The Herpesvirus Saimiri Replication and Transcription Activator Acts Synergistically with CCAAT Enhancer Binding Protein Alpha To Activate the DNA Polymerase Promoter

Plasmid constructs.In order to generate an ORF 9 promoter reporter gene construct, the ORF 9 gene promoter was PCR amplified with primers 5′-CGG GGT ACC GTT AAT GTA GCA AGC GGC GC and 5′-CTA GCT AGC GTC AAG ACA GCA ACT CAG. These oligonucleotides incorporated KpnI and NheI restriction sites, respectively, for convenient cloning of the PCR product. PCR (30 cycles [1 cycle consists of 1 min at 92°C, 1 min at 58°C, and 2 min at 72°C]) was performed with 4 units of Pfu (Stratagene). This fragment was inserted upstream of the luciferase coding region in pGL3-basic (Promega) to derive pORF9-Luc. The 5′ ORF 9 promoter deletion series was constructed by PCR amplification using the forward primer Δ1 (5′-CGG GGT ACC GTT CAC ATT TCT ATT AAT AGG), Δ2 (5′-CGG GGT ACC CCC AGA TGT TGA GAA ATC), Δ3 (5′-CGG GGT ACC GGA ATG CAT AAC ATG CAG), Δ3.5 (5′-CGG GGT ACC GAG CTG CTA GAC CGT C), and Δ4 (5′-CGG GGT ACC GCG TAA GCG ATC TGG TTA); these oligonucleotides incorporated KpnI restriction sites for the convenient cloning of the PCR products. Each fragment was inserted into the luciferase reporter vector, pGL3-basic to derive pORF9Δ1 to pORF9Δ4 (see Fig. 2a). All constructs were confirmed by DNA sequencing (data not shown).

In order to further assess the putative ORF 50 RE within the ORF 9 promoter comprising 20307 to 20335 bp of the published sequence, oligonucleotides encompassing this region, 5′-AAG CAT ATA AGA AAA AGA AGA ACA AAG A and 5′-TCT TTG TTC TTC TTT TTC TTA TAT GCT T, were cloned into pGL3-promoter (Promega), which contains the luciferase reporter gene under the control of a minimal simian virus 40 (SV40) promoter. The oligonucleotides were annealed to form blunt-ended fragments and then cloned directly in either orientation into pGL3-promoter previously digested with SmaI, to derive pGL3-9RE1 and pGL3-9RE2.

In order to generate an ORF 9 promoter construct lacking the putative C/EBPα response element, a deletion construct lacking the sequence between 20286 and 20314 bp was constructed by a PCR-based method. The ORF 9 promoter was PCR amplified in two fragments, thereby removing the C/EBPα binding site, using the following sets of primers: for fragment 1, primer A (5′-CGG GGT ACC GTT AAT GTA GCA AGC GGC GC) and primer B (TTC CCC GGG CCA AGC AAA ATT TGT TTT AAT TG); for fragment 2, primer C (5′ TTC CCC GGG GCA AGA AGC ATA TAA GAA AAA AAG) and primer D (5′-CTA GCT AGC GTC AAG ACA GCA ACT CAG). Primer A and D oligonucleotides incorporated KpnI and NheI and primer B and C incorporated SmaI restriction sites, respectively, for convenient cloning of the PCR product. PCR (30 cycles [1 cycle consists of 1 min at 92°C, 1 min at 50°C, and 2 min at 72°C]) was performed with 4 units of Pfu (Stratagene). These fragments were inserted by triple-way ligation upstream of the luciferase coding region in pGL3-basic (Promega) to derive pORF9ΔC/EBP-Luc.

Plasmids encoding C/EBPα and C/EBPβ were kindly provided by C. Heckman, Stanford University School of Medicine. These constructs contained the respective cDNAs in plasmid pcDNA3.1 (Invitrogen); to generate a myc-tagged version, the coding region was excised from pC/EBPα as a HindIII-XbaI fragment and cloned into pcDNA3.1-myc (Invitrogen), deriving pC/EBPα-myc. Plasmid constructs pEGFP, p50GFP, p50GFPΔAT-hook, pGL3-50RE, and pGL3-6RE have been previously reported (36).

Cell culture, viruses, and transfections.293T cells were maintained in Dulbecco modified Eagle medium (Life Technologies) supplemented with 10% fetal calf serum. Plasmids used in the transfections were prepared using QIAGEN plasmid kits according to the manufacturer's directions. 293T cells were seeded at 5 × 10⁵ cells per 35-mm-diameter petri dish 24 h prior transfection. Transfections were performed using Lipofectamine (Invitrogen) as described by the manufacturer, using 2 μg of the appropriate DNAs. HVS (strain A11) was propagated in owl monkey kidney (OMK) cells which were maintained in Dulbecco modified Eagle medium (Life Technologies) supplemented with 10% fetal calf serum.

Luciferase reporter assay.Transfected 293T cells (1 × 10⁶) were harvested in 200 μl of passive lysis buffer (Promega). Quantitation of relative light units was determined using the dual-luciferase Stop & Glo reagent using the manufacturer's directions (Promega) and a Berthold luminometer (EG& G Berthold) with a dual-injector system. All assays were performed in triplicate, and each experiment was repeated a minimum of three times.

RT-PCR analysis.RNA was extracted from 293T cells 24 h posttransfection. Cells were lysed using Trizol reagent (Invitrogen). Chloroform (0.2 ml) was then added, and the solution was vortex mixed for 20 s and stored at 20°C for 15 min. Samples were centrifuged for 15 min at 4°C, and the aqueous phase containing nucleic acids was precipitated using 0.5 ml of isopropanol. After the pellet was washed with 70% ethanol, it was resuspended in 50 μl of water and stored at −70°C. First-strand cDNA was reverse transcribed using Superscript II reverse transcriptase (Life Technologies) and an oligo(dT) primer. Reverse transcription-PCR (RT-PCR) was then performed using the following sets of forward and reverse primers (indicated by the f or r letter at the end of the primer designation, respectively): ORF50f, 5′-CTG GAA TAG TCT CTA CAA CAT TAG CA; ORF50r, 5′-CCG CTC GAG GTT TGG ATC TAT GTT GCG ACT CTG; LUCf, 5′-GCG CCA TTC TAT CCG CTG; LUCr, 5′-ATC GAA GGA CTC TGG CAC; hGAPDHf, 5′-CCA CCC ATG GCA AAT TCC ATG GCA; and hGAPDHr, 5′-TCT AGA CGG CAG GTC AGG TCC ACC]). PCR (30 cycles [1 cycle consists of 1 min at 92°C, 1 min at 55°C, and 1 min at 72°C]) was performed with 4 units of Taq polymerase (Promega).

Gel retardation analysis.The following sets of fluorescein-labeled oligonucleotides, set 1 (5′-GGA ATG CAT AAC ATG CAG CAA GAA GCA T and 5′-ATG CTT CTT GCT GCA TGT TAT GCA TTC C) and set 2 (5′-AAG CAT ATA AGA AAA AGA AGA ACA AAG A and 5′-TCT TTG TTC TTC TTT TTC TTA TAT GCT T) (Invitrogen) were annealed and then incubated with nuclear extracts of untransfected cells or cells transfected with pORF50a or p50GFPΔAT-hook prepared by the method of Andrews and Faller (2). The binding reactions were performed in 20 μl of 100 mM KCl, 20 mM HEPES (pH 7.3), 1% glycerol, 0.2 mM EDTA, 5 mM MgCl₂, 4 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride with 1 μg of poly(dI-dC) as a nonspecific competitor. The protein-nucleic acid complexes were separated on a 5% polyacrylamide gel, run in 1% Tris-borate-EDTA buffer, and detected using a Typhoon 9410 variable-mode imager (Amersham).

In vitro transcription/translation.Protein expression from ORF 50, C/EBPα, and C/EBPβ was performed by in vitro transcription/translation using the TNT system (Promega) according to the manufacturer's instructions. Transcription was initiated from the bacteriophage T7 promoter situated upstream of the cloned fragments.

