Evidence for a Functional Glucocorticoid Responsive Element in the Epstein-Barr Virus Genome
Catherine Schuster, Sylvette Chasserot-Golaz, Gilbert Urier, Gisele Beck, and Alain Sergeant Institut de Biologie Moleculaire et Cellulaire du CNRS (C.S., S.C.-G., G.B.) 67084 Strasbourg Cedex, France Laboratoire de Virologie Moleculaire Ecole Normale Superieure de Lyon (G.U., A.S.) 69364 Lyon Cedex 07, France
Glucocorticoids induce the expression of EpsteinBarr virus early antigens in latently infected Daudi cells. By sequence analysis, we found that fragment C of the BamHI digested Epstein-Barr virus B95-8 genome contains a region with a large degree of homology to the glucocorticoid responsive element of known glucocorticoid-regulated genes. By transfection experiments in Daudi and HeLa cells, different lengths of this region, cloned in front of the bacterial chloramphenicol acetyl transferase linked to the Herpes Simplex virus thymidine kinase promoter (pBLCAT.2), were assayed for their responsiveness to dexamethasone; our results led us to the conclusion that the hormonal effect observed was mediated by a minimal sequence of 15 base pairs presenting 85% homology with the consensus glucocorticoid responsive element sequence. (Molecular Endocrinology 5: 267-272, 1991)
detected. Those include the early antigens (EA) which are expressed in the early steps of the viral lytic cycle, and the viral capsid antigens corresponding to structural viral proteins. Various agents are able to induce the EBV EA synthesis. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (3), n-butyrate (4), or infection with the EBV defective strain P3HR1 (5) are known to disrupt latency, whereas interferon (6), transforming growth factor-/? (7) and steroids (8) were only shown to induce EBV EA synthesis. Such inducers constitute ideal tools to study viral gene expression. In a previous work, we observed that dexamethasone (DXM) induces the synthesis of EBV EA in Daudi lymphoma cells (8). Glucocorticoids are known to induce transcription of cellular and viral genes. In Mouse Mammary Tumor virus, hormone receptor complexes bind to a glucocorticoid responsive element (GRE) (9, 10) and thereafter up-regulate the expression of both autologous and heterologous genes (11, 12). Such sequences were also found in the long terminal repeat of several other retroviruses such as the Moloney Murine Leukemia virus (13) and the Moloney Murine Sarcoma virus (14, 15). Recently, it has been shown that genes from DNA viruses, like the Human Papilloma virus-16 (HPV-16) (16) and Hepatitis B virus (HBV) (17), might also be under glucocorticoid control. The mechanism of hormonal induction of transcription involves binding of the glucocorticoid receptor to the GRE. The comparison of numerous GREs has led to the identification of a 15 base pair (bp) consensus sequence which includes an incomplete palindrome of the hexanucleotide5'-TGTYCT-3'(18).
INTRODUCTION The Epstein-Barr virus (EBV) is a double stranded DNA virus that belongs to the Herpes virus family. This virus is directly involved in several human diseases. Primary viral infection often results in infectious mononucleosis, a benign lymphoproliferative human disease. However, EBV, in association with genetic and environmental factors, is also responsive for two types of malignant tumors, targeting B lymphoid cells in childhood lymphomas, i.e. Burkitt's lymphomas, and poorly differentiated epithelial cells in nasopharyngeal carcinomas. In vitro, EBV immortalizes B lymphocytes; in this way it allows perpetual cell proliferation in culture (2). In transformed cells, the virus persists in the nucleus as an episome and only a few genes are expressed to maintain latency. In producer cell lines, other classes of viral antigens are
To determine whether the increased EBV EA synthesis by glucocorticoids in Daudi cells is due to a direct action of the hormone on the EBV genome, a computer search was performed to identify putative GREs in EBV. Since GREs were found 3' to the BCRF1 Open Reading Frame (ORF), we examined the effects of DXM on gene expression in recombinant plasmids containing different segments of the BCRF1 ORF region using the chlor-
0888-8809/91 /0267-0272$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
267
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 November 2015. at 23:46 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 2
MOL ENDO-1991 268
amphenicol acetyl transferase (CAT) system developed by Gorman etal. (19).
