1 School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK
2 Institute of Cardiovascular Research, University of Leeds, Leeds LS2 9JT, UK
3 Molecular and Cellular Biology Research Group, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK
Correspondence
Adrian Whitehouse
a.whitehouse{at}leeds.ac.uk
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ABSTRACT |
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Present address: Department of Microbiology, Columbia University, New York, NY 10032, USA.
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MAIN TEXT |
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Gene expression in HVS is modulated by the two major transcriptional regulating genes, encoded by open reading frames (ORFs) 50 and 57 (Nicholas et al., 1988, 1991
; Whitehouse et al., 1997a
, 1998a
, b
). The ORF 57 gene product encodes a multifunctional protein that is capable of both transactivation and repression of viral gene expression at a post-transcriptional level (Whitehouse et al., 1998a
, b
; Goodwin et al., 1999
). The ORF 50 gene produces two transcripts, termed ORF 50a and b. The larger, spliced ORF 50a gene product is the major transactivating protein. It activates transcription following direct binding to promoters containing a specific sequence motif, CCN9GG, termed the ORF 50 response element (RE) (Nicholas et al., 1991
; Whitehouse et al., 1997a
, b
, 1998a
). This RE has significant similarity to the EBV Rta RE consensus sequence, GNCCN9GGNG (Gruffat et al., 1990
, 1992
; Gruffat & Sergeant, 1994
). Recently, we have identified a DNA-binding domain within ORF 50a that has a high degree of similarity to the DNA-binding domain encoded by mammalian high-mobility group A (HMGA) chromosomal proteins, termed an AT hook (Reeves & Nissen, 1990
). This domain is required for ORF 50 transactivation and binding to the ORF 50 RE (Walters et al., 2004
). Once bound, HVS ORF 50a recruits and interacts with the TATA binding protein (TBP) (Hall et al., 1999
), suggesting that it recruits components of the TFIID complex and allowing transcription initiation by RNA polymerase II.
Studies in EBV, KSHV, MHV-68 and HVS have shown that ORF 50 proteins are key regulators in the switch from the latent state to the lytic-replication cycle (Zalani et al., 1996; Lukac et al., 1998
, 1999
; Ragoczy et al., 1998
; Sun et al., 1998
; Ragoczy & Miller, 1999
; Wu et al., 2000
; Goodwin et al., 2001
). Studies have shown that the Rta promoters of EBV (Zalani et al., 1992
, 1995
, 1996
, 1997
; Glaser et al., 1998
; Chang & Liu, 2000
; Ragoczy & Miller, 2001
) and KSHV (Deng et al., 2000
; Chen et al., 2001
; Sakakibara et al., 2001
; Brown et al., 2003
; Haque et al., 2003
; Liang & Ganem, 2003
; Wang et al., 2003
; Damania et al., 2004
) are activated by many cellular factors and, more significantly, by the Rta proteins themselves. Therefore, in this report, we investigated whether HVS ORF 50a was capable of regulating its own promoter.
To determine whether ORF 50a was capable of stimulating its own promoter, the putative ORF 50a promoter situated between positions 68320 and 69878 of the HVS genome was PCR-amplified by using the primers DelFor1 (5'-ACATGCATGCGAACAGACATAACGCGTAGA-3') and DelRev (5'-ACATGCATGCTGTTGTTGCTTAAGAGCTCAG-3'). These primers incorporated SphI and SalI sites, respectively, and the resulting products were cloned into pCAT-Basic (Promega), previously digested with SalI and SphI, deriving pCAT-DEL1. 293T cells were transfected by using Lipofectamine (Invitrogen) with 1 µg of either pEGFP, p50GFP or p50GFPAT-hook (which contains a deletion of the ORF 50 DNA-binding domain) (Walters et al., 2004
) in the presence of 1 µg pCAT-DEL1. Cells were harvested 30 h post-transfection and the protein concentration of each sample was calculated by using a DC Protein Assay kit (Bio-Rad). Cell extracts corresponding to 10 µg protein were assayed for chloramphenicol acetyltransferase (CAT) activity by standard methods (Gorman et al., 1982
). Levels of chloramphenicol converted to acetylated products were calculated by using a FUJIX BAS1000 Bio-Imaging analyser (Fuji Photo Film Co.). Raw data produced from the Image Reader BAS-1000 software were subsequently quantified by using Advanced Image Data Analyser (AIDA) version 2.31 software and represented as fold increase in CAT activity compared to vector control (pEGFP). Results showed that p50GFP was able to stimulate the ORF 50a promoter to high levels (36-fold), suggesting that the ORF 50a protein can stimulate its own promoter. In contrast, stimulation was reduced dramatically when using p50GFP
AT-hook (Fig. 1a
), suggesting that ORF 50a stimulates its own promoter via direct binding of the promoter. To confirm that the transfection efficiency of these experiments was normalized, immunofluorescence was undertaken to ensure that a comparable number of cells were transfected. In addition, 100 µg cell extract was analysed by Western blot using a primary monoclonal green fluorescent protein (GFP) antibody (Clontech) to demonstrate comparable expression between assays (data not shown).
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293T cells were transfected with 1 µg of either pEGFP, p50GFP or p50GFPAT-hook in the presence of 1 µg pCAT-DEL27. Cells were assayed for CAT activity and the data were normalized as described previously. p50GFP was capable of stimulating DEL2 to levels comparable with those for DEL1; however, stimulation decreased from 32-fold to 23·4-fold for DEL3. Fold stimulation then increased to 28 for DEL4, with a subsequent drop to 17·8-fold for DEL5. These data suggest the presence of positive- and negative-acting elements within the ORF 50a promoter between positions 69237 and 68587 of the published sequence (GenBank accession no. X64346). ORF 50a stimulation levels remained similar for DEL5 and DEL6; however, removal of the region between DEL6 and DEL7 resulted in a drop in stimulation from 15·6-fold to 5·8-fold, reducing the CAT activity levels to those of p50GFP
AT-hook. This suggests that the region between DEL6 and DEL7 is important for the stimulatory effect of ORF 50a on its own promoter.
To fine-map the ORF 50a-responsive element, further deletions of this region were generated (Fig. 1d). The promoter fragments were PCR-amplified by using the forward primers DelFor89 (DelFor8, 5'-ACATGCATGCCGGAAGGTTCTCTACATAC-3'; DelFor9, 5'-ACATGCATGCGTAAGCCTAGTGTCCGAC-3') and the reverse primer DelRev, and subsequently cloned into pCAT-Basic as described previously, deriving pCAT-DEL6.1 and pCAT-DEL6.2. 293T cells were then transfected with 1 µg p50GFP in the presence of 1 µg pCAT-DEL6 or pCAT-DEL7 and assayed for CAT activity as described previously (Fig. 1e
). The results demonstrated that p50GFP was capable of stimulating DEL6 and DEL6.1 to similar levels. However, deletion of the region between DEL6.1 and DEL6.2 resulted in ORF 50a stimulation returning to levels comparable to those obtained with DEL7. This, therefore, suggests that the element(s) responsible for ORF 50a stimulation lie between DEL6.1 and DEL6.2 of the ORF 50a promoter.
To assess whether the ORF 50a protein binds directly to this region encompassing DEL6.1 to DEL6.2 of the ORF 50a promoter, gel-retardation experiments were performed by using three sets of overlapping oligonucleotides spanning the region, termed sets (I)(III) (Fig. 2a). The oligonucleotides were annealed and labelled by using a DIG Gel Shift kit (Roche) according to the manufacturer's protocol. The digoxigenin (DIG)-labelled oligonucleotides were then incubated with 2·5 µl nuclear extract from untransfected and p50GFP-transfected 293T cells, prepared by using a NucBuster Protein Extraction kit (Novagen). The proteinnucleic acid complexes were separated on a 4 % polyacrylamide gel and detected by immunoblotting using the DIG Gel Shift kit protocol (Fig. 2b
). Results show the formation of a retarded complex with p50GFP-transfected cell extracts when incubated with oligonucleotide set (I). No other complexes were identified with oligonucleotide sets (II) and (III), suggesting that ORF 50a binds to the ORF 50a promoter directly between positions 69526 and 69560 of the published sequence (GenBank accession no. X64346). Moreover, to demonstrate that the retarded complex detected is specific for ORF 50a, two further assays were performed. A supershift assay was undertaken by using a monoclonal GFP antibody (Clontech) and the set (I) oligonucleotides. GFP antibody (2 µg) was added to the binding reaction (described previously) and incubated for 15 min. A clear shift of the p50GFP-retarded complex was detected, demonstrating specificity for ORF 50a. Secondly, competition assays were performed by using increasing quantities of unlabelled oligonucleotides spanning the ORF 50 RE present within the ORF 6 promoter (Whitehouse et al., 1997b
). This out-competed the binding reaction with labelled set (I) oligonucleotides, demonstrating that this retarded complex is specific for the ORF 50a protein (Fig. 2b
).
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ACKNOWLEDGEMENTS |
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Received 26 October 2004;
accepted 30 November 2004.