Coimmunoprecipitation assays.293T cells were transfected using 2 μg of the appropriate DNAs. After 24 h, cells were harvested and lysed with lysis buffer (0.3 M NaCl, 1% Triton X-100, 50 mM HEPES buffer, pH 8.0) containing protease inhibitors (leupeptin and phenylmethylsulfonyl fluoride). For each immunoprecipitation, 50 μl of the anti-ORF 50 polyclonal antibody (17) was incubated with protein A-Sepharose beads (Pharmacia Biotech) for 16 h at 4°C. The beads were then pelleted and washed four times in phosphate-buffered saline (PBS). Each cell lysate was then added to the beads and incubated for 16 h at 4°C. The beads were then pelleted and washed four times in lysis buffer and resuspended in Laemmli buffer, and precipitated polypeptides were resolved on a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel.

Immunoblot analysis.Polypeptides were resolved on 12% SDS-polyacrylamide gels and then soaked for 10 min in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol [vol:vol]). The proteins were transferred to nitrocellulose membranes by electroblotting for 3 h at 250 mA. After transfer, the membranes were soaked in PBS and blocked by preincubation with 2% (wt/vol) nonfat milk powder for 2 h at 37°C. Membranes were incubated with a 1:2,000 dilution of anti-myc (Sigma) antibody or 1:1,000 dilution of anti-green fluorescent protein (GFP) (Clontech) antibody, washed with PBS, and then incubated for 1 h at 37°C with a 1:1,000 dilution of secondary immunoglobulin conjugated with horseradish peroxidase (Dako) in blocking buffer. After five washes with PBS, the nitrocellulose membranes were developed using an ECL kit (Pierce).

Chromatin immunoprecipitation (ChIP) assays.OMK cells seeded at 5 × 10⁵ cells per 35-mm-diameter petri dish, were infected at a multiplicity of infection of 1. After 24 h, the cells were harvested and ChIP assays performed using the ChIP assay kit (Upstate Biotechnology). Chromatin extraction, cross-linking, sonication, immunoprecipitation, agarose bead elution, and protein removal were carried out according to the manufacturer's protocol. DNA recovered from immunoprecipitates with anti-ORF 50, anti-C/EBPα (Santa Cruz), or anti-ORF 57 monoclonal antibody was used as a template for PCR amplifications. The following specific primers for the HVS ORF 9 promoter were used: 5′-CGG GGT ACC CCC AGA TGT TGA GAA ATC and 5′-CTA GCT AGC GTC AAG ACA GCA ACT CAG. These generated a 291-bp region promoter encompassing 20216 and 20507 bp of the published sequence. Primers ORF73f (5′-CGC GGA TCC ATG GAA GCA GGA CCA AGT ACT CCA) and ORF73r (5′-CCG CTC GAG CCT TCT ATA GGC AGG CTT TTG CT-3), specific for the HVS ORF 73 coding region, were used as a negative control to detect a nonpromoter region. The PCR products were analyzed by electrophoresis on a 1.2% agarose gel.

The ORF 50a protein transactivates the DE ORF 9 promoter and requires the AT-hook DNA binding domain.We have previously shown that the HVS ORF 50a protein can transactivate a range of DE HVS promoters which contain a consensus ORF 50 RE, such as the ORF 6 and ORF 57 promoters (41, 42). However, not all DE genes contain such a consensus RE. We have also shown that the ORF 50a protein can stimulate its own promoter via binding to an AT-rich response element (37). Therefore, we aimed to determine whether the ORF 50a protein can activate DE gene promoters which contain AT-rich elements with their promoter elements. One such promoter is the DE ORF 9 DNA polymerase gene promoter.

To determine whether ORF 50a was capable of stimulating the ORF 9 gene promoter, the putative ORF 9 promoter situated between 20034 and 20507 bp of the HVS genome (1) was PCR amplified and cloned upstream of the luciferase reporter gene to derive pORF9-Luc. 293T cells were then transfected with either 1 μg of pEGFP, p50GFP, or p50GFPΔAT-hook (which contains a deletion of the ORF50 DNA binding domain) (36) in the presence of 1 μg of pORF9-Luc. Cells were harvested 30 h posttransfection, and the protein concentration of each sample was calculated using the DC protein assay kit (Bio-Rad). Moreover, to confirm that the transfection efficiency of these experiments was normalized, immunofluorescence was undertaken to ensure that a comparable number of cells was transfected. In addition, 100 μg of cell extracts was analyzed by Western blotting using a primary monoclonal GFP antibody (Clontech) to demonstrate comparable expression between assays (data not shown). An equal amount of cell extracts was then assayed for luciferase activity by standard methods. Results showed that p50GFP was able to stimulate the ORF 9 promoter to high levels, suggesting that the ORF 50a protein can activate a DE promoter which does not contain a consensus ORF 50 RE (Fig. 1). In contrast, stimulation was drastically reduced, by approximately 95%, using p50GFPΔAT-hook, suggesting that ORF 50a stimulates the ORF 9 gene via direct binding to the promoter.

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FIG. 1.

ORF 50 transactivation of the ORF 9 DNA polymerase promoter requires the AT-hook DNA binding domain. 293T cells were cotransfected with pORF9-Luc in the presence of pEGFP, p50GFP, or p50GFPΔAT-hook. Cells were harvested 30 h posttransfection, and cell lysates were assayed for luciferase activity by standard methods. The variations between three replicate assays are indicated. All luciferase data are presented as a percentage of luciferase activity compared to the p50GFP level on the pORF9-Luc promoter construct, with the p50GFP level representing 100% activity. WT, wild type.

Mapping the minimal domain within the ORF 9 promoter that is required for ORF 50a stimulation.To map the ORF 50a responsive regions within the ORF 9 promoter, a series of further promoter deletions were generated (Fig. 2a). The promoter fragments were PCR amplified, and resulting products were cloned upstream of the luciferase reporter gene, deriving pORF9Δ1 to pORF9Δ4.

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FIG. 2.

Mapping the minimal domain required for ORF 50 stimulation of the ORF 9 promoter. (a) Schematic representation of the ORF 9 promoter deletion series cloned upstream of the luciferase reporter gene. (b) Effect of the ORF 50 wild-type protein on the stimulation of the ORF 9 promoter deletion series. 293T cells were cotransfected with each ORF 9 promoter-luciferase construct in the presence of pEGFP or p50GFP. Cells were harvested 30 h posttransfection, and cell lysates were assayed for luciferase activity by standard methods. The variations between three replicate assays (each performed twice) are indicated. The increase in stimulation is shown as a value above each bar. (c) Schematic representation of more-refined ORF 9 promoter deletion cloned upstream of the luciferase reporter gene. (d) Effect of the ORF 50a protein on the stimulation of the refined ORF 9 promoter deletion series. 293T cells were cotransfected with pORF9-Luc or pORF9Δ3 to pORF9Δ4 in the presence of pEGFP or p50GFP. Cells were harvested 30 h posttransfection, and cell lysates were assayed for luciferase activity by standard methods. The variations between three replicate assays (each performed twice) are indicated. All luciferase data are presented as a percentage of luciferase activity compared to the p50GFP level on the pORF9-Luc promoter construct, with the p50GFP level representing 100% activity. The increase in stimulation is shown as a value above each bar.

293T cells were transfected with either 1 μg of pEGFP or p50GFP in the presence of 1 μg of pORF9Δ1 to pORF9Δ4. Cells were assayed for luciferase activity, and the data were normalized as described above. p50GFP was capable of stimulating pORF9Δ1 to pORF9Δ3 to levels comparable to that with the full-length promoter; however, stimulation drastically decreased between Δ3 and Δ4. The luciferase activity of pORF9Δ4 is only 15% of the activity of pORF9Δ3 (Fig. 2b). These data suggest that the region required by ORF 50a is within the 100 bp between Δ3 and Δ4, comprising 20286 to 20381 bp of the published sequence (1).

To finely map the ORF 50a RE within the ORF 9 promoter, a further deletion within the mapped region was generated (Fig. 2c). The promoter fragment was PCR amplified and subsequently cloned upstream of the luciferase reporter gene as previously described, deriving pORF9Δ3.5. 293T cells were then transfected with 1 μg of p50GFP in the presence of 1 μg of pORF9Δ3, pORF9Δ3.5, or pORF9Δ4 and assayed for luciferase activity as described previously (Fig. 2d). The results demonstrated that p50GFP was capable of stimulating pORF9Δ3 to levels similar to those previously reported. However, deletion of the region between Δ3 and Δ3.5 resulted in the level of ORF 50a stimulation returning to a level comparable to that with Δ4, approximately 15% of wild-type levels. This, therefore, suggests that the element(s) responsible for ORF 50a stimulation lies between Δ3 and Δ3.5, comprising 20286 to 20335 bp of the published sequence within the ORF 9 promoter.

The ORF 50a protein binds directly to an AT-rich sequence within the ORF 9 promoter.To assess whether the ORF 50a protein binds directly to this region encompassing Δ3 and Δ3.5 of the ORF 9 promoter, gel retardation experiments were performed using two sets of overlapping oligonucleotides spanning the region, termed set I-II (Fig. 3a). The fluorescein-labeled oligonucleotides were incubated with 2.5 μl of nuclear extracts from untransfected or p50GFP- or p50GFPΔAT-hook-transfected 293T cells. The protein-nucleic acid complexes were then separated on a 4% polyacrylamide gel and detected by using a variable-mode imager (Fig. 3b). Results show the formation of a retarded complex with p50GFP-transfected cell extracts when incubated with oligonucleotide set II. No complexes were identified in the presence of 50GFPΔAT-hook, the mutant ORF 50a protein which lacks the DNA binding domain, suggesting that ORF 50a directly binds the ORF 9 promoter between 20307 and 20335 bp of the published sequence. Moreover, to demonstrate that the retarded complex detected is specific for ORF 50a, three further assays were performed. Firstly, the electrophoretic mobility shift assays (EMSAs) were repeated in the presence of 2.5 μl of control reticulocyte lysate or in vitro-translated ORF 50. A retarded complex was detected, demonstrating that ORF 50 directly binds to a RE contained within set II oligonucleotides (Fig. 3bii). Secondly, a supershift assay was undertaken using a monoclonal GFP antibody (Clontech) and a control B23 antibody (Santa Cruz) and the set II oligonucleotides. Two micrograms of GFP or control antibody was added to the binding reaction mixture described above and incubated for 15 min. A clear shift of the p50GFP retarded complex was detected (Fig. 3biii), in contrast to the control antibody (Fig. 3biv), demonstrating specificity for ORF 50a. However, a complete supershift of the ORF 50-GFP antibody complex was not observed; this was probably due to insufficient amounts of antibody being used in the assay. Thirdly, a competition assay was performed using increasing quantities of unlabeled oligonucleotides spanning the ORF 50 RE present within the ORF 6 promoter (42) or an unlabeled random oligonucleotide. The ORF 6 promoter competed out the binding reaction with labeled set II oligonucleotides (Fig. 3bv), in contrast to the random oligonucleotides (Fig. 3bvi), demonstrating that this retarded complex is specific for the ORF50a protein.

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FIG. 3.

ORF 50 is capable of binding to a 2-bp AT-rich sequence within the ORF 9 promoter. (a) Schematic representation of the oligonucleotide primers spanning the ORF 50 RE within the ORF 9 promoter. (b) (i) EMSAs were performed using fluorescein-labeled oligonucleotides spanning the ORF 9 promoter. Each labeled set of oligonucleotides was incubated with nuclear extracts prepared from untransfected or p50GFP- or p50GFPΔAT-hook-transfected 293T cells. (ii) To demonstrate that ORF 50 interacts directly with set II oligonucleotides, oligonucleotides were incubated with control reticulocyte lysate or in vitro-translated ORF 50 and separated on a polyacrylamide gel. (iii) To demonstrate the specificity of ORF 50 binding, EMSAs were performed by the addition of primary monoclonal GFP antibody (Ab) (rightmost lane). (iv) To show the specificity of the GFP antibody, EMSAs were repeated with a control antibody (Ab). (v) To demonstrate the specificity of the ORF 50 interaction, increased quantities of unlabeled oligonucleotides spanning the ORF 50 RE present within the ORF 6 promoter were used to compete out the binding reaction. (vi) To show that the competition was specific to the ORF 6 oligonucleotides, EMSAs were repeated with a random oligonucleotide.

The ORF 50 response element within the ORF 9 promoter can function as an enhancer element.To further confirm the specificity of ORF 50a binding, the sequences between 20307 and 20335 bp of the published sequence (1) were cloned upstream of a heterologous promoter to determine whether they can confer ORF 50a responsiveness. Set II oligonucleotides were cloned by blunt-end ligation in both possible orientations into pGL3-promoter, which contains the luciferase reporter gene under the control of a minimal SV40 promoter, deriving pGL3-9REI and pGL3-9RE2 (Fig. 4a). Moreover, to compare the specificity of the ORF 9 promoter RE to previously identified REs, similar constructs were produced containing the REs from the ORF 6 and ORF 50 promoters (37). 293T cells were then transfected with 1 μg of pGL3-promoter, pGL3-50RE1, pGL3-9RE2, pGL3-6RE, or pGL3-9RE in the presence of 1 μg of pEGFP or p50GFP and assayed for luciferase activity by standard methods (Fig. 4b). Results demonstrated that p50GFP was able to increase luciferase activity to significant levels from all four RE constructs. This demonstrates that the sequences with the ORF 9 promoter RE are able to confer ORF 50a responsiveness to a heterologous promoter in either orientation and may function as an enhancer sequence. Levels of activity were similar between constructs containing the REs identified within the ORF 50 and ORF 9 promoter, which both contain AT-rich REs. In contrast, activation levels were 70% higher using the construct containing the canonical ORF 6 promoter RE. This variation in activity of ORF 50-responsive genes may have a role as a further regulatory step of the HVS lytic temporal cascade.

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FIG. 4.

The 28-bp AT-rich sequence can confer ORF 50 responsiveness to a heterologous promoter. (a) Schematic representation of the insertion of one copy of the 28-bp AT-rich sequence upstream of the enhancerless SV40 promoter in either orientation to generate pGL3-ORF9RE1 and pGL3-ORF9RE2. (b) 293T cells were cotransfected with pGL3-promoter, pGL3-ORF9REI, pGL3-ORF9REII, pGL3-ORF50RE, or pGL3-ORF6RE in the presence of pEGFP or p50GFP. Cells were harvested 30 h posttransfection, and cell lysates were assayed for luciferase activity by standard methods. The variations between three replicate assays are indicated. All luciferase data are presented as a percentage of luciferase activity compared to the p50GFP levels on the pORF9-Luc promoter construct, with the p50GFP level representing 100% activity.

Rta and C/EBPα proteins synergistically activate the ORF 9 promoter.Further analysis of the ORF 9 promoter using the transcription factor element-searching algorithm Matinspector (http://www.genomatix.com) identified a number of potential transcription factor binding sites. Interestingly, multiple CCAAT enhancer binding protein alpha elements within the promoter and specifically two within the close vicinity of the ORF 50 RE were identified (Fig. 5a). Recent reports have demonstrated that C/EBPα is involved in KSHV ORF 50-mediated transactivation of DE promoters (38, 39). Therefore, the potential involvement of C/EBPα on the ORF 50a stimulation of the ORF 9 promoter was further investigated. To this end, 293T cells were transfected with either 1 μg of pEGFP, p50GFP, or pC/EBPα in the presence of 1 μg of pORF9-Luc. Cells were assayed for luciferase activity, and the data were normalized as described above (Fig. 5b). As a further control, RT-PCR was performed to demonstrate that ORF 50a was expressed in similar quantities in all transfected cells (Fig. 5c). Results showed that no significant increase in luciferase activity was observed in the presence of C/EBPα alone; in contrast, ORF50a activated the ORF 9 promoter to levels similar to those previously shown. However, upon coexpression of ORF 50a and C/EBPα, ORF 9 promoter activity was significantly enhanced to levels of approximately 160% of activity compared to ORF 50a alone (Fig. 5b). Therefore, this suggests that ORF 50a and C/EBPα may have a synergistic effect on the activation of the ORF 9 promoter.

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FIG.5.

ORF 50 and C/EBPα synergistically activate the ORF 9 promoter. (a) Schematic representation of potential C/EBPα transcription factor binding sites within the ORF 9 promoter. (b) 293T cells were cotransfected with pORF9-Luc in the presence of pEGFP, p50GFP, pC/EBPα, or combinations of these. Cells were harvested 30 h posttransfection, and cell lysates were assayed for luciferase activity by standard methods. The variations between three replicate assays (each performed twice) are indicated. All luciferase data are presented as a percentage of the luciferase activity compared to the p50GFP level on the pORF9-Luc promoter construct, with the p50GFP level representing 100% activity. The increase in stimulation is shown as a value above each bar. (c) RT-PCR analysis to demonstrate that ORF 50 and C/EBPα were expressed in similar quantities in cells cotransfected with pORF9-Luc in the presence of pEGFP, p50GFP, pC/EBPα, or combinations of these. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (d) RT-PCR analysis of luciferase expression in cells cotransfected with pORF9-Luc in the presence of pEGFP, p50GFP, pC/EBPα, or combinations of these.

To ascertain whether this synergistic activation of the ORF 9 promoter in the presence of ORF 50a and C/EBPα is due to an increase in the levels of luciferase mRNA, RT-PCR analysis was performed. Total RNA was isolated from 293T cells transfected with either 1 μg of pEGFP, p50GFP, or pC/EBPα in the presence of 1 μg of pORF9-Luc, harvested 24 h posttransfection, and used in RT-PCR analysis to determine the quantities of luciferase mRNA (Fig. 5d). Results show that in the presence of ORF 50a, luciferase mRNA levels are increased; this is further enhanced upon expression of ORF50a and C/EBPα. This demonstrates that the transactivation of luciferase reporter gene from the ORF 9 promoter is due to an increase in mRNA levels.

In vitro-translated C/EBPα protein binds directly to the ORF 9 promoter, and binding is required for its transactivation. To determine whether C/EBPα binds directly to the ORF 9 promoter, gel retardation experiments were performed using the two sets of oligonucleotides spanning the region between Δ3 and Δ3.5, namely, set I-II; set I contains a putative C/EBPα binding site identified by Matinspector (Fig. 6a). The fluorescein-labeled oligonucleotides were incubated with 2.5 μl of control reticulocyte lysate or in vitro-translated C/EBPα or C/EBPβ. The protein-nucleic acid complexes were then separated and detected by using a variable-mode imager. Results showed the formation of a retarded complex with in vitro-translated C/EBPα when incubated with oligonucleotide set I (Fig. 6bi). No other complexes were identified, suggesting that C/EBPα directly binds the ORF 9 promoter between 20286 and 20314 bp of the published sequence (1). To confirm that the retarded complex detected is specific for C/EBPα, a competition assay was performed using increasing quantities of unlabeled oligonucleotides spanning a known C/EBPα binding previously identified in the KSHV Rta promoter site or a randomized oligonucleotide (39). The C/EBPα oligonucleotides competed out the binding reaction with labeled set I oligonucleotides (Fig. 6bii), in contrast to the random oligonucleotide, demonstrating that this retarded complex is specific for the C/EBPα protein.

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FIG. 6.

C/EBPα binds upstream of the ORF 50 RE within the ORF 9 promoter. (a) Schematic representation of the oligonucleotide primers spanning the potential C/EBPα binding site adjacent to the ORF 50 RE within the ORF 9 promoter. The potential C/EBPα binding site is boxed. (b) (i) EMSAs were performed using fluorescein-labeled set I and II oligonucleotides spanning the potential C/EBPα binding site. Each labeled set of oligonucleotides was incubated with control reticulocyte lysate or in vitro-translated C/EBPα or C/EBPβ and separated on a polyacrylamide gel. (ii) To demonstrate the specificity of the C/EBPα interaction, increased quantities of unlabeled oligonucleotides specific for the C/EBPα binding recognition sequence were used to compete out the binding reaction. EMSAs were repeated with a random oligonucleotide.

To further assess the role of C/EBPα on the activation of the ORF 9 promoter, a mutant promoter was generated that contained a deletion within the putative C/EBPα recognition sequence, termed pORF9ΔC/EBPα-Luc. 293T cells were then transfected with 1 μg of either pORF9-Luc or pORF9ΔC/EBPα-Luc in the presence of 1 μg of pEGFP, p50GFP, or pC/EBPα and assayed for luciferase activity as described previously (Fig. 7). The results demonstrated that p50GFP was capable of stimulating both pORF9-Luc or pORF9ΔC/EBPα-Luc to levels similar to those previously described. However, deletion of the putative C/EBPα region resulted in no enhancement of ORF 9 promoter activity in the presence of C/EBPα, in contrast to the wild-type ORF 9 promoter. This, therefore, suggests that the element responsible for the synergistic response of the ORF 9 promoter in the presence of C/EBPα comprises 20286 to 20314 bp of the published sequence within the ORF 9 promoter.

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FIG. 7.

C/EBPα binding is required for the synergistic response on the ORF 9 promoter. 293T cells were cotransfected with pORF9-Luc or p ORF9ΔC/EBPα-Luc in the presence of pEGFP, p50GFP, or pC/EBPα and various combinations of these. Cells were harvested 30 h posttransfection, and cell lysates were assayed for luciferase activity by standard methods. The variations between three replicate assays are indicated. All luciferase data are presented as a percentage of the luciferase activity compared to p50GFP levels on the pORF9-Luc promoter construct, with the p50GFP level representing 100% activity.

ORF 50a and C/EBPα interact with the ORF 9 promoter in vivo.To elucidate whether ORF 50a and C/EBPα interact with the ORF 9 promoter in vivo, ChIP assays were performed. Initially, HVS-infected OMK cells were harvested 24 h postinfection. Then ORF 50 and C/EBPα were immunoprecipitated using an anti-ORF 50 and anti-C/EBPα monoclonal sera, respectively, from the cross-linked protein-DNA complexes. After the removal of the proteins, the recovered DNA was used as a template for the specific amplification of the ORF 9 promoter and a HVS genomic control. Results demonstrate both ORF 50 and C/EBPα proteins were found to be specifically associated with the ORF 9 promoter (Fig. 8). In contrast, the negative controls with anti-ORF 57 antiserum failed to precipitate any ORF 9 promoter. This demonstrates that the ORF 50 and C/EBPα interact with the ORF 9 promoter in vivo.

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FIG. 8.

ORF 50 and C/EBPα associate with the ORF 9 promoter in vivo. (i) PCR amplification of the ORF 9 promoter from input, no-antibody controls (No Ab), immunoprecipitates with anti-ORF 57 antisera (ORF 57 Ab), immunoprecipitates with anti-ORF 50 antisera (ORF 50 Ab), or immunoprecipitates with anti-C/EBPα antisera (C/EBPα Ab). (ii) As a negative control, PCR amplification was performed on nonpromoter fragments, specifically the ORF 73 gene from input (lane 1), no-antibody controls (No Ab), immunoprecipitates with anti-ORF 57 antisera (ORF 57 Ab), immunoprecipitates with anti-ORF 50 antisera (ORF 50 Ab), or immunoprecipitates with anti-C/EBPα antisera (C/EBPα Ab).

ORF 50a interacts with C/EBPα.Gel retardation analysis has demonstrated that both ORF 50a and C/EBPα bind the ORF 9 promoter in close proximity. Therefore, to investigate whether ORF 50a and C/EBPα interact directly with each other, coimmunoprecipitation studies were performed. Control untransfected 293T cells were compared with cells transfected with pEGFP, p50GFP, and a myc-tagged expression construct for C/EBPα. After 24 h, the cells were harvested, and cell lysates were utilized in coimmunoprecipitation analysis using an anti-ORF 50 antiserum (17). Polypeptides precipitated from cellular extracts were then analyzed by Western blotting with anti-myc-specific antiserum (Fig. 9). The results demonstrate that C/EBPα coimmunoprecipitates in the presence of both ORF 50a and the mutant ORF 50a construct lacking its DNA binding domain. This result suggests that the HVS ORF 50a can specifically interact with C/EBPα and that the AT-hook DNA binding domain is not required for this interaction.

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FIG. 9.

ORF 50 and C/EBPα physically interact. 293T cells were cotransfected with pC/EBPα-myc in the presence of pEGFP, p50GFP, or p50GFPΔAT-hook. (a) Cell lysates were immunoprecipitated using anti-ORF 50 antiserum. Bound proteins were resolved by SDS-polyacrylamide gel electrophoresis, and the presence of C/EBPα-myc was detected using anti-myc antiserum (b). As a control, Western blot analysis was carried out on the supernatants before immunoprecipitation using a anti-GFP antibody to confirm that GFP, 50GFP, and 50GFPΔAT-hook were expressed in all samples. Untrans, untranslated.