RESULTS DXM Effect on the EBV Genome in Daudi Lymphoma Cells In order to define the region responsive for the glucocorticoid effect in the EBV genome, we subjected the viral DNA sequence to a computer search for homologies with the consensus GRE sequence—GNNACAANNTGTYCT—which functions as a specific glucocorticoid receptor binding site. Such sequences have been identified in several glucocorticoid regulated genes including the rat tryptophane oxygenase gene and the Metallothionein IIA gene (20, 21). In the fragment C of the EBV B95-8 SamHI digested genome, four hexanucleotides—TGTYCT—were found in a closely related region of 156 bp (Fig. 1); these hexanucleotides are highly conserved motifs of the steroid receptor binding sites (22). Thereafter, we tested whether this region may be able to stimulate the transcriptional activity of a linked heterologous promoter after hormone administration. Initially, we inserted the DNA fragment spanning nucleotide 9134 to nucleotide 10439 (containing the putative GRE sequences) into the polylinker of piasmid pBLCAT.2 upstream of the Herpes Simplex virus thymidine kinase (HSV-TK) promoter. This promoter is commonly used for its responsiveness to glucocorticoid regulatory elements (12,23). This construct, called pC1.3CAT, contains both the coding region of the CAT gene and a viral ORF coding for the hypothetical BCRF1 protein in inverted orientation. Thus, the
Jh
GRE sequence was placed upstream of the TK-CAT gene and downstream of the putative promoter for an hypothetical ORF (Fig. 1). In Fig. 2, an example of the effect of DXM on Daudi cells transfected with pC1.3CAT is presented. A very low CAT activity was detected in cells transfected with pC1.3CAT without hormonal administration (lane 1). In hormonal treated cells, a 3-fold increase of CAT activity was observed (lane 2) as compared to the control (lane 1). The DXM effect is inhibited by a simultaneous equimolar addition of the potent antiglucocorticoid RU486 (lane 3); with RU486 alone the CAT activity remained at the basal level (lane 4). From these results we concluded that glucocorticoids may act on the EBV genome through a GRE region localized in the Aval digested C EBV fragment. Thus, it was interesting to investigate if this GRE region found in the EBV genome was also functional in non-EBV-positive cells and to define the minimal sequence required to detect a hormonal effect. Functional analysis of the EBV GRE sequence Transfection experiments were performed in HeLa cells with the pC1.3CAT construct co-transfected with an expression vector coding for the human glucocorticoid receptor (HG1). An 11 -fold induction of the CAT activity was observed in DXM-treated cells as compared to untreated cells (Fig. 3B). In the absence of glucocorticoid receptors no hormonal effect was observed (data not shown). Next, deletion mutants of pC1.3CAT were prepared, as described in Materials and Methods, creating plasmids pC1 .OCAT and pC0.25CAT (Fig. 3A). Each piasmid tested in HeLa cells were able to confer a strong DXM inducibility of CAT expression through
i
B OKFUCKFl
GRB' 10439
pC 1.3 CAT
c
Fig. 1. Linear representation of the EBV B95-8 SamHI digested genome and of the piasmid pC1.3CAT A, Sequence organization of the EBV B95-8 SamHI digested genome. B, Enlargement of the Ava\ restriction fragment. The location of the GRE sequence {black block) and of ORF BCRF1 are indicated. C, Detailed linear representation of the piasmid pC1.3CAT containing the Ava\ fragment inserted in front of the CAT coding region controlled by the TK promoter. Restriction sites abbreviations: S = Sail; A = Aval.
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269
Glucocorticoid Action on Epstein-Barr Virus Genome
CAT inductibility in the presence of DXM. Moreover, every induction observed required the presence of functional glucocorticoid receptors and was at least inhibited to 70% by simultaneous addition of the antiglucocorticoid RU486.
