase assay. A20 effectively blocked K13-inducedNF-�B activity but had only a minor effect of Tax inducedNF-�B activity (Fig. 3, A and B). Furthermore, A20 co-expres-sion led to near complete inhibition of NF-�B reporter activityin 293T cells that had been transfected with the K13-ERTAM

construct and treated with 4OHT (Fig. 3C). A20 contains twoubiquitin-editing domains, an N-terminal deubiquitinatingdomain of the OTU family, and a C-terminal ubiquitin ligasedomain that contains seven zinc fingers (3). Both these domainshave been shown to play a role in inhibiting NF-�B activity. Todetermine the role of these domains in K13-induced NF-�Bactivity, we tested the ability of A20 deletion mutants contain-ing its N-terminal (1–380) and C-terminal (373–790) domainsto block K13-induced NF-�B activity. We observed that bothdomains of A20 contributed to inhibition of K13-inducedNF-�B activity (Fig. 3D). Essentially similar results wereobtained when the experiment was repeated using the K13-ERTAM construct (Fig. 3E).A20 Blocks K13-induced CCL20 and IL-8 Production—We

and others have shown that K13 induces the expression of alarge number of proinflammatory chemokines and cytokinesthat have been implicated in the pathogenesis of Kaposi sar-coma (15, 18–19, 35–37). Because A20 is known to block theinflammatory response, we examined the effect of A20 on K13-induced promoter activation of two inflammatory chemokines,CCL20 and IL-8. As shown in Fig. 4, A–D, luciferase-basedreporter assays showed that A20 blocks the induction of CCL20and IL-8 by K13 by blocking the transcriptional activation of

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their promoters. Furthermore, co-expression of A20 blockedthe induction of both these chemokines by K13 as measured byELISA (Fig. 4, E and F).A20 Blocks K13-induced Cellular Transformation—Anchor-

age-independent growth is a characteristic feature of trans-formed cells. We have demonstrated previously that ectopicexpression of K13 in Rat1 fibroblast cells confers on them theability to grow in an anchorage-independent manner and formcolonies in soft agar. To examine the effect of A20 on K13-induced cellular transformation, we generated stable popula-tions of Rat1-K13 cells expressing an empty vector and A20(Fig. 4G). We next used a soft agar colony formation assay totest the effect of A20 on K13-induced cellular transformation.

As shown in Fig. 4H, we observed greater than 4-fold reductionin soft agar colony formation in the Rat1-K13 cells transfectedwith the A20 expression vector compared with those trans-fected with the control vector.A20 Blocks K13-induced NF-�B Activity by Blocking p65

Nuclear Translocation and I�B� Phosphorylation—Themech-anism by which A20 blocks NF-�B is not entirely clear, andcontradictory results have been reported on the effects of A20on NF-�B nuclear translocation and DNA binding. For exam-ple, it has been reported that A20 blocks the TNF-inducedNF-�B luciferase activity in HEK293 cells without blockingNF-�B nuclear localization or DNA binding (38). To under-stand the mechanism by which A20 blocks K13-induced

FIGURE 3. A20 inhibits K13-induced NF-�B activity. A and B, 293T cells were transfected with control vector, K13, or Tax along with an NF-�B-Lucconstruct (75 ng/well) and a pRSV/LacZ (�-galactosidase) reporter construct (75 ng/well) in the presence or absence of A20. Cells were lysed 20 h aftertransfection for reporter assays. The values shown are the averages (mean � S.E.) of one representative experiment of three in which each transfectionwas performed in duplicate. The expression of the transfected A20 and FLAG-tagged K13 and Tax proteins was demonstrated by immunoblotting (lowerpanel). C, A20 inhibits K13-ERTAM-induced NF-�B activity. The experiment was performed essentially as described in 2A. The values shown are averages(mean � S.E.) of one representative experiment of three in which each transfection was performed in duplicate. D and E, 293T cells were transfected withK13 or K13-ERTAM along with NF-�B-Luc and pRSV/LacZ reporter constructs in the presence or absence of the indicated A20 constructs. The cellstransfected with K13-ERTAM were treated with 4OHT (20 nM) for 24 h prior to cell lysis and measurement of reporter activities. The values shown are theaverages (mean � S.E.) of one representative experiment of three in which each transfection was performed in duplicate. Lower panel shows theexpression of the transfected proteins as determined by Western blotting with a FLAG antibody. Asterisks indicate statistical significance at levels ofp � 0.05.

