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Previous Article | Next Article 
Journal of Virology, April 2004, p. 4248-4267, Vol. 78, No. 8
0022-538X/04/$08.00+0 DOI: 10.1128/JVI.78.8.4248-4267.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Viral Oncology Program of the Sidney Kimmel Comprehensive Cancer Center,1 Molecular Virology Laboratories, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231-10002
Received 18 August 2003/ Accepted 3 December 2003
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ABSTRACT |
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) is induced in TPA-treated PEL cells and contributes to transactivation of the promoters for all of these genes through both direct binding and cooperative interactions with RTA and RAP targeted to upstream C/EBP sites. However, little is known about how RTA expression is triggered initially at the earliest stages after TPA induction when the C/EBP levels are still limited. Treatment with TPA proved to significantly induce both AP1 DNA-binding activity and levels of activated phosphorylated cJUN in PEL cells and ectopic expression of cJUN-plus-cFOS-induced RTA protein expression in PEL cells. Cotransfected cJUN plus cFOS or TPA treatment transactivated the KSHV RTA, RAP, and MTA promoters in an AP1-binding site-dependent manner in all three promoters. Chromatin immunoprecipitation assays confirmed that cJUN associates with these KSHV target promoters in PEL cells as early as 4 h after TPA treatment. Furthermore, the KSHV RTA and RAP proteins both interact with cJUN or both cJUN and cFOS in vitro or by coimmunoprecipitation from induced PEL cells and enhance cJUN-plus-cFOS-mediated transactivation of these viral promoters. Both increased phosphorylated cJUN and AP1 DNA-binding activity was detected as early as 1 h after TPA treatment in PEL cells, suggesting that AP1 activity may be crucial for very early activation of the RAP, MTA, and RTA promoters during the KSHV lytic cycle. Finally, expression of RTA alone increased cJUN protein levels severalfold in DG75 cells but did not induce cJUN phosphorylation. Therefore, we suggest that the initiating effects of TPA via the AP1 pathway in PEL cells need to be amplified by RTA for full lytic-cycle induction. |
INTRODUCTION |
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KSHV RTA is a highly diverged homologue of the Epstein-Barr virus (EBV) DNA-binding transactivator RTA or BRLF1 (23, 40, 60), which can also trigger the lytic-cycle program in cells latently infected with EBV. Both are DNA-binding proteins with classical C-terminal acidic activation domains (7, 22, 24, 34, 46, 52). Several KSHV early lytic genes that have been identified so far as directly responding targets for RTA include the replication-associated protein (RAP or K8, a positional homologue of EBV ZTA), MTA (or ORF57, a homologue of EBV MTA), and PAN (or T1.1 polyadenylated nuclear RNA) genes, as well as the vIL6, K12 (Kaposin) and vOX2/GPCR genes (7, 11, 16, 34-36, 46, 49, 52). We have previously identified one or more functional CCAAT/enhancer-binding protein (C/EBP) binding sites in each of the first three promoters listed above (54, 55). At least two types of RTA-responsive elements (RREs) have been characterized, of which only the type I RRE found in the PAN promoter binds RTA directly with high affinity (7, 34, 48, 49, 54, 55). Although more-complex oligomeric interactions may occur (31), the defined type II RREs found in the RAP and MTA promoters themselves have low direct binding affinity for RTA in electrophoretic mobility shift assay (EMSA) experiments, but both encompass strong binding sites for C/EBP, which mediates cooperative transactivation probably via DNA-bound C/EBP-RTA protein complexes (54, 55). In the MTA promoter, RTA activates gene expression through interaction with the cellular CBF1 (CSL or RBP-J
) protein at a CBF1 binding site just downstream from the C/EBP binding site within the RRE (29, 30); however, no consensus CBF1 motif is present within the RAP promoter. Although RTA transactivates the PAN promoter through direct binding to the type I RRE, mutations in the C/EBP sites outside the originally defined RRE also lead to a partial decrease in RTA activation of the PAN promoter (55). It has been suggested that, besides C/EBP and CBF1, cellular factors such as OCT, CREB, K-RBP, and SP1 play roles in RTA transactivation on certain target promoters (21, 44, 53, 61).
RTA has also been reported to positively autoregulate its own promoter (15, 44). However, in our experience this occurs only indirectly through the interaction of RTA with C/EBP at one or more C/EBP-responsive binding sites identified within the proximal RTA promoter (55). Therefore C/EBP mediates RTA activation on all four of these early KSHV promoters during viral reactivation. Furthermore, strong induction of C/EBP protein levels in those TPA-treated PEL cells that enter the lytic cycle is also observed, as judged by double-label indirect immunofluorescence assays (IFA) for RTA and RAP expression. This appears to create a self-reinforcing loop, because RTA and RAP, which also interact with each other (26, 54), both enhance C/EBP-mediated transcriptional autoregulation of the C/EBP promoter. They also both stabilize preexisting levels of short-half-life C/EBP through protein-protein interactions (55, 59). In chromatin immunoprecipitation (ChIP) assays, C/EBP, RTA, and RAP have all been detected in association with the C/EBP, RTA, RAP, MTA, and PAN promoters in vivo in PEL cells, but not until at least 8 to 12 h after TPA treatment (54, 55, 59).
The bZIP (basic region leucine zipper) group of transcription factors include the JUN family, the FOS family, the CREB/ATF family, and the C/EBP family as well as the two gammaherpesvirus-encoded proteins EBV ZTA and KSHV RAP (9, 27, 32, 33, 47, 56). The most potent transcriptional activator in the JUN family is cJUN, and cFOS also contains a transcriptional activation domain. However, cFOS cannot homodimerize and instead forms stable heterodimers with JUN family proteins, a process which enhances their DNA-binding activity (47). Classic AP1 transcriptional activating complexes comprise cJUN and cFOS subunits that specifically bind to the DNA sequence 5'-TGA(G/C)TCA-3', which is known as an AP1 site or a TPA-responsive element, known as TRE (2, 47). This same consensus AP1 motif is also a strong target recognition motif for EBV ZTA but is not bound by KSHV RAP (9, 59).
The phosphorylation and activation of cJUN are regulated by cJUN N-terminal kinase (JNK), which can be activated by TPA via protein kinase C (PKC) or mitogen-activated protein kinase pathways in numerous human cell lines (62). It has been previously observed that high levels of AP1 DNA-binding activity are induced upon TPA treatment of both EBV-positive Raji cells (32, 42) and KSHV-positive BCBL1 PEL cells (28). Therefore, in this study, we asked whether the induction of AP1-binding activity by TPA might contribute to KSHV lytic-cycle gene activation through AP1-mediated transcriptional regulation. The KSHV RTA and RAP proteins, which have both been shown to interact with and cooperate with C/EBP in our previous work, also proved to functionally associate with cJUN-plus-cFOS transcriptional activating complexes and to enhance the AP1-mediated transactivation of KSHV early gene expression.
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MATERIALS AND METHODS |
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Plasmid pHC125C expresses Flag-tagged full-length human C/EBP driven by the simian virus 40 (SV40) promoter-enhancer (SV2); plasmids pHC91A, pHC66, and pcJUN-DBD (kindly provided by Michael Birrer, National Cancer Institute) express SV2-driven wild-type cFOS, Flag-tagged wild-type cJUN, and a cJUN DNA-binding domain (DBD) mutant protein JUN(A
D265In265), respectively. The last contains an alanine-to-glutamic acid point mutation at amino acid 265 and an adjacent insertion of three acidic amino acids (2). Expression plasmids carrying full-length KSHV RTA(1-691) cDNA and Flag-tagged KSHV RAP(1-237) effector genes in pJX15 and pCJC581a, respectively, were described previously (54, 55). Plasmids pSEW-R21 and pFYW-07 express glutathione S-transferase (GST) fusion versions of the full-length RTA and RAP proteins. Plasmid pSEW-P01 contains the RAP(-190/+10)-LUC reporter gene as described previously (54). Three mutated reporter clones, RAP(PM1)-LUC (with TGT at positions -87 to -85 replaced with ATC), expressed by plasmid pSEW-P02, RAP(PM2)-LUC (with ACA at positions -71 to -69 replaced with TTC), expressed by pSEW-P03, and RAP(PM1+2)-LUC (with the two combined mutations), expressed by pSEW-P04, were generated from pSEW-P01. Plasmids pSEW-MP5 and pSEW-MP6 express reporter genes for KSHV MTA(-130/+10)-LUC and MTA(-130/+10)AP1m-LUC (in which TGAG at -121 to -118 has been changed to GATC), respectively. Reporter plasmids pGL3-FL(-914) (55), pSEW-RP1, pDH404, and pDH405 contain the KSHV RTA(-914/+34)-LUC, RTA(-587/+20)-LUC, RTA(-134/+20)-LUC, and RTA(-76/+20)-LUC reporter genes, respectively. Plasmid pSEW-RP8 contains the KSHV RTA(-587/+20 OCTm)-LUC reporter gene, in which ATCT at -214 to -211 has been changed to GGTA to destroy the OCT-binding site, and plasmid pSEW-RP9 contains the KSHV RTA(-134/+20)AP1m-LUC reporter gene in which ACTCA at -85 to -81 has been changed to GATCC to destroy the AP1-binding site.
DNA transfection and LUC assays. Transfection of HeLa cells was performed with 5 x 105 cells for each 1 µg of DNA sample in six-well plates using Lipofectamine (Invitrogen). The BCBL1 PEL cell line and DG75 B-lymphoblast cell line as well as U937 cells (107 cells/sample) were transfected with 5 to 10 µg of DNA by electroporation (Gene-pulser; Bio-Rad) at 300 V and 950 µF in 0.5 ml of RPMI medium as described previously (53, 54). In all cases, cells were harvested at 48 h posttransfection for LUC assays, which were carried out in triplicate using a LUC assay kit (Promega). Results are presented as means and ranges. All samples also received an added 0.2 µg of cytomegalovirus (CMV) ß-galactosidase (ß-Gal) expression plasmid DNA as an internal control for transfection variability. Activity of ß-Gal in the cell lysate was measured with o-nitrophenol-ß-D-galactopyranoside (ONPG) as the substrate. For TPA treatment of DNA-transfected HeLa cells, a final concentration of 60 ng/ml was added at 24 h posttransfection, followed by incubation for another 24 h before harvesting the cells. To normalize for TPA-induced cell apoptosis, protein assays were carried out to determine the total protein levels in TPA-treated and untreated cell lysates using bicinchoninic acid protein assay reagents (Pierce, Rockford, Ill.), although less than 20% divergence was observed before and after TPA treatment.
EMSA.
C/EBP, cJUN, and cFOS protein samples used for EMSA were in vitro translated with the T7-TNT quick-coupled transcription-translation system (Promega) according to the manufacturer's procedures using corresponding expression plasmids pHC125C, pHC66, and pHC91A as templates. Annealed double-stranded oligonucleotides with 4-bp 5' overhangs were radiolabeled with [-32P]dCTP during a Klenow DNA polymerase fill-in reaction. For EMSA, approximately 50,000 cpm of the 32P-labeled probe and 2 µl of in vitro-translated proteins or of BCBL1 nuclear extract were incubated for 30 min at 20°C in a binding system containing 10 mM HEPES (pH 7.5), 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 5% glycerol, and 2 µg of poly(dI/dC). For supershift experiments, 0.5 µl of mouse anti-Flag monoclonal antibody (MAb; catalog no. F-3165; Sigma, St. Louis, Mo.), rabbit anti-cJUN polyclonal antibody (PAb; catalog no. sc-1694; Santa Cruz Biotechnology, Santa Cruz, Calif.), or C/EBP rabbit antiserum (54) was added to the mixture after 30 min and allowed to incubate for another 30 min before gel loading. Samples were separated on a 4.5% polyacrylamide gel in 1x HEE buffer (10 mM HEPES [pH 7.5], 1 mM EDTA, 0.5 mM EGTA) at 150 V and 4°C as described previously (10). The gels were dried and subjected to autoradiography with Kodak X-ray film.
All oligonucleotides used were purchased from Qiagen Operon (Alameda, Calif.). Nucleotide sequences of the annealed DNA probes for AP1 consensus, RAP-PWT, RAP-PM1, RAP-PM2, RAP-PM1+2, MTA-AP1, MTA-AP1m, RTA-AP1, and RTA-AP1m are indicated in the figures.
Recombinant protein expression and in vitro GST affinity binding assay. GST fusion protein expression was induced in Escherichia coli (BL21 strain) with 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) for 4 h at 30°C. Bacterial pellets were resuspended in ice-cold phosphate-buffered saline (PBS; 150 mM NaCl, 16 mM Na2HPO4, 4 mM NaH2PO4, pH 7.3) and sonicated for 30 s. After centrifugation, clarified lysates were immobilized on the glutathione-Sepharose 4B beads (Amersham Pharmacia). Input [35S]methionine-labeled proteins were synthesized in vitro using the T7-TNT quick-coupled transcription-translation system as described above. Recombinant GST fusion proteins immobilized on beads were pretreated with 0.2 U of DNase I and 0.2 µg of RNase A per µl for 30 min at 20°C in pretreating buffer (50 mM Tris-HCl [pH 8.0], 5 mM MgCl2, 2.5 mM CaCl2, 100 mM NaCl, 5% glycerol, 1 mM dithiothreitol). The beads were washed twice with binding buffer (20 mM Tris-Cl [pH 7.5], 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol) and blocked in the same buffer containing 10 mg of bovine serum albumin (BSA)/ml for 30 min at 4°C. After being blocked, the bead-immobilized GST fusion protein was resuspended in binding buffer containing 1 mg of BSA/ml, and labeled proteins were subsequently added and allowed to incubate for 1 h at 4°C. The beads were then washed five times in binding buffer at 10-min intervals, resuspended in 20 µl of 2x sodium dodecyl sulfate (SDS)-gel loading buffer, and boiled for 5 min before being loaded onto SDS-polyacrylamide gel electrophoresis (PAGE) gels. After electrophoresis, the gels were fixed in 50% methanol-40% H2O-10% acetic acid for 30 min and dried for X-ray autoradiography.
In vivo coimmunoprecipitation from PEL cell lysates. Nuclear extracts of BCBL1 cells were prepared for coimmunoprecipitation by procedures similar to those described previously (54, 57). Approximately 108 cells were harvested at 30 h or at various time points after TPA induction (as indicated in Fig. 1B8, 9A9A, 9C, and 12), washed once with PBS, and gently resuspended in 10 ml of cold hypotonic buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate). The mixture was allowed to swell on ice for 30 min, and NP-40 was then added to a final concentration of 0.62%. The mixture was vortexed vigorously for 10 s, and the nuclear pellet was collected after centrifuging for 3 min at 1,200 x g at 4°C. The nuclear pellet was resuspended in 3 ml of cold immunoprecipitation buffer (50 mM Tris-Cl [pH 7.9], 50 mM NaCl, 0.1 mM EDTA, 1% glycerol, 0.2% NP-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate) and sonicated for 2 s at the minimal setting. The samples were then centrifuged at 15,000 x g for 1 min at 4°C, and the supernatant was collected and stored at -70°C. For coimmunoprecipitation, 400 µl of prepared nuclear extract was pretreated with 0.2 U of DNase I and 0.2 µg of RNase A per µl and precleared with 5 µl of rabbit preimmune serum and 100 µl of a 50% slurry of protein A/G (20%:80%)-Sepharose beads for 1 h. After preclearing, 3 µg of anti-RAP or anti-RTA rabbit PAb or rabbit preimmune serum was added to the precleared nuclear extracts and incubated for 1 h at 4°C; then 100 µl of a 50% slurry of protein A/G (20%:80%)-Sepharose beads blocked with 5% BSA in PBS was added to the nuclear extracts and incubated for 1 h at 4°C. The beads were then washed three times with cold immunoprecipitation buffer at 15-min intervals, resuspended in 2x SDS-gel loading buffer, and boiled for 5 min before being loaded onto SDS-PAGE gel.
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Immunohistochemical staining. Immunohistochemical staining was performed with TPA-induced or uninduced BCBL1 cells. Cells were washed three times in 1x PBS and fixed in 4% paraformaldehyde in 1x PBS for 10 min, followed by boiling in 10 mM sodium/citrate buffer (pH 6.0) for 10 min. After cells were quenched in freshly prepared ethanol-PBS-H2O2 (77:20:3) for 1 h in blocking solution (1% BSA and 10% normal goat serum in 1x PBS with 1% Tween 20), the primary rabbit phospho-JUN(Ser73) PAb was applied first at 1:200 dilution in blocking solution for 12 h at 20°C and detected with a biotinylated goat anti-rabbit immunoglobulin (Ig) antibody plus the streptavidin-peroxidase-ABC complex (Dako Corporation, Carpinteria, Calif.), followed by 3,3'-diaminobenzidine peroxidase substrate (Sigma). For double staining, after cells were washed in 1x PBS and treated with an avidin-biotin blocking kit (Vector Labs, Burlingame, Calif.), the secondary rabbit PAb against the KSHV vMIR2 (ZMP-A or K5) protein (11, 12) was applied at a 1:500 dilution for 1 h at 20°C and then detected with the biotinylated goat anti-rabbit Ig antibody plus the streptavidin-peroxidase-ABC complex, followed by True Blue peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md.).
IFA. IFA was performed 40 h after BCBL1 cells were transfected by the acetone-methanol (1:1) fixation method (54, 55). Primary antibodies included rabbit antipeptide antisera against KSHV RTA, KSHV vMIR2, and KSHV vIL6 (K2) (1:200 dilution) (3, 12) and a mouse anti-Flag MAb (Sigma; 1:100 dilution). Secondary donkey- or goat-derived rhodamine- or fluorescein isothiocyanate (FITC)-conjugated anti-rabbit or anti-mouse IgG (Jackson Pharmaceuticals, West Grove, Pa.; both at a 1:100 dilution) was used to detect the primary antibodies.
ChIP. BCBL1 PEL cells were treated with TPA, and 106 cells were harvested at different time points for ChIP assays using a ChIP assay kit (Upstate Biotechnology) in accordance with the manufacturer's protocol. Histones were cross-linked to DNA by adding formaldehyde directly to the culture medium to a final concentration of 1% and incubating for 10 min at 37°C. Cells were then washed twice with ice-cold PBS containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg of aprotinin/ml, and 1 µg of pepstatin A/ml), resuspended in 200 µl of SDS lysis buffer containing the protease inhibitors, and incubated for 10 min on ice. DNA was sheared by sonicating samples on ice, and samples were centrifuged to collect the supernatants. The supernatants were then diluted in 1,800 µl of ChIP dilution buffer before 80 µl of salmon sperm DNA-protein A agarose slurry was added, followed by agitation for 30 min at 4°C. After the agarose beads were spun down, precleared samples were aliquoted, antibodies against cJUN or CHOP10 (Santa Cruz Biotechnology; catalog no. sc-7351) were added to each aliquot of 500 µl, and samples were rotated for 2 h at 4°C. Sixty microliters of salmon sperm DNA-protein A agarose slurry was then added to each sample, and samples were rotated for another hour at 4°C. The beads were subsequently washed in low-salt immune complex wash buffer, high-salt immune complex wash buffer, and LiCl immune complex wash buffer and then washed twice in Tris-EDTA (TE) buffer. After the washing steps, 250 µl of freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3) was added to the beads and samples were vortexed and incubated for 15 min at 20°C by rotation. The supernatants were collected by centrifugation, and the elution was repeated with another 250 µl of elution buffer. Then 20 µl of 5 M NaCl was added to each sample, and samples were incubated at 65°C for 12 h before proteinase K treatment, phenol-chloroform extraction, and ethanol precipitation. DNA pellets were resolved in 20 µl of TE buffer, and 2 µl was used as the template for PCR. Primers LGH3641 and LGH4362 (54), specific for a 164-bp region in the KSHV RAP promoter from -160 to +4, primers LGH4980 and LGH4987 (55), specific for a 168-bp region in the KSHV MTA promoter from -160 to +8, and primers LGH4354 and LGH4355 (55), specific for a 261-bp region in the KSHV RTA promoter from -241 to +20, were used for PCR detection (30 cycles). The PCR products were fractionated on a 2% agarose gel and detected by ethidium bromide staining.
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RESULTS |
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We first investigated the effect of AP1 on the KSHV RAP gene promoter by cotransfecting cJUN and cFOS expression plasmids either alone or in combination with the RAP(-190/+10)-LUC target reporter gene. Transfected HeLa cells expressing either cJUN or cFOS alone showed little increase in LUC expression compared to control cells transfected with the empty vector DNA, whereas cells cotransfected with equal amounts of cJUN and cFOS produced up to 142-fold activation of RAP-LUC activity in a dose-responsive manner (Fig. 1B). A cJUN mutant DBD with a disrupted basic region (Fig. 1A) that is unable to recognize consensus AP1 DNA-binding sites (2) was also used as a specificity control in place of the wild-type cJUN in some cotransfection experiments. Activation of the RAP promoter by a combination of JUN(DBD) and cFOS reduced transactivation from 109-fold (0.25 µg each) to 13-fold (Fig. 1B), suggesting that direct binding to specific DNA sites by the AP1 transcription factor complex rather than potential alternative indirect effects is crucial here.
The proximal upstream RAP(-190/+10)-LUC promoter contains two potential AP1-like sequences, located at positions -87 to -81 (5'-TGTGACT-3') and -75 to -69 (5'-TGAAACA-3'), that diverge from the 7-bp AP1 consensus binding sequence 5'-TGA(G/C)TCA-3' at one or two positions, respectively (Fig. 2A). The sequence at -75 to -69 overlaps with the previously identified strong C/EBP site and the RRE. To test the potential of these AP1-like sequences to mediate AP1 transactivation, we introduced point mutations into the RAP(-190/+10)-LUC reporter gene to destroy either one or both of the two sites (Fig. 2A). Mutation at -87 to -81 in RAP(PM1)-LUC led to a 3-fold decrease in cJUN-plus-cFOS-mediated activation from 109-fold down to 37-fold (Fig. 2B). However, mutation of the other site at -75 to -69 in RAP(PM2)-LUC, which abolished most of the C/EBP and the RTA responsiveness (55), had little effect on AP1 responsiveness, although activation of the double-mutant gene fell further to 28-fold. None of the mutations had any significant effect on the basal level of expression. These results indicated that the sequence at positions -87 to -81 may be one of possibly several AP1-responsive elements within the RAP promoter.
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as expected, whereas RAP-PM2 retained AP1-binding ability but lost most of the C/EBP binding (Fig. 2F). The double-mutant probe RAP-PM1+2 failed to bind to either of the two proteins. Therefore, these EMSA results are consistent with our cotransfection data and indicate that cJUN plus cFOS transactivate the KSHV RAP gene promoter by a mechanism that includes direct binding to the moderate-affinity AP1 site at the position -87 to -81. JUN and FOS transactivate the KSHV MTA promoter through an AP1-binding site mapping at -121 to -115. The KSHV MTA promoter is also induced during the early stages of viral reactivation and responds to transactivation by KSHV RTA through a type I RRE, which is partially homologous to that in the RAP promoter and which also does not directly interact with RTA but encompasses a strong functional C/EBP site as well as an adjacent CBF1 site (55). Therefore, we were interested in whether or not AP1 also transactivates the MTA promoter. We recognized only one potential AP1 site within the first 500 bp upstream of the MTA TATA box, represented by the consensus sequence 5'-TGAGTCA-3' mapping at positions -121 to -115, adjacent to the previously identified RRE and C/EBP sites. To test the contribution of this potential high-affinity AP1 site in the MTA promoter, we cotransfected HeLa cells with either a wild-type MTA(-130/+10)-LUC reporter gene or with a mutated version [MTA(-130/+10)AP1m-LUC] that was designed to destroy the AP1 site (Fig. 3A). Plasmids expressing cJUN or cFOS proved to have little effect on MTA promoter activity when transfected alone but in combination were again able to activate wild-type MTA-LUC by up to 48-fold (Fig. 3B). The point mutant MTA reporter gene showed a 2.3-fold decrease in responsiveness to cJUN plus cFOS (down to 21-fold) but also had a 4-fold reduction in basal-level expression. In addition, when the cJUN(DBD) mutant protein was used in the cotransfection to replace wild-type cJUN, the activation level was reduced to 15-fold (Fig. 3B). Therefore, direct DNA binding by AP1 at this site contributes to both basal and cJUN-plus-cFOS-mediated activation of the MTA promoter, but again there are likely to be additional weaker target sites.
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JUN and FOS transactivate the KSHV RTA promoter through an AP1-binding site mapping at -87 to -81.
In the RTA promoter, three C/EBP sites that both contribute to C/EBP transactivation and mediate enhanced positive autoregulation by RTA, apparently through cooperative formation of RTA protein complexes with C/EBP bound at these sites, have been identified (55). Although addition of C/EBP alone can activate endogenous RTA expression, the highest levels of induction in PEL cells appear to require cooperative interaction by C/EBP and RTA activating both the cellular C/EBP and viral RTA promoters, as well as a stabilization effect on C/EBP protein through physical interactions with both RTA and RAP (55, 59). We were interested in addressing whether TPA-triggered AP1 induction contributes to the rapid accumulation of the RTA IE protein at even earlier stages during viral reactivation, prior to initiation of the self-regulating positive-feedback loop mediated by C/EBP and RTA on both the C/EBP and RTA promoters. An OCT site located at -220 to -213 has also been reported to contribute to RTA induction (44), and we included a promoter with a mutation at this site in our studies (Fig. 4A).
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To test the contribution by this site to AP1-mediated activation of the RTA promoter, as well to evaluate the possibility of other potential AP1 sites, we examined the AP1 responsiveness of five RTA reporter genes with deletions or mutations, namely, the RTA(-587/+20)-LUC, RTA(-587/+20)OCTm-LUC, RTA(-134/+20)-LUC, RTA(-76/+20)-LUC, and RTA(-134/+20)AP1m-LUC genes, in HeLa cells (Fig. 4A). The RTA(-587/+20)-LUC gene was activated by cotransfected cJUN plus cFOS at a level similar to that of the full-length RTA (RTA(-914/+34)-LUC) promoter, whereas the RTA(-587/+20)OCTm-LUC and RTA(-134/+20)-LUC genes each gave a 20 to 40% reduction in AP1 responsiveness without reduced basal activity (Fig. 4E). This suggests that the OCT site at -220 to -213 may have a small indirect contribution to AP1 activation of the RTA promoter in HeLa cells. In contrast, either mutation of the confirmed AP1-binding site at -87 to -81 in the RTA(-134/+20)AP1m-LUC gene or its deletion in the RTA(-76/+20)-LUC gene gave a twofold loss of responsiveness compared to that for the wild-type RTA(-134/+20)-LUC gene (Fig. 4E). This deletion also lowered the basal activity fourfold compared to only a small effect for the AP1 site point mutation, although there are no other obvious recognizable motifs within this region. Use of the DBD mutant form of cJUN in combination with cFOS reduced residual transactivation of the latter two AP1 site-negative targets by only 35%, whereas for the wild-type RTA(-134/+20)-LUC target gene, which retained the AP1 site, the effect was more than twofold (Fig. 4E). Therefore, we infer from these results that what causes the rather surprisingly high residual cJUN-plus-cFOS responsiveness may be somewhat indirect.
Exogenously introduced JUN plus FOS trigger endogenous RTA and vIL6 protein expression in PEL cells. Cotransfected cJUN plus cFOS strongly transactivate the KSHV RTA promoter in transient reporter gene assays in HeLa cells. To investigate whether activation of the RTA promoter also occurs in KSHV-infected PEL cells, resulting in an induction of RTA protein levels, double-label IFA experiments were performed on BCBL1 cells cotransfected with SV2-Flag-cJUN plus SV2-cFOS. Spontaneous expression of the endogenous RTA protein was highly limited in latently infected BCBL1 cells and could be detected in less than 1% of the cells (55). However, in cJUN-plus-cFOS-cotransfected cells, 20% of the cells expressed exogenous Flag-tagged cJUN in the nucleus and 83% of these cJUN-positive cells were also positive for RTA protein expression in the nucleus (Fig. 5A). Therefore, expression of exogenous cJUN plus cFOS can activate RTA expression in PEL cells from a latent state, and because RTA alone is sufficient to trigger progression of the entire lytic cycle, cJUN plus cFOS are potentially capable of fully reactivating PEL cells from latency into the lytic cycle.
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Both RTA and RAP enhance JUN-plus-FOS-mediated transactivation of KSHV promoters.
KSHV RTA activates expression of the RTA, RAP, MTA, and PAN promoters either directly through binding to type II RREs or indirectly by associating with other transcription factors, such as C/EBP and CBF1, that bind to type I RREs (29, 54, 55). Similarly, KSHV RAP, although it does not itself bind to or transactivate any of these promoters, can also enhance the transactivation by RTA and C/EBP on both their own and other target promoters severalfold, apparently through strong protein-protein interactions with DNA-bound C/EBP (54, 55, 58, 59). To ask whether the AP1 transactivator complex behaves similarly to C/EBP, we performed cotransfection experiments together with RTA and RAP using the same three wild-type KSHV promoter driven reporter genes that had been shown to respond to cJUN-plus-cFOS transactivation. On all three promoters investigated, both RTA and RAP were able to cooperate with cJUN and cFOS to significantly stimulate transactivation, and the effects were more than additive compared to transactivation in samples that were transfected with equivalent amounts of either the cJUN-plus-cFOS, RTA, or RAP expression plasmid alone (Fig. 6). Essentially equivalent results were obtained with transfected HeLa cells (Fig. 6A) and electroporated DG75 B lymphocytes (Fig. 6B), except that 20 times as many cells and 10 times as much DNA were used in the latter case.
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, there may also be interactions between cJUN or cFOS and RTA or RAP that lead to further recruitment of viral and cellular transcription factors at the AP1 sites in these early-lytic-cycle promoters. RTA and RAP interact with the JUN or FOS proteins both in vitro and intracellularly. To detect a potential physical association between the individual AP1 protein subunits and RTA or RAP, we carried out GST affinity-binding assays using bacterially expressed GST-RTA or GST-RAP recombinant proteins. In vitro-translated cJUN and cFOS proteins were labeled with [35S]methionine and incubated with the GST fusion proteins immobilized on glutathione-Sepharose beads. These experiments revealed that GST-RTA was able to interact efficiently with cJUN, but had little affinity for cFOS (Fig. 7A, second panel from the top, lanes 1 and 2). However, when cJUN and cFOS were either admixed together or cotranslated before addition to the system, the interaction between cJUN and cFOS evidently allowed GST-RTA to retain both proteins on the beads (Fig. 7A, second panel, lanes 3 and 4). In contrast, in the same experiments GST-RAP displayed a high affinity for both cJUN and cFOS individually, as well as after the proteins were mixed or cotranslated (Fig. 7A, third panel). None of the labeled cJUN or cFOS samples bound to GST alone (Fig. 7A, fourth panel).
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Induction of AP1 DNA-binding activity early after TPA treatment in PEL cells. The upregulation of several KSHV lytic-cycle promoters by AP1 in cotransfection assays suggests that AP1 may have an important role in reactivating the viral lytic replication cycle from latency in vivo through targeting to the promoters of viral regulatory genes such as the RTA, RAP, and MTA genes. Cultured PEL cell lines that carry multicopy latent KSHV episomes can be induced to undergo lytic-cycle infection and viral DNA replication in response to triggering by adding TPA to the medium. To evaluate how early induced AP1 DNA-binding ability can be detected after TPA treatment in PEL cells, we prepared nuclear extracts from BCBL1 cells at 0, 1, 3, 12, and 24 h after TPA treatment and performed EMSA experiments using these nuclear extracts with the 32P-labeled consensus AP1 oligonucleotide probe. Shifted bands migrating at the same positions as in vitro-translated AP1-bound DNA complexes as a positive control were detected as early as 1 h after TPA treatment and continued to increase in abundance up to at least 24 h (Fig. 8, left). In comparison, a parallel experiment using the PAN-RRE probe showed that RTA did not start to associate with its target DNA-binding site until later than 3 h (Fig. 8, right), suggesting that AP1 DNA-binding activity may be induced significantly earlier than when RTA DNA-binding activity can be first detected.
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To examine the time course of cJUN activation in BCBL1 cells at different time points, we examined protein extracts harvested at 0, 1, 3, 12, and 24 h after TPA treatment for the levels of phosphorylated cJUN by immunoblotting with a PAb that detects only the phosphorylated form of cJUN. Phosphorylated cJUN was barely detectable at 0 h but started to appear as early as 1 h after TPA treatment and continued to accumulate up to 24 h (Fig. 9C, top, lanes 1 to 5). A large increase in phosphorylated cJUN was also evident at 24 h after TPA induction in HeLa cells (lanes 6 and 7). Therefore, induction of a phosphorylated form of cJUN that is active for DNA binding correlates with the rapid appearance of the AP1 DNA-binding activity in EMSA experiments, and both assays detected induction within 1 h upon TPA treatment in PEL cells.
We subsequently performed immunocytochemical staining using the PAb against the phosphorylated form of cJUN. Before TPA treatment 5% of the BCBL1 cells showed positive staining in the nucleus (Fig. 9D, a), whereas after TPA treatment for 24 h, 15% of the cells were positive and had significantly stronger staining (b). To further address the question of whether the phosphorylation and activation of cJUN observed in both Western immunoblotting and immunocytochemical staining occur in PEL cells undergoing KSHV lytic-cycle replication, we carried out a double-label immunohistochemical procedure with BCBL1 cells at either 0 or 24 h after TPA treatment using a PAb against vMIR2 as well as the phosphorylated cJUN antibody. As expected, more of the cells (10%) expressed cytoplasmic vMIR2 (blue) after TPA treatment (Fig. 9D, d) than before TPA treatment (1%; c). However, among the TPA-treated BCBL1 cells, at least 60 to 70% of the vMIR2-positive cells were also positive for nuclear phosphorylated cJUN (brown) by double-stain immunohistochemical procedures (d; see inset). Therefore, most cells undergoing KSHV lytic-cycle replication were also positive for phosphorylated cJUN staining in the nucleus, suggesting that the phosphorylation of cJUN is highly related to lytic-cycle replication in these cells.
Addition of RTA alone induces the levels of endogenous unphosphorylated JUN protein.
Our evidence above that RTA physically interacts with cJUN-cFOS complexes raised the possibility that, similar to the situation with C/EBP, this may result in increased overall levels of AP1 components in RTA-expressing cells. To examine this question, we first carried out Western blotting for total cJUN protein levels in Flag-RTA-transfected DG75 and HeLa cells. Indeed, as shown in Fig. 9B, the addition of RTA alone induced a large increase in the level of unphosphorylated cJUN (middle, lanes 2 to 4), although there was no increase in the slower-migrating phosphorylated form.
To further support these results, we also carried out double-label IFA experiments after transfection of BCBL1 and DG75 cells with Flag-RTA by electroporation (Fig. 10). The results confirmed that, although a small fraction (1.2%) of the untreated BCBL1 cells were spontaneously positive for nuclear cJUN, virtually all of the 10% of cells in the transfected culture that expressed exogenous nuclear Flag-RTA (Fig. 10A, FITC, green) also became positive for endogenous nuclear cJUN protein expression (Fig. 10B, rhodamine, red, and C, merge, yellow). In the KSHV-negative DG75 culture, again almost all of the 3% of transfected cells that expressed either endogenous cJUN or Flag-RTA were positive for both proteins (Fig. 10D to F). These results suggest that, once RTA is induced early in the KSHV lytic cycle, it is capable of in turn activating cJUN expression either directly or indirectly by an as-yet-unknown mechanism.
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on the RAP, MTA, and RTA promoters.
AP1 is thought to be one of the most important pathways through which TPA stimulates target gene promoters. When transfected into HeLa cells, all three KSHV lytic-cycle-promoter-driven reporter genes studied in this work, the RAP(-190/+10)-LUC, MTA(-130/+10)-LUC, and RTA(-914/+34)-LUC genes, were responsive to TPA treatment after 24 h, showing 7.2-, 6.7-, and 8.5-fold-increased LUC activity, respectively (Fig. 11A). When these promoters were both cotransfected with the cJUN plus cFOS plasmids and then treated with TPA to analyze the dual effect by AP1 and TPA, activation by AP1 was stimulated three- to fourfold by TPA compared to that for untreated AP1-transfected samples (Fig. 11A). However, in parallel experiments TPA treatment had no added effect on the transactivation levels achieved by C/EBP on these same promoters.
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Interestingly, in both sets of experiments we observed that, compared to the wild-type promoters, the promoters with AP1 site mutations (especially the mutation in the MTA promoter) also gave some reductions in basal activity, even without exogenous cJUN-plus-cFOS coexpression. This may result from relatively high endogenous AP1 expression in HeLa cells, giving basal activation of the AP1 sites of the wild-type promoters but not of the mutated promoters. To address the possibility of cell type-specific effects, we also performed the same transfection and TPA treatment experiments with the U937 premonocytic cell line, used successfully previously for measuring TPA responsiveness in the CMV MIE promoter (6) (Fig. 11C). However, essentially identical effects on TPA responsiveness as well as on the basal activities of these reporter genes by mutations in the AP1 sites were also observed in U937 cells.
Association of the JUN protein with KSHV target promoters by ChIP assay during early-lytic-cycle reactivation. The above experiments indicated that cotransfected cJUN and cFOS can transactivate the expression of the KSHV RAP, MTA, and RTA promoters primarily through the AP1-binding sites that we identified above and that the phosphorylated form of cJUN and AP1 DNA-binding activity itself are both induced as early as 1 h after TPA treatment in PEL cells. To investigate whether and when AP1 protein complexes can be detected in association with the three viral promoters during the early stages of lytic-cycle reactivation in PEL cells, we carried out a time course ChIP assay using BCBL1 cell lysates prepared at 0, 4, 8, and 24 h after TPA treatment. Antibodies against either cJUN or CHOP10 (as a negative control) were used to immunoprecipitate sonicated chromatin and any associated DNA fragments (Fig. 12). Following PCR amplification, DNA regions representing all three promoters (RAP, MTA, and RTA promoters) were detected in the cJUN immunoprecipitates as early as 4 h after TPA treatment (Fig. 12, lanes 7 to 9) and for MTA and RTA the levels increased greatly by 24 h. Importantly, none of these promoter DNAs were detected at any time point in parallel immunoprecipitates obtained with the CHOP10 antibody (lanes 10 to 13). Furthermore, as expected, little or no cJUN-bound promoter DNA was detected in the uninduced (0-h) BCBL1 cell extracts (Fig. 12, lanes 6).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interacts with two important KSHV-encoded lytic-cycle regulatory proteins RTA and RAP (54, 58, 59) and mediates KSHV early-lytic-cycle gene regulation by directly binding to and transactivating at least four viral promoters, including those for RTA, RAP, MTA, and PAN (54, 55). Both the RTA and RAP proteins cooperate with C/EBP through these protein-protein interactions to augment target gene expression, including having positive autoregulatory effects on their own promoters. The expression of C/EBP itself is also strongly induced in TPA-treated PEL cells, and both RTA and RAP reciprocally contribute to this effect by enhancing positive autoregulation of the C/EBP promoter as well as by stabilizing the C/EBP protein (54, 58, 59). As an IE protein, RTA is one of the first viral proteins expressed upon primary infection or viral reactivation, and the initial induction of RTA mRNA does not depend on new protein synthesis (45, 51, 63). Despite the fact that ectopic expression of C/EBP induces RTA and RAP expression in latently infected PEL cells, we knew that C/EBP protein levels are not induced by TPA treatment in the absence of RTA or RAP. As shown here, C/EBP transactivation is also not induced or enhanced by TPA. Therefore, some other cellular factors that are regulated through signal transduction pathways and by protein modification at the posttranslational level must be responsible for the initial induction of RTA mRNA expression by TPA, which is then subsequently boosted by the RTA and RAP transcriptional autoregulatory loop mediated by C/EBP. The activity of the AP1 transcriptional activating complexes consisting of cJUN-plus-cFOS heterodimers is known to be significantly induced by TPA in various cell lines through induction of the JNK pathway that activates cJUN by phosphorylation. In this study, we have shown that TPA indeed induces AP1 DNA-binding activity and phosphorylated cJUN in PEL cells and that the three KSHV lytic-cycle promoters tested, including the RTA promoter itself, are all responsive to both TPA and cJUN-plus-cFOS transactivation. We also identified specific AP1-binding sites that contributed to these effects in all three promoters. The strongest of these sites (the classical AP1 motif in the MTA promoter) also contributed to basal-level expression. Even after mutation of the identified AP1 sites, each promoter still retained significant additional cJUN-plus-cFOS responsiveness, suggesting the presence of other unidentified target sites. However, some of this residual activity, including that involving the OCT site in the RTA promoter, appears to be relatively indirect, because it was less dependent on the presence of the DBD of cJUN than was the responsiveness mediated by the AP1 sites themselves.
Addition of exogenous RTA or Flag-tagged RTA increased overall cJUN protein levels detectable by Western blotting and double-label IFA of PEL cells, as well as of KSHV-negative DG75 and HeLa cells, but did not increase phosphorylated cJUN levels. Although this effect closely resembles that for KSHV RTA and RAP as well as for EBV ZTA on c/EBP and p21 protein levels (55, 57, 58, 59), we do not know at present whether RTA itself has direct effects on the cJUN promoter or whether this is a consequence of protein stabilization associated with the binding of RTA to cJUN and cFOS protein complexes or both. In contrast, TPA alone induced just the phosphorylation of cJUN in an RTA-independent manner in DG75 and HeLa cells but had no effect on overall levels of unphosphorylated cJUN protein.
Nevertheless, in TPA-treated KSHV-positive PEL cells, increases in total cJUN protein levels were detected at 24 h, cJUN association with the viral promoters was detectable by ChIP assay within 4 h, and both increased activated phosphorylated cJUN and increased AP1 DNA-binding activity occurred within 1 h. Furthermore, introduction of exogenous Flag-tagged cJUN plus cFOS into PEL cells (in the absence of TPA) induced endogenous RTA protein expression in the majority of the cells (83%) that expressed Flag-cJUN, as detected by double-label IFA. Therefore, although activation of the JNK pathway and phosphorylation of cJUN to activate AP1 DNA-binding activity by TPA might be responsible for (or at least participate in) the initial activation of the RTA promoter, subsequent positive amplification of cJUN-plus-cFOS levels by RTA may also be critical. This may be accomplished by the interaction of newly synthesized RTA with cJUN-plus-cFOS complexes bound to AP1 sites, which results in a cooperative stimulation of further RTA promoter activity and enhances the levels of both the RTA and cJUN proteins, followed by the switching on of the RAP promoter and other early promoters. Later, when RTA also stimulates C/EBP induction, all of these promoters, which are independently responsive to both C/EBP and to cJUN-plus-cFOS activation, are also further stimulated by the newly synthesized RAP protein, in turn cooperatively enhancing transactivation of both its own promoter and those for the RTA gene and the other early genes by a combination of interactions with RTA, C/EBP, and cJUN plus cFOS.
We have shown previously that RTA and RAP both reciprocally enhance transcriptional autoregulation of the cellular C/EBP promoter and that both also stabilize the C/EBP protein through direct protein-protein contacts. Although we demonstrate here that RTA and RAP both coimmunoprecipitate cJUN and bind to either or both cJUN and cFOS in vitro, we have yet to address whether either of those transcriptional or posttranslational mechanisms also applies to cJUN or cFOS.
The effects of TPA treatment on all three viral early promoters in HeLa cells ranged from only 6.7- to 8.5-fold compared to 27- to 109-fold for cotransfected cJUN plus cFOS, but, importantly, the identified AP1 sites proved to play more significant roles in the TPA responsiveness than they did in activation by cJUN-plus-cFOS cotransfection, with point mutations in each giving 3- to 5-fold reductions in cJUN-plus-cFOS responsiveness and 8- to 10-fold reductions in overall activity for the TPA-treated MTA and RTA promoters. Interestingly, addition of TPA together with cotransfection by cJUN plus cFOS increased overall activation of all three wild-type promoters by 3- to 4-fold to between 95- and 430-fold over basal levels. Curiously, addition of TPA also boosted RTA responsiveness of the wild-type promoters three- to fourfold in HeLa cells (data not shown) but had no effect on C/EBP responsiveness of the same promoters in parallel experiments in either the presence or absence of RTA. The synergistic effects of TPA on basal-level activity, on cJUN-plus-cFOS-induced activity, and on RTA-induced activity can all be reasonably well explained by the observed increase in phosphorylation of cJUN over the preexisting basal or activated levels, but we have no explanation at present for the apparent ability of C/EBP to block, prevent, or counteract the added effects of TPA in the transient reporter gene assays.
AP1 sites were originally defined in the SV40 T-antigen promoter-enhancer region, where they were also recognized as a subset of TREs (13). They also occur in the human CMV MIE enhancer (25) and in the herpes simplex virus type 1 IE110 first intron (20). However, there are also other targets of TPA responsiveness, such as the ZI and ZII sites in the upstream EBV ZTA promoter (17) and the classical CRE, NF-B, and overlapping SRE/ETS sites within the CMV MIE enhancer (6). Therefore, it is not particularly surprising that cJUN and cFOS and activated AP1 DNA-binding activity play a role in TPA induction in B lymphocytes, but few studies have addressed the JUN/FOS/AP1 pathway even in the EBV system, and no previous studies have addressed these issues specifically for the KSHV l