DISCUSSION
Fig. 2. Effect of DXM on the pC1.3CAT construct in Daudi
cells. CAT assays were performed essentially as described in Materials and Methods Lane 1, Extract of cells transfected with pC1.3CAT in absence of steroid; lane 2, extract of cells transfected with pC1.3CAT in presence of 3 IXM DXM; lane 3, extract of cells transfected with pC1.3CAT in presence of 3 ^M DXM and 3 ^M RU486; lane 4, extract of cells transfected with pC1.3CAT in presence of 3 HM RU486.
the TK promoter (Fig. 3B) and the induction measured was about three times over the induction rate obtained with the pC1.3CAT construct. One of the hexanucleotide —TGTYCT— found in the EBV genome was included in a DNA sequence forming an imperfect repeat showing a 85% homology with the GRE sequence. To define the contribution of this sequence —AGCACATTTTGTTCT— to the induction ratio observed with the total GRE zone, we carried out a construction pGREEBVCAT by inserting a 27 bp long oligonucleotide immediately upstream of the TK promoter. By transfecting HeLa cells with pGRE • EBVCAT, we measured also an equally 28-fold DXM induction. These experiments showed that the GRE region found in the EBV genome was able to confer DXM inducibility through the heterologous TK promoter in EBV negative nonlymphoma cells, such as HeLa cells. We also determined that the minimal GRE sequence —AGCACATTTTGTTCT— was able to confer maximal
In various systems, including viral models, glucocorticoids were found to regulate specific gene expression. The comparison of all the sequences involved in the glucocorticoid action led to the determination of a consensus GRE sequence —GNNACAANNTGTYCT— essential for the relaying of the hormonal message (18). A computer search in the EBV genome revealed a similar sequence in the C fragment of the BamH\ digested EBV B95-8 genome. This sequence had 85% homology with the consensus GRE. Moreover, we found three times the highly conserved motif — TGTTCT— in surrounding sequences. A restriction fragment spanning nucleotide 9104 to nucleotide 10439, including this putative GRE region, linked to the heterologous promoter TK (plasmid pC1.3CAT) conferred DXM responsiveness to the TK-CAT chimeric gene in Daudi cells. GRE are known to act as hormonal regulated enhancers and confer gene induction without regard to the orientation and the distance separating them from the target promoters (21, 24). In the pC1.3CAT, the GRE is located downstream of the ORF BCRF1 promoter and upstream of the TK promoter. A priory, one might think that this GRE would regulate these two independent promoters; however, glucocorticoids were found to act only through the TK promoter by inducing the transcription of the CAT gene in this construct (data not shown). With regard to the EBV cell specificity, restricted to B lymphoid cells and to poorly differentiated epithelial cells, it was interesting to test whether the glucocorticoid CAT induction observed in EBV positive lymphoma cells, such as Daudi cells, also occurs in other cell lines. The pC1.3CAT construct was therefore transfected in HeLa cells. These cells, known to be EBV negative, contain a low level of endogenous glucocorticoid receptors. In order to study the glucocorticoid responsiveness in these cells, it was necessary to transfect the CAT reporter genes together with a glucocorticoid receptor expressing vector, HG1. After DXM treatment, we measured a strong inducibility of the CAT expression in transfected HeLa cells. Maximal CAT induction was already obtained at a 5.10"8 M DXM concentration; this is in agreement with the saturation concentration obtained for other GREs (25, 26). However, in Daudi cells, the amount allowing a maximal effect was about 3.10~6 M. The difference may be relevant to the steroid metabolism occurring in lymphoma cells, which contain more hydroxylases than HeLa cells. Cotransfection with a glucocorticoid receptor expressing vector was essential to obtain hormonal induction; moreover, the CAT
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 November 2015. at 23:46 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 2
MOL ENDO-1991 270
pC 1J CAT
ORF BCRF1 "*1
GRE IB -1335
ITK [ V / ^ C J M ' V V / V
lp.l
A
TK r x ' x ' x O&T N ' N ' s ' N '*'
pC 1.0 CAT •364
•1335
pC
Catherine Schuster, Sylvette Chasserot-Golaz, Gilbert Urier, Gisele Beck, and Alain Sergeant Institut de Biologie Moleculaire et Cellulaire du CNRS (C.S., S.C.-G., G.B.) 67084 Strasbourg Cedex, France Laboratoire de Virologie Moleculaire Ecole Normale Superieure de Lyon (G.U., A.S.) 69364 Lyon Cedex 07, France