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NF-�B, we examined its effect on K13-induced nuclear trans-location of p65/RelA subunit of NF-�B. For this purpose, wetransfected 293T cells with an EGFP-p65 fusion construct inthe presence and absence of co-transfected K13 and A20 andexamined the cellular localization of EGFP-p65 by fluores-cence microscopy. Consistent with the fact that under basalconditions p65 is retained in the cytosol by its interactionwith I�B�, the cells transfected with the EGFP-p65 constructalone primarily showed cytoplasmic localization of EGFP-p65 (Fig. 5A). On the other hand, EGFP-p65 was localized in

the nucleus in a significant proportion of cells cotransfectedwith K13 (Fig. 5A). However, K13-induced nuclear localiza-tion of EGFP-p65 was markedly reduced upon coexpressionof A20 (Fig. 5A).K13 activates NF-�B by inducing phosphorylation of I�B

which results in its degradation by the ubiquitin-proteasomepathway, allowing the NF-�B subunits to enter the nucleus(28). To examine the mechanism by which A20 blocks K13-induced nuclear translocation of p65/RelA, we examined thephosphorylation status of I�B� in 293T cells that have been

FIGURE 4. A20 blocks K13-induced up-regulation of CCL20 and IL-8 and cellular transformation. A–D, A20 blocks K13-induced up-regulation ofCCL20 and IL-8. 293T cells were transfected with an empty vector, K13, or K13-ERTAM along with luciferase reporter constructs driven by CCL20(CCL20-Luc) or IL-8 promoters (IL-8-Luc) (75 ng/well) and a pRSV/LacZ (�-galactosidase) reporter construct (75 ng/well) in the presence or absence ofA20. The cells transfected with K13-ERTAM were subsequently treated with 4OHT (20 nM) for 24 h and the luciferase reporter assay performed essentiallyas described in Fig. 2A. Lower panels demonstrate the expression of the transfected proteins as measured by immunoblotting with the indicatedantibodies. The values shown are the averages (mean � S.E.) of one representative experiment of three in which each transfection was performed induplicate. E and F, cellular supernatants from 293T cells transfected, as explained in A–D, were collected before doing luciferase assay and used tomeasure the secretion of CCL20 and IL-8 by ELISA. The values shown are averages (mean � S.E.) of one representative experiment of three in which thelevel of CCL20 or IL-8 secretion was measured in duplicate. Asterisks (*) indicate significance at levels of p � 0.05. G, immunoblot shows increased A20expression in Rat1-K13 cells transfected with an A20 expression construct. H, soft agar colony formation by Rat1-K13 cells transfected with an A20expression vector was reduced compared with those transfected with an empty vector. The values shown are mean � S.D. of two independentexperiments performed in triplicate. *, p � 0.05 versus vector cells.

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transfected with K13 in the presence or absence of A20. Asshown in Fig. 5B, transfection of K13-induced significantphosphorylation of I�B� compared with empty vector-transfected cells. However, phosphorylation of I�B� wasmarkedly reduced in cells in which K13 was co-transfectedwith A20 (Fig. 5B).K13-induced NF-�B Activity Is Enhanced in A20�/� MEFs—

To confirm the physiological relevance of the above studies, wegenerated stable pools ofA20�/� andA20�/�MEFs expressingFLAG-K13-ERTAM and confirmed equivalent expression of thetransduced protein by Western blotting (Fig. 5C). Consistentwith our previous results, treatment of A20�/�K13-ERTAM

cells with 4OHT resulted in up-regulation of A20 expression,which was evident as early as 6 h after treatment and continuedto increase up to 12 h after treatment (Fig. 5C). More impor-tantly, 4OHT-induced phosphorylation of I�B� was sub-stantially increased in the A20�/�K13-ERTAM MEFs com-pared with the A20�/� K13-ERTAM MEFs (Fig. 5C).Furthermore, 4OHT-induced a greater increase in NF-�Bluciferase activity in A20�/� K13-ERTAM cells comparedwith A20�/� K13-ERTAM cells, as measured by the lucifer-ase-based reporter assay (Fig. 5D). Collectively, these resultsdemonstrate that A20 negatively regulates K13-inducedNF-�B activity by blocking phosphorylation of I�B�.A20 Silencing Enhances NF-�B Activity in BC1 PEL Cells—

To demonstrate that A20 also controls NF-�B activity in PELcells, we took advantage of BC1 cells that are naturally infected

with KSHV and express high levels of K13 endogenously (Fig.1A). We generated stable populations of these cells expressingan NF-�B-driven luciferase reporter construct using lentiviral-mediated gene transfer. We next used siRNA-mediated genesilencing to study the effect of A20 depletion on theNF-�B-Lucactivity in the BC1-NF-�B-Luc cells. Lamin A/C and p65siRNAs were used as negative and positive controls, respec-tively. As shown in Fig. 5E, we observed increased NF-�B-Lucactivity in theA20-depleted cells comparedwith those depleted oflamin A/C, whereas reduced NF-�B-Luc activity was observed inthe p65-depleted cells. These results demonstrate that A20 nega-tively regulates NF-�B activity in the KSHV-infected PEL cells.A20 Interacts with K13—To examine the mechanism by

which A20 blocks K13-induced I�B� phosphorylation, we useda co-immunoprecipitation assay to examine the interactionbetween the two proteins.We immunoprecipitated FLAG-K13fromK13-expressing BC3 cells using FLAGor control antibodybeads and examined the presence of endogenously expressedA20 in the immunoprecipitated samples by Western blotting.As shown in Fig. 6A, A20 was readily detected in the samplesimmunoprecipitated with the FLAG antibody. A strong inter-action between K13 andNEMOwas also observed (Fig. 6A). Toconfirm the results, we repeated the experiment in the K13-expressing A20�/� and A20�/� MEFs. A band correspondingtoA20was readily detected in the samples immunoprecipitatedwith the FLAG antibody fromK13-expressingA20�/� cells butnot the A20�/� cells (Fig. 6B).

FIGURE 5. A20 blocks K13-induced NF-�B activation by blocking I�B� phosphorylation and p65 nuclear translocation. A, 293T cells were trans-fected with an EGFP-p65 fusion construct along with the indicated expression constructs, and cellular localization of EGFP-p65 was examined byfluorescence microscopy. K13-induced nuclear accumulation of EGFP-p65 is blocked by co-expression of A20. B, 293T cells were transfected with K13 inthe presence or absence of A20, and 24 h after transfection, total cell lysates were prepared and used to examine the effect of A20 on K13-inducedphosphorylation of I�B�. The expression of total I�B� is shown in the lower panel. C, immunoblot shows increase in phosphorylation of I�B� inK13-ERTAM-expressing A20�/� MEFs compared with the corresponding A20�/� MEFs. A20�/� and A20�/� MEFs expressing FLAG-K13-ERTAM weretreated with 4OHT for the indicated time points, and the phosphorylation of I�B� and A20 expression was measured using the indicated antibodies.Equivalent expression of the FLAG-tagged K13-ERTAM protein and equal protein loading was confirmed by blotting with FLAG and tubulin antibodies,respectively. D, A20�/� and A20�/� MEFs expressing FLAG-K13-ERTAM were transfected with NF-�B promoter luciferase construct (75 ng/well) and aRenilla reporter construct (75 ng/well, normalization control) using Lipofectamine-mediated transfection as described previously (33). The transfectedcells were subsequently treated with 4OHT (20 nM) for 48 h, and the luciferase reporter assay was performed essentially as described in Fig. 2A. Thevalues shown are the averages (mean � S.E.) of one representative experiment of three in which each transfection was performed in duplicate. Asterisks(*) indicate significance at levels of p � 0.05. E, upper, BC1-NF�B-Luc cells were transfected with a control siRNA (lamin A/C) or siRNAs directed againstA20 or p65, respectively. Approximately 72 h after transfection, cell lysates were prepared and used for the measurement of luciferase activity. The datarepresent mean � S.E. of a representative of two independent experiments with similar results. *, p � 0.05 versus control cells. E, lower, immunoblotconfirms specific knock-down of A20 and p65 proteins following siRNA-mediated gene silencing.

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To confirm that KSHV-derived endogenous K13 also inter-acts with endogenously expressed A20, we immunoprecipi-tated K13 from BC1 and BC3 cells using 8F6, a monoclonalantibody against K13, and examined its interaction with A20and IKK�/� (positive control) by Western blotting. As shownin Fig. 6C, A20 and IKK�/�were readily detected in the samplesimmunoprecipitated with the 8F6mAb but not in those immu-noprecipitated with a control mAb.Finally, we examined whether the K13-A20 interaction will

interfere with the known interaction of K13 with the IKK com-plex. For this purpose, we transfected 293T cells with FLAG-tagged K13, either alone or in combination with A20, and stud-ied the effect of A20 on the interaction between K13 andendogenously expressed IKK complex subunits by co-immuno-precipitation assay. As shown in Fig. 6D, co-expression of A20did not impair the interaction between K13 and IKK complexsubunits. These results demonstrate that A20 blocks K13-induced NF-�B without physically disrupting the K13-IKKcomplex.

DISCUSSION

A20 is known to act as a tumor suppressor in several subtypesof non-Hodgkin and Hodgkin lymphomas, including marginal

zone lymphoma, diffuse large B cell lymphoma, mantle celllymphoma, mucosa-associated lymphoid tissue lymphoma,primary mediastinal lymphoma, Burkitt lymphoma, naturalkiller cell lymphoma, adult T cell leukemia/lymphoma, andclassical Hodgkin lymphoma (3, 39–41). A20 expression is lostin a substantial number of these lymphomas by gene deletion,promoter methylation, frameshift mutations, and/or pointmutations (3). Because A20 is a negative regulator of NF-�Bactivity, its inactivation is believed to contribute to develop-ment of these lymphomas by promoting unchecked NF-�Bactivity and enhancing cell survival (3). However, KSHV-in-fected PEL cells are known to possess an alternativemechanismfor activating the NF-�B pathway, namely the expression ofKSHV-encoded K13 protein which is a potent activator of theIKK complex (28, 42). Consistentwith their lack of requirementfor A20 inactivation to activate NF-�B, we observed A20expression, albeit of varying magnitude, in all four PEL-derivedcell lines tested in the current study, which correlated closelywith their level of K13 expression. Interestingly, similar to K13,EBV-encoded LMP1 results in constitutive NF-�B activation(43), and a recent study revealed that A20 mutation frequencyand mutation patterns in classical Hodgkin lymphoma differedsignificantly according to their EBV status (40). Thus, whereasA20 genetic alterations were relatively rare among 16 EBV-positive classical Hodgkin lymphomas, they were quite fre-quent among the EBV-negative classical Hodgkin lymphomasand frequently involved both copies of the TNFAIP3 gene (40).Taken collectively with this study, our results suggest that A20inactivation is not a common feature of KSHV and EBV-asso-ciated lymphomas due, in part, to the constitutive NF-�B acti-vation induced by the K13 and LMP1 proteins.In this report we demonstrate that K13 up-regulates A20

expression via NF-�B activation. The involvement of K13-in-duced NF-�B pathway in up-regulating A20 expression is sup-ported by the following lines of evidence. First, the expressionof A20 correlated closely with their level of K13 expression andtheir previously reported level of NF-�B activity (21). Thus, theBC1 cell line, which possesses high level K13 expression andconstitutive NF-�B activity (21, 28, 42), showed high A20expression, whereas the BCBL1 cell line, which has low K13expression and NF-�B activity (21, 28, 42), showed weakA20 expression. Furthermore, A20 expression was up-regu-lated in the BCBL1-K13 cells, which possess increased NF-�Bactivity (21). Second, we observed a strong correlation betweenthe ability of vFLIPs to activate NF-�B and their ability to acti-vate A20 promoter. Thus, whereas the wild-type K13 stronglyactivated A20 promoter, its NF-�B-defective mutants failed todo so. Similarly, no A20 promoter activation was observedupon expression of the vFLIP E8 that lacks NF-�B activity (24).Third, K13 failed to activate an A20 promoter construct con-taining mutations in the two NF-�B sites. Finally, K13-inducedA20 promoter activity was blocked by genetic (i.e. I�B� S32A/S36A, �NI�B�, IKK1-KM, and IKK2-KM) and pharmacologi-cal (i.e.Bay-11-7082, PS1145, and arsenic trioxide) inhibitors ofthe NF-�B pathway.We observed that A20 overexpression effectively blocked

K13-induced NF-�B activity, induction of NF-�B-responsivegenes, such as CCL20 and IL-8, andK13-induced cellular trans-

FIGURE 6. K13 interacts with A20. A, cell lysates (C.L.) prepared from BC3 cellsexpressing an empty vector or FLAG-tagged K13 were immunoprecipitated(I.P.) using control (C) or FLAG (F) beads, and the presence of interacting A20and NEMO proteins was detected by Western blotting with the indicatedantibodies. B, co-immunoprecipitation assay shows interaction of K13 withA20 in A20�/� MEFs. The experiment was performed essentially as describedfor A. A tubulin blot shows equal protein loading. C, cell lysates (C.L.) preparedfrom BC1 and BC3 cells were immunoprecipitated using control antibody or8F6 antibody raised against K13, and the presence of interacting A20 and IKK�/� proteins was detected by Western blotting with the indicated antibodies.D, A20 does not block the interaction between K13 and IKK complex subunits.293T cells were transfected with an empty vector or a vector expressingFLAG-tagged K13, either alone or with A20, and a co-immunoprecipitationassay was performed essentially as described for A. A tubulin blot shows equalprotein loading.

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formation. The physiological relevance of these studies wasconfirmed by results showing that K13-induced I�B� phosphor-ylation and NF-�B transcriptional activity are increased inA20�/� cells. The N-terminal region of A20 contains a deubiq-uitinating domain of the OTU family that deubiquitinates Lys-63-polyubiquitinated proteins, whereas the C-terminal regioncontains 7Zn fingers that catalyze the addition of Lys-48-linkedpolyubiquitin chains on the target proteins, targeting them forproteasomal degradation (3, 44). A recent study showed thatA20, together with the regulatory molecule TAX1BP1, inter-acted with E2 ubiquitin-conjugating enzymes Ubc13 andUbcH5c, triggering their ubiquitination and proteasome-de-pendent degradation, which in turn blocked IL-1�-inducedNF-�B by blocking Lys-63-linked polyubiquitination (45). Boththe OTU and zinc finger 4 domains of A20 were found to beimportant for binding to Ubc13 and UbcH5c and for inhibitionof IL-1�-induced NF-�B by A20 (45). In the present study, wediscovered that both the N-terminal and C-terminal domainsof A20 are involved in inhibition of K13-induced NF-�B activ-ity. Therefore, it is conceivable that similar to its inhibition ofIL-1� signaling, A20 blocks K13-induced NF-�B by inducingdegradation of Ubc13 and UbcH5c. We also observed thatalthough A20 physically interacted with K13, it did not blockK13-IKK interaction, thereby arguing against the possibilitythat A20 blocks K13-induced NF-�B by interfering with theassembly of the K13-IKK complex. Finally, we have observedthat co-expression of A20 and K13 sometimes results in theappearance of their high molecular weight products uponimmunoblotting (e.g. Fig. 6, A and B). Studies are currently inprogress to examine whether these slow migrating products ofA20 and K13 represent their polyubiquitinated forms and theirrole in the regulation of K13-induced NF-�B activity.

Similar to K13, human T cell lymphotropic virus 1 Tax acti-vates NF-�B by directly binding to NEMO (45). However, Tax-induced NF-�B has been reported to be relatively resistant toA20 (7, 45), a finding confirmed in the current study. Tax isbelieved to circumvent A20 inhibition by binding to Tax1BP1and disrupting the interaction among A20, Tax1BP1, andUbc13, thereby preventingUbc13 degradation (45). Our resultsshowing that K13-induced NF-�B can be blocked by A20suggest that although both K13 and Tax require binding toNEMO for NF-�B activation, they employ distinct underly-ing mechanisms.What is the physiological significance of these results?

K13-induced NF-�B activation is known to play a key role inthe natural history of KSHV infection and in the pathogen-esis of KSHV-associated malignancies (46). K13 has beenshown to promote cellular proliferation, transformation,cytokine secretion, and viral latency (13–20). However,unchecked activation of the NF-�B pathway has the poten-tial of resulting in uncontrolled inflammatory response. Byblocking K13-induced NF-�B activation, A20 may serve toattenuate the inflammatory response and cellular transfor-mation and thereby help maintain a balance between thevirus and the host. Finally, in addition to its role in blockingthe inflammatory response, A20 is known to serve a cytopro-tective role and has been shown to protect vascular endothe-lial cells against TNF�, Fas, and natural killer cell-induced

cell death (47). Although based on its structural homology tothe prodomain of caspase 8/FLICE, K13 was originallybelieved to protect against apoptosis induced by the deathreceptors of the TNF family by directly binding to FADDand/or caspase 8/FLICE (12), subsequent studies have ques-tioned this interaction as the basis of its cytoprotective abil-ity (16). In contrast, it is now generally believed that K13exerts its antiapoptotic and cytoprotective effect primarilyvia NF-�B activation (46). However, the NF-�B-responsivegenes responsible for this effect have not been identified.Based on our results, it is conceivable that K13-induced A20up-regulation may protect the KSHV-infected vascularendothelial cells against death receptor-induced apoptosisand thereby contribute to the pathogenesis of Kaposisarcoma.

Acknowledgments—We thank Dr. Averil Ma for A20�/� MEFs, Dr.Dikstein for A20 luciferase reporter constructs, Dr. Lin Rongtuan forwild-type and mutant A20 expression constructs, and Dr. CiarenGraham for critical reading of the manuscript.

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