JOURNAL OF VIROLOGY, Feb. 2010, p. 1722–1730 Vol. 84, No. 40022-538X/10/$12.00 doi:10.1128/JVI.01918-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Transcriptional Regulation of BK Virus by NuclearFactor of Activated T Cells�

Joslynn A. Jordan,1 Kate Manley,2 Aisling S. Dugan,2 Bethany A. O’Hara,2 and Walter J. Atwood2*Graduate Program in Pathobiology, Brown University, Providence, Rhode Island 02912,1 and Department of

Molecular & Cell Biology and Biochemistry, Brown University, Providence, Rhode Island 029122

Received 10 September 2009/Accepted 23 November 2009

The human polyomavirus BK virus (BKV) is a common virus for which 80 to 90% of the adult populationis seropositive. BKV reactivation in immunosuppressed patients or renal transplant patients is the primarycause of polyomavirus-associated nephropathy (PVN). Using the Dunlop strain of BKV, we found that nuclearfactor of activated T cells (NFAT) plays an important regulatory role in BKV infection. Luciferase reporterassays and chromatin immunoprecipitation assays demonstrated that NFAT4 bound to the viral promoter andregulated viral transcription and infection. The mutational analysis of the NFAT binding sites demonstratedcomplex functional interactions between NFAT, c-fos, c-jun, and the p65 subunit of NF-�B that togetherinfluence promoter activity and viral growth. These data indicate that NFAT is required for BKV infection andis involved in a complex regulatory network that both positively and negatively influences promoter activity andviral infection.

Polyomavirus-associated nephropathy (PVN) is prevalent in5 to 10% of kidney transplants, resulting in a 50% or greaterincidence of allograft loss or dysfunction in immunosuppressedrenal transplant recipients (19, 40, 42). PVN is associated withthe human polyomavirus BK virus (BKV). BKV is a member ofthe Polyomavirdae family, which also includes JC virus (JCV)and simian virus 40 (SV40). Polyomaviruses are characterizedby small (40 to 50 nm in diameter), nonenveloped virusescontaining circular double-stranded DNA (18, 23). The modeof transmission is unknown, but initial infection takes place inearly childhood and is restricted to the epithelium of renaltubules, ureter, and urinary bladder (16, 50). BKV initiates anasymptomatic, lifelong persistent infection in approximately 80to 90% of healthy human adults (6, 27, 48). The reactivation ofBKV occurs during immunosuppression following kidney orbone marrow transplantation (17, 24). Transplant recipientsreceiving more intensive immunosuppressive regimens are at ahigher risk of viral reactivation (3). This can permit high levelsof viral replication, resulting in renal damage and ultimatelygraft failure (18, 20, 40). Currently, the treatment of PVNincludes a reduction in immunosuppressive therapy in combi-nation with the antiviral agents leflunomide and cidofovir (11,26, 45, 54).

Like those of other members of the polyomavirus family, theBKV life cycle is highly regulated. The virus binds to cells viaan N-linked glycoprotein with �(2,3)-linked sialic acid andgangliosides GT1b and GD1b (29) and is internalized viacaveola-mediated endocytosis (8, 10). The genome is com-prised of three functional regions. The noncoding control re-gion (NCCR) mediates viral replication and transcriptionwhile dividing the genome into the early gene products small

and large T antigens and the late gene products viral capsidproteins VP1, VP2, and VP3 (47). In the archetype strain(WW), the NCCR is a highly conserved bidirectional promotercomposed of five sequence blocks labeled in alphabetical orderas O, P, Q, R, and S. The O block contains a palindrome of twoinverted repeat sequences, a 20-bp A/T region, and the startcodon for early genes, P, Q, and R are three transcriptionfactor binding blocks, followed by the S block, which containsthe start codon for agnoprotein (38). Any deletions, duplica-tions, or rearrangements in the NCCR are classified as rear-ranged forms compared to the archetype (39). The occurrenceof rearrangements is not well understood, and they are notconsidered unique strains but rather adaptations in variablecellular environments and tissue culture systems (1). The re-arranged NCCR of the BKV Dunlop strain is composed oftriplicate P blocks (NCCR structure, O-P-P1-7;26-68-P1-64-S)where Q and R blocks have been deleted (7, 51).

Previous research in our laboratory identified nuclear factorof activated T-cells (NFAT) as an important transcription fac-tor in the regulation of both JCV and SV40 infection (34, 35).The NFAT family of transcription factors is composed of fiveproteins, of which NFAT1 (NFATc2), NFAT 2 (NFATc1),NFAT 3 (NFATc4), and NFAT4 (NFATc3) are regulated bycalcium (5, 22, 30, 31). The N terminus of the protein is highlyconserved, containing the NFAT homology region and theDNA binding domain (21, 30, 44). These proteins are ex-pressed in various immune cells and have been detected in avariety of tissues, such as kidney, thymus, and spleen (21, 37).NFAT is phosphorylated in resting cells; dephosphorylationoccurs by the calcium activation of a calmodulin-dependentserine phosphatase calcineurin, which exposes the nuclear lo-calization signal (31, 44, 46, 49). NFAT translocates to thenucleus, where it regulates the expression of various genes (25,46). During T-cell activation, NFAT promotes gene transcrip-tion by syngerstic interactions with other transcription factors,such as activator protein 1 (AP-1).

* Corresponding author. Mailing address: Department of MolecularBiology, Cell Biology and Biochemistry, Brown University, Providence,RI 02912. Phone: (401) 863-3116. Fax: (401) 863-9653. E-mail: Walter_Atwood@brown.edu.

� Published ahead of print on 2 December 2009.

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In this study, we examined the role of NFAT in BKV infec-tion and transcription. Luciferase reporter assays and chroma-tin immunoprecipitation assays (ChIP) revealed that NFAT4binds to the Dunlop promoter and regulates promoter activityin Vero cells. Mutational analysis demonstrates that all threeNFAT sites are required for transcriptional activity. However,mutations of NFAT site 1 or NFAT site 2 increased earlypromoter activity and viral propagation. These two sites canboth activate and repress transcription, depending on whetherthe AP1 or NF-�B site also is bound by fos/jun or p65, respec-tively.

MATERIALS AND METHODS

Cells, virus, and VIVIT inhibitor peptide. Vero cells were maintained inminimum essential medium (E-MEM: Mediatech Inc., Herdon, VA) containing5% heat-inactivated fetal bovine serum (Mediatech Inc.) at 37°C in a 5% CO2

incubator. The BKV Dunlop strain used in these experiments was purchasedfrom the American Type Culture Collection (ATCC; Manassas, VA). Cell-permeable NFAT inhibitor peptide (11R-VIVIT) was purchased from Calbio-chem (San Diego, CA). The NFAT inhibitor peptide sequence was HRRRRRRRRRRRGGGMAGPHPVIVITGPHEEOH (41).

Indirect immunofluorescent analysis. Cells were grown on coverslips to 70%confluence and infected with BKV at a multiplicity of infection (MOI) of 5 in thepresence of 2% fetal bovine serum for 1 h at 37°C. Seventy-two hours postin-fection, cells were fixed with 2% paraformaldehyde for 20 min at room temper-ature and permeabilized for 20 min in 0.5% Triton X-100. Cells were stainedwith monoclonal antibody PAB 416 (Ab-2) to detect BKV large T-antigen(T-Ag) (Oncogene Research Products, Cambridge, MA). Late viral protein VP-1was detected with monoclonal antibody PAB 597 made against SV40, whichcross-reacts with BKV and JCV. Secondary antibody Alexa Fluor 488-labeledgoat anti-mouse antibody was purchased from Invitrogen (Carlsbad, CA). Cellswere mounted on slides using Vectashield with 4�,6�-diamidino-2-phenylindole(DAPI) (Vector Labs, Burlingame, CA) and observed using a Nikon epifluores-cence microscope (Eclipse E800; Nikon Inc.).

Constructs and mutagenesis. pluc2/DunlopE and pluc2/DunlopL were madeby the PCR amplification of the promoter region of the pBR322 Dunlop con-struct (ATCC) with BglII and XhoI sites at either end of the primers to insert thepromoter into the pGL4.10 luciferase construct (Promega, Madison, WI) ineither the early or late orientation. The phMGFP construct expresses only greenfluorescent protein (GFP) driven by the cytomegalovirus (CMV) promoter and

was purchased from Promega. The pGFP-VIVIT construct contains the NFATinhibitor peptide sequence (MAGPHPVIVITGPHEE) subcloned into pEGFPat the N terminus (2). The pRL-TK plasmid was purchased from Promega andexpresses the Renilla luciferase from the thymidine kinase promoter of herpessimplex virus. The pluc2/Dunlop reporter constructs and BKV/puc19 plasmidwere mutated using a Stratagene QuikChange mutagenesis kit. NFAT sequencesin the viral promoter were mutated from GGAAA to TAGAT (28). To confirmmutations, samples were sent to Genewiz, South Plainfield, NJ, for sequencingwith RVprimer3 (Promega). The primers used to mutate BKV/puc19 are thefollowing: NFAT site 1 forward, 5�-CCATGACCTCAGGAATAGATGTGCATGACTCACAGGGGAATGCAG-3�; reverse, 5�-GCTGCATTCCCCTGTGAGTCATGCACATCTATTCCTGAGGTCATGG-3�; NFAT site 2 forward, 5�-CCATGACCTCAGGAATAGATGTGCATGACTCACAGGGAGGAGCTGC-3�;reverse, 5�-GCAGCTCCTCCCTGTGAGTCATGCACATCTATTCCTGAGGTCATGG-3�; and NFAT site 3 forward, 5�-CCAAACCATGACCTCAGGAATAGATGTGCATGACAGACATGTTTTGC-3�; reverse, 5�-GCAAAACATGTCTGTCATGCACATCTATTCCTGAGGTCATGGTTTGG-3� (nucleotides in bolddenote NFAT binding site mutations).

ChIP. To assess NFAT4 binding during infection, Vero cells were grown to70% confluence in 75-cm2 flasks and infected with BKV at an MOI of 5 in thepresence of 2% fetal bovine serum for 1 h at 37°C. Cells were maintained in

FIG. 1. Inhibition of NFAT reduces BKV infection. Vero cellswere pretreated with increasing concentrations of a cell-permeableNFAT peptide inhibitor (11R-VIVIT). The cells were infected withBKV in the presence of the same concentrations of 11R-VIVIT asthose used for pretreatment. Peptide-containing medium was changedat 24 and 48 h postinfection due to the half-life of the 11R-VIVITpeptide. At 72 h postinfection, cells were fixed and infection wasscored by indirect immunofluorescence for early viral protein (T-anti-gen)-positive or late viral protein (V-antigen)-positive cells. Cells werecounted and compared to the percentage of the positive control ofuntreated BKV-infected cells. The error bars are the calculations ofthe standard deviations of three independent experiments.

FIG. 2. NFAT activates the viral promoter. Luciferase assays wereused to determine the effect of the NFAT inhibitor peptide (VIVIT)on viral transcription. Vero cells were cotransfected with either theearly promoter (pluc2/DunlopE) or the late promoter (pluc2/DunlopL)and a construct that expresses the NFAT inhibitor peptide sequence(pGFP-VIVIT) or a control construct expressing only GFP(phMGFP). NFAT was stimulated with 2 �M ionomycin and 80 ng/mlPMA, and luciferase activity was measured. Luciferase results werenormalized to TK-Renilla (pRL-TK). Error bars represent the stan-dard deviations.

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medium with 5% serum for 3 and 9 days postinfection. Chromatin immunopre-cipitation (ChIP) was conducted by following the Santa Cruz Biotechnologyprotocol. Early promoter-containing NFAT mutants and wild-type (WT) earlypromoters were transfected in Vero cells using Fugene (Roche, Branchburg, NJ).Cells were treated with 2 �M ionomycin and 80 ng/ml PMA 48 h posttransfec-tion. ChIP was carried out by following the protocol obtained from Santa CruzBiotechnology. Cells were harvested by mechanical scraping and were washedtwice with 1� phosphate-buffered saline (PBS). DNA was crossed-linked using1% formaldehyde for 8 min at room temperature. To terminate cross-linking,cells were treated with 0.25 M glycine for 5 min on ice. Cells were resuspendedthree times with lysis buffer (Santa Cruz Biotech, Inc.), and crude nuclearextracts were resuspended in 1 ml of high-salt lysis buffer (Santa Cruz Biotech-nology, Inc.). Nuclear extracts were sonicated using a Branson Sonifier 150 at apower setting of 2 six times for 1 min each on ice. A concentration of 500 �g ofchromatin was precleared by protein A/G Plus-agarose (Santa Cruz Biotechnol-ogy, Inc.). Chromatin was incubated with primary antibody overnight at 4°C.Primary antibodies were purchased from Santa Cruz Biotechnology, Inc. ProteinA/G Plus-agarose was added at 4°C for 2 h to harvest immune complexes. Beadswere resuspended and washed with lysis buffer twice, followed by three washeswith wash buffer (Santa Cruz Biotechnology, Inc.). Samples were eluted usingelution buffer (Santa Cruz Biotechnology, Inc.) and incubated at 67°C overnight.DNA was extracted using a Qiagen PCR purification kit according to the man-ufacturer’s protocol. SYBR green quantitative PCR (Applied Biosystems, Foster

FIG. 3. NFAT4 binds to the viral promoter during infection. Verocells were infected with BKV, cells were harvested 3 and 9 days postin-fection, and chromatin immunoprecipitation was performed. DNA wasimmunoprecipitated with an anti-NFAT4 antibody and compared toresults for an anti-NFAT3 antibody and a nonspecific IgG antibody.Quantitative PCR was performed to analyze the DNA and thresholdcycle (CT) values of DNA immunoprecipitated with control antibody;IgG results were normalized to a value of 1. The graph representsaverages from three independent experiments. Error bars representthe standard deviations from three independent experiments.

FIG. 4. Schematic of the Dunlop early promoter. The promoter contains three NFAT binding site sequences located in triplicate P regions. Thefirst schematic is of the wild-type promoter containing an NF-�B subunit p65 binding site, two AP-1 binding sites, and all three NFAT binding sites,labeled in numerical order (site 1, site 2, and site 3), from the A/T-rich region (TATA box). Mutations of the NFAT sites are shown by schematicsof the early promoter with mutations (highlighted in red) of individual NFAT binding sites or combinations of multiple NFAT binding sites. TheNFAT binding sites in the early promoter reporter constructs were mutated from GGAAA to TAGAT.

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City, CA) was used to amplify the immunoprecipitated DNA. The primers usedto detect the Dunlop promoter were (antisense) 5�-TGAGCTCCATGGATTCTTCC-3� and (sense) 5�-ATTCCTAGGCTCGCAAAACA-3�.

Transfection and luciferase assay. Transient transfections were achieved usingFugene (Roche, Branchburg, NJ). Plasmids were cotransfected with the controlluciferase plasmid Renilla (pRL-TK) from Promega. Cells were treated with 2�M ionomycin and 80 ng/ml PMA 48 h posttransfection. Luciferase sampleswere measured using a Berthoid Lumat LB9501 luminometer. Luciferase resultsrepresent data from three independent transfections. The results of the fireflyluciferase measurements were normalized using Renilla luciferase measure-ments.

Viral growth assay. Vero cells were transfected with 1 �g of linearized WT orviral promoter containing NFAT mutant DNA as previously described (9).Transfected cells were fixed and stained for V antigen (V-Ag) 4, 7, 10, 13, 16, and19 days posttransfection. Virus-containing supernatants were collected at day 19.Vero cells were treated with supernatant as previously described (9). Cells werefixed and stained for V antigen expression 72 h postinfection.

RESULTS

Inhibition of NFAT reduces BK virus infection. To deter-mine whether NFAT activity is required for BKV infection, weexamined the effects of a cell-permeable NFAT inhibitor pep-tide, 11R-VIVIT, on BKV infection (2, 41). The NFAT inhib-itor peptide binds to the calcium-dependent phosphatase, cal-cineurin, and inhibits its ability to dephosphorylate NFAT butdoes not disrupt the calcineurin phosphatase activity (2, 41).Vero cells were pretreated with the 11R-VIVIT peptide for 6 hand then infected with the BKV Dunlop strain for 1 h. Cellswere maintained in medium containing the 11R-VIVIT pep-tide for 72 h. Infectivity was assessed by indirect immunofluo-rescence using antibodies specific for early viral protein T-Agand late viral protein V-Ag (Fig. 1). We observed a dose-dependent decrease of infection in the presence of the 11R-VIVIT peptide. This indicates that NFAT contributes to BKVinfection.

NFAT regulates BKV transcription. NFAT has been shownpreviously to regulate JCV and SV40 transcription (34, 35). Todetermine whether NFAT mediates BKV transcription, Verocells were cotransfected with either the early promoter (pluc2/DunlopE) or the late promoter (pluc2/DunlopL) and a con-struct that expresses the NFAT inhibitor peptide sequence(pGFP-VIVIT) or a control construct expressing only GFP(phMGFP). Forty-eight hours posttransfection, cells werestimulated with ionomycin and phorbol 12-myristate 13-ace-tate (PMA) to activate the calcineurin-NFAT pathway (Fig.2A and B). In the presence of pGFP-VIVIT there was a sig-nificant decrease in both early and late promoter activity.These results demonstrate that NFAT contributes to the reg-ulation of BKV transcription.

NFAT4 binds to the viral promoter during infection. TheNFAT family is composed of five proteins, of which NFAT1 toNFAT4 are regulated by calcium signaling. As Vero cells ex-press endogenous NFAT4 (35), we asked whether NFAT4could bind to the viral promoter during infection using chro-matin immunoprecipitation assays. Chromatin from infectedcells was immunoprecipitated with an NFAT4 antibody at 3and 9 days postinfection. NFAT4 bound to the promoter witha 3.5-fold increase at 3 days postinfection and a 9-fold increaseat 9 days postinfection compared to that of DNA immunopre-cipitated with an NFAT3 antibody or a nonspecific IgG controlantibody (Fig. 3). This suggests that NFAT4 binds to the viralpromoter during infection.

NFAT sites in the viral promoter have diverse roles in thetranscriptional regulation of the promoter. The Dunlop pro-moter contains a palindrome, two inverted repeat sequences,and a 20-bp A/T region followed by triplicate P regions en-compassing three NFAT binding sites. To determine whetherthese sites are required for BKV transcription, we mutated theNFAT binding sites in the early promoter (pluc2/DunlopE) tobe nonfunctional. The NFAT binding sequence was changedfrom GGAAA to TAGAT by site-directed mutagenesis (Fig.4) (28). Mutations of individual NFAT binding sites or variouscombinations of NFAT sites as well as mutations of all threesites on the promoter were constructed. Mutational analysisdemonstrated that mutations of these NFAT binding sites dis-played a diverse range of promoter activity. Vero cells weretransfected with either the wild-type Dunlop early promoter ormutant early promoter. We expected that mutations of theseNFAT binding sites would result in a decrease in early pro-

FIG. 5. NFAT has a diverse role in the regulation of early tran-scriptional activity. (A) Vero cells were transfected with either thewild-type early promoter (WT) or the early promoter containing mu-tations of the NFAT binding sites. The mutated sites are representedby an X. NFAT was stimulated with 2 �M ionomycin and 80 ng/mlPMA, and luciferase activity was measured. Luciferase results werenormalized to TK-Renilla (pRL-TK). The graph represents the exper-iment performed in triplicate. (B) ChIP analysis of NFAT binding tothe mutated viral promoter in the early orientation. Vero cells weretransfected with either the WT early promoter or the mutant earlypromoter. Cells were treated with ionomycin and PMA to activateNFAT and then immunoprecipitated with an anti-NFAT4 antibody.WT and NFAT mutants were compared to a nonspecific IgG antibody.Quantitative PCR was used to analyze DNA. CT values of IgG controlDNA were normalized to a value of 1. The graph represents theaverage fold change of NFAT mutants compared to the IgG control ofthree independent experiments.

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moter activity; however, mutations in NFAT site 1 demon-strated a 20-fold increase in early promoter activity comparedto that of the WT (Fig. 5A). Interestingly, mutations in NFATsite 2 demonstrated a greater increase in early promoter ac-tivity, increasing 54-fold compared to results for the WT (Fig.5A). Mutations of both NFAT site 1 and NFAT site 2 also ledto enhanced promoter activity compared to that of the wildtype. These findings suggest that the negative regulation of thepromoter by NFAT has been disrupted by these mutations.Early promoter activity was reduced when NFAT site 3 wasmutated (Fig. 5A). Mutations of NFAT site 1 and NFAT site3 showed wild-type activity. Mutations of site 2 and 3 showedsignificantly reduced activity compared to wild type. When allthree NFAT binding sites were mutated, early promoter activ-ity was abolished.

As luciferase data showed enhanced promoter activity withmutations in NFAT site 1 or NFAT site 2, we tested whetherNFAT still could bind to the mutated viral promoters. Cellswere transfected with wild-type early promoter or the relevantearly promoter containing NFAT binding site mutations, and

chromatin immunoprecipitation assays were performed. ChIPassays confirmed that NFAT4 bound to the wild-type promoterwith a 7-fold increase compared to results for the IgG control(Fig. 5B). When site 1 was mutated, NFAT binding to thepromoter was increased (Fig. 5B). When site 2 was mutated,NFAT binding was similar to binding to the wild-type pro-moter (Fig. 5B). However, when site 3 was mutated, NFATbinding to the promoter again was increased (Fig. 5B). Thesedata suggest a hierarchy of NFAT binding, with functionalNFAT site 1 being the dominant site and functional NFAT site2 being a possible repressor site. Wild-type levels of NFATbinding when NFAT site 2 is mutated suggests that NFATbinds to NFAT site 1 and, possibly, site 3 under these condi-tions. Interestingly, NFAT4 could not bind to an NFAT sitewhen site 1 and site 3 were mutated, suggesting that anothertranscription factor binds to NFAT site 2.

NFAT mutations in the viral genome enhance viral propa-gation. Luciferase assays and chromatin immunoprecipitationassays demonstrate that mutations of the NFAT binding sitesin the early promoter enhance promoter activity and increase

FIG. 6. NFAT mutations accelerate viral propagation. (A) Vero cells were transfected with either the WT Dunlop genome or NFAT mutants.Viral propagation was measured by scoring for late viral protein expression (V-Ag). The graph depicts a viral growth curve for each NFAT mutantvirus compared to results for the WT for a duration of 19 days posttransfection. (B) Representative indirect immunofluorescence images ofV-Ag-positive cells (green) and counterstained with DAPI on day 19 postinfection. Panel I, WT; II, mutant NFAT site 1; III, mutant NFAT site2; IV,; mutant NFAT site 3; V, Puc19. (C) Infectious titers of WT and NFAT mutant viruses. On day 19, supernatants were collected from theexperiments represented in panel A and used to infect Vero cells. At 72 h postinfection, cells were scored for V-antigen. The graph depicts theexperiment in triplicate, and error bars represent standard deviations.

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NFAT binding. To further investigate these effects, mutationsof NFAT binding sites were generated in the viral genome totest the effect on viral spread and infectivity. Mutated viralgenomes were transfected into cells, and cells were stained forlate viral protein expression (V-Ag) (Fig. 6A). Cells were fixedand stained at 3-day intervals 4 days posttransfection for aduration of 19 days (Fig. 6A). At 4 days posttransfection,mutations of NFAT site 1 or NFAT site 2 demonstrated anincreased level of viral spread compared to that of the WT,while mutations in NFAT site 3 showed a slight decrease.These data correlate with the luciferase data (Fig. 5A), sug-gesting that any disruption of NFAT binding site 1 or NFATbinding site 2 results in enhanced viral promoter activity andviral spread at time points 2 to 4 days posttransfection. Muta-tions in NFAT site 1 or site 2 accelerated viral spread at latertime points up to 19 days posttransfection (Fig. 6A). Imagesdepict the accelerated viral spread of mutations in NFAT bind-ing site 1 (panel II) and NFAT binding site 2 (panel III)compared to that of the WT (panel I). The viral spread ofmutated viral genome in NFAT binding site 3 was similar tothat of the wild type at day 19 (Fig. 6A and B, panel IV). Toassess the infectivity of each mutated viral genome, superna-tants were collected at day 19 posttransfection and used toinfect Vero cells. Mutations in NFAT site 1 or NFAT site 2were shown to be more infectious than the wild type (Fig. 6C).Taken together, viral propagation, luciferase, and ChIP dataindicate that any disruption of NFAT binding site 1 or NFATbinding site 2 results in elevated levels of viral spread andenhanced promoter activity.

Synergism between NFAT and other transcription factors inthe transactivation of the early viral promoter. To elucidatethe mechanisms of increased promoter activity due to NFATbinding site mutations on the viral promoter, we investigatedthe binding of AP-1 and subunit p65 of NF-�B to their recog-nized consensus sites on the early promoter. Functional syn-ergy between NFAT and AP-1 has been shown in NFAT-dependent promoters in various immune cells (22, 32). It hasbeen established previously that AP-1 and subunit p65 activateBKV early transcription (12, 36). In the Dunlop promoterthere are AP-1 binding sites following NFAT site 1 and NFATsite 2 and a binding site for the subunit p65 of NF-�B upstreamfrom the initiation start site (12). To test the effect of muta-tions of the NFAT binding sites in the viral promoter on AP-1and NF-�B activation, cells were transfected with wild-typeearly promoter or early promoter containing NFAT bindingsite mutations, and ChIP was performed. Chromatin was im-munoprecipitated for heterodimer c-Fos or c-Jun or for sub-unit p65, and results were compared to those for the IgGcontrol antibody. As shown in Fig. 7A and B, the level of thebinding of c-Jun and c-Fos increased when NFAT site 1 orNFAT site 2 was mutated. Interestingly, results demonstratedthe increased binding of subunit p65 when NFAT site 1 orNFAT site 2 was mutated (Fig. 7C). Mutations in NFAT site 3did not result in any changes in the binding of c-Jun, c-Fos, orp65 to the promoter (Fig. 7A, B, and C). These data positivelycorrelate with the luciferase data in Fig. 5A, suggesting that thetransactivation of the early viral promoter is due to the en-hanced activity of AP-1 and NF-�B when NFAT site 1 or site2 is mutated.

DISCUSSION

The clinical importance of NFAT in BKV infection is high-lighted by its dependence on calcineurin for activation. Cal-cineurin inhibitors and the immunosuppressive agents tacroli-mus (FK506) and cyclosporine A are commonly used duringkidney and bone marrow transplantations to reduce the risk ofacute rejection (13, 33). The reactivation of BKV occurs underthese conditions, resulting in the onset of the disease polyoma-virus-associated nephropathy. Previous studies have demon-strated that NFAT contributes to the regulation of polyoma-viruses, JC virus, and SV40 (34, 35).

In this study, we investigated the role of NFAT in the reg-ulation of BKV infection. We found that a cell-permeable

FIG. 7. NFAT mutations enhance AP-1 and NF-�B activity. Verocells were transfected with either the WT early viral promoter or oneof the NFAT mutants. ChIP was performed and DNA was immuno-precipitated with an antibody specific for c-Jun (A), c-Fos (B), or p65(C). Results were compared to DNA immunoprecipitated with a non-specific IgG control antibody. Quantitative PCR was performed, andthe graph depicts the averages from three independent experiments.CT values of control DNA immunoprecipitated with IgG were normal-ized to a value of 1.

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NFAT inhibitor peptide, 11R-VIVIT, dose dependently inhib-its the infection of Vero cells by BKV. The NFAT inhibitorpeptide specifically targets NFAT activity and does not inhibitAP-1 or NF-�B activation (2, 41), indicating that the reductionin infection is through a calcium-calcineurin-dependent path-way. Previous work has shown that Vero cells express calcium-regulated isoform NFAT4 (35). Our findings indicated thatNFAT4 bound to the viral promoter during infection in Verocells.

In the Dunlop promoter, we identified three NFAT bindingsites located within each transcription factor binding triplicateP regions. A common feature of NFAT-dependent promoteractivity is the presence of multiple NFAT binding sites. This isseen in the interleukin-2 (IL-2), interleukin-4 (IL-4), andCD95 promoters (4, 28, 43, 52). We analyzed the function ofeach NFAT binding site by mutating the consensus sequencefrom GGAAA to TAGAT, mutating each individual site aswell as all possible combinations of sites. When all three siteswere mutated, the promoter was nonfunctional. However, mu-tations of NFAT site 1 or NFAT site 2 resulted in a significantincrease in promoter activity, suggesting that one or both ofthese sites negatively influenced promoter activity (Fig. 8B andC). These mutations led to the increased binding of fos/junheterodimers and p65 to AP1 and NF-�B sites, respectively,and were correlated with the increased activity of the pro-moter. Mutations in NFAT site 3 slightly reduced promoteractivity and led to enhanced NFAT binding to the promoter,

presumably to sites 1 and 2 (Fig. 8D). In the context of themutations in NFAT site 3, AP-1 and p65 binding to the pro-moter was not increased.

NFAT proteins synergistically interact with the transcriptionfactor AP-1. AP-1 heterodimers c-Fos and c-Jun are activatedby the mitogen-activated protein kinase pathway to translocateto the nucleus, form NFAT–AP-1 complexes, and promotegene transcription. AP-1 and NF-�B have been shown to con-tribute to the transcriptional regulation of BKV (12, 36). Ourfindings show that enhanced promoter activity by mutations ofNFAT site 1 or NFAT site 2 is due to the increased activity ofNFAT, AP-1, and subunit p65 of NF-�B. The disruption of apotential repressor site may contribute to the increase inNFAT, AP-1, and NF-�B binding. In addition, promoter ac-tivity was abolished when all NFAT sites were mutated, indi-cating that NFAT sites are required for promoter activity andcooperation with these transcription factors.

Taken together, these data suggest that the BKV promoteris sensitive to changes in the relative levels of activators andthat different combinations of these factors can both negativelyand positively influence BKV transcription and infection. Theregulation of the promoter likely is much more complex andmay involve other factors. Previous studies have shown thatBKV large T antigen binds to retinoblastoma family membersregulating cellular proliferation in host cells by disrupting pRb-E2F repression (15). The phosphorylation of pRb disrupts thepRb-E2F complex and promotes the transcription of E2F fam-

FIG. 8. Model of BKV transcriptional activity in the presence of NFAT binding site mutations. (A) The Dunlop promoter contains three NFATbinding sites in each P region, AP-1 binding sites following NFAT site 1 and NFAT site 2 and a subunit p65 binding site upstream of the earlyinitiation site. Under wild-type conditions NFAT binds to functional NFAT sites, and AP-1 and p65 bind to their consensus sequences to activatethe promoter. A repressor site is intact, and transcription is regulated. (B) Mutation of NFAT site 1 demonstrated increased promoter activity andenhanced binding of NFAT, AP-1, and p65. The mutation of NFAT site 1 may have removed a repressor site to promote enhanced promoteractivity. (C) Mutations of NFAT site 2 may have disrupted the repressor from binding and regulating transcriptional activity. Mutant NFAT site2 demonstrated the enhanced binding of NFAT, AP-1, and p65 and increased promoter activity. (D) NFAT mutant of site 3 depicts a level ofpromoter activity similar to that of the wild type. NFAT binds to functional site 1, and the recruitment of AP-1 and p65 is at wild-type levels. Therepressor is intact to regulate transcriptional activity.

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ily members E2F1 to E2F4 and E2F6 (14, 53). These factorsbind to sequences that are similar to the NFAT binding se-quences that we identified in the BKV promoter (53).

In summary, this study demonstrates that NFAT contributesto the regulation of BK virus infection and viral transcription.Multiple NFAT binding sites in the Dunlop promoter supportthe hypothesis that BKV early viral promoter activity is NFATdependent. Additionally, NFAT cooperates with transcriptionfactors AP-1 and NF-�B to regulate viral transcription. Indi-vidual NFAT sites in the viral promoter are activators andrepressors of transcription to promote a low level of viralreplication. Mutations of these sites disrupt the coordinatedregulation of BKV transcription by NFAT, AP-1, and NF-�Band reveal important regulatory interactions between thesefactors. These mutations may mimic viral reactivation uponimmunosuppression after organ transplantation. Future stud-ies will determine whether other factors, such as E2F familymembers, bind to the viral promoter via NFAT binding sitesand participate in the transcriptional regulation of BKV.

ACKNOWLEDGMENTS

We thank all of the Atwood laboratory members for their criticaldiscussion during the course of this work. We also thank Tammy Glass,Wendy Virgadamo, and Jamie Rees for their administrative assistance.

This project was supported by a grant from the National CancerInstitute (R01 CA71878).

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