Glucocorticoids induce the expression of EpsteinBarr virus early antigens in latently infected Daudi cells. By sequence analysis, we found that fragment C of the BamHI digested Epstein-Barr virus B95-8 genome contains a region with a large degree of homology to the glucocorticoid responsive element of known glucocorticoid-regulated genes. By transfection experiments in Daudi and HeLa cells, different lengths of this region, cloned in front of the bacterial chloramphenicol acetyl transferase linked to the Herpes Simplex virus thymidine kinase promoter (pBLCAT.2), were assayed for their responsiveness to dexamethasone; our results led us to the conclusion that the hormonal effect observed was mediated by a minimal sequence of 15 base pairs presenting 85% homology with the consensus glucocorticoid responsive element sequence. (Molecular Endocrinology 5: 267-272, 1991)
detected. Those include the early antigens (EA) which are expressed in the early steps of the viral lytic cycle, and the viral capsid antigens corresponding to structural viral proteins. Various agents are able to induce the EBV EA synthesis. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (3), n-butyrate (4), or infection with the EBV defective strain P3HR1 (5) are known to disrupt latency, whereas interferon (6), transforming growth factor-/? (7) and steroids (8) were only shown to induce EBV EA synthesis. Such inducers constitute ideal tools to study viral gene expression. In a previous work, we observed that dexamethasone (DXM) induces the synthesis of EBV EA in Daudi lymphoma cells (8). Glucocorticoids are known to induce transcription of cellular and viral genes. In Mouse Mammary Tumor virus, hormone receptor complexes bind to a glucocorticoid responsive element (GRE) (9, 10) and thereafter up-regulate the expression of both autologous and heterologous genes (11, 12). Such sequences were also found in the long terminal repeat of several other retroviruses such as the Moloney Murine Leukemia virus (13) and the Moloney Murine Sarcoma virus (14, 15). Recently, it has been shown that genes from DNA viruses, like the Human Papilloma virus-16 (HPV-16) (16) and Hepatitis B virus (HBV) (17), might also be under glucocorticoid control. The mechanism of hormonal induction of transcription involves binding of the glucocorticoid receptor to the GRE. The comparison of numerous GREs has led to the identification of a 15 base pair (bp) consensus sequence which includes an incomplete palindrome of the hexanucleotide5'-TGTYCT-3'(18).
INTRODUCTION The Epstein-Barr virus (EBV) is a double stranded DNA virus that belongs to the Herpes virus family. This virus is directly involved in several human diseases. Primary viral infection often results in infectious mononucleosis, a benign lymphoproliferative human disease. However, EBV, in association with genetic and environmental factors, is also responsive for two types of malignant tumors, targeting B lymphoid cells in childhood lymphomas, i.e. Burkitt's lymphomas, and poorly differentiated epithelial cells in nasopharyngeal carcinomas. In vitro, EBV immortalizes B lymphocytes; in this way it allows perpetual cell proliferation in culture (2). In transformed cells, the virus persists in the nucleus as an episome and only a few genes are expressed to maintain latency. In producer cell lines, other classes of viral antigens are
To determine whether the increased EBV EA synthesis by glucocorticoids in Daudi cells is due to a direct action of the hormone on the EBV genome, a computer search was performed to identify putative GREs in EBV. Since GREs were found 3' to the BCRF1 Open Reading Frame (ORF), we examined the effects of DXM on gene expression in recombinant plasmids containing different segments of the BCRF1 ORF region using the chlor-
0888-8809/91 /0267-0272$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
267
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 November 2015. at 23:46 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 2
MOL ENDO-1991 268
amphenicol acetyl transferase (CAT) system developed by Gorman etal. (19).
RESULTS DXM Effect on the EBV Genome in Daudi Lymphoma Cells In order to define the region responsive for the glucocorticoid effect in the EBV genome, we subjected the viral DNA sequence to a computer search for homologies with the consensus GRE sequence—GNNACAANNTGTYCT—which functions as a specific glucocorticoid receptor binding site. Such sequences have been identified in several glucocorticoid regulated genes including the rat tryptophane oxygenase gene and the Metallothionein IIA gene (20, 21). In the fragment C of the EBV B95-8 SamHI digested genome, four hexanucleotides—TGTYCT—were found in a closely related region of 156 bp (Fig. 1); these hexanucleotides are highly conserved motifs of the steroid receptor binding sