The Herpesvirus saimiri replication and transcription activator acts synergistically with CCAAT enhancer binding protein alpha to activate the DNA polymerase promoter

Abstract

The open reading frame (ORF) 50 gene product, also known as the replication and transcription activator (Rta), is an immediate-early gene which is well conserved among all gamma-2 herpesviruses and plays a pivotal role in regulating the latent-lytic switch. Herpesvirus saimiri (HVS) ORF 50a functions as a sequence-specific transactivator capable of activating delayed-early (DE) gene expression via binding directly to an ORF 50 response element (RE) within the respective promoter. Analysis of the ORF 50 REs have identified two distinct types within HVS gene promoters. The first comprises a consensus sequence motif, CCN(9)GG, the second an AT-rich sequence. Here we demonstrate that ORF 50a is capable of transactivating the DE ORF 9 promoter which encodes the DNA polymerase. Deletion analysis of the ORF 9 promoter mapped the ORF 50 RE to a 95-bp region situated 126 bp upstream of the initiation codon. Gel retardation analysis further mapped the RE to a 28-bp fragment, which was able to confer ORF 50 responsiveness on an enhancerless simian virus 40 minimal promoter. Furthermore, sequence analysis identified multiple CCAAT enhancer binding protein alpha (C/EBPalpha) binding sites within the ORF 9 promoter and specifically two within the close vicinity of the AT-rich ORF 50 RE. Analysis demonstrated that the HVS ORF 50a and C/EBPalpha proteins associate with the ORF 9 promoter in vivo, interact directly, and synergistically activate the ORF 9 promoter by binding to adjacent binding motifs. Overall, these data suggest a cooperative interaction between HVS ORF 50a and C/EBPalpha proteins to activate the DNA polymerase promoter during early stages of the lytic replication cycle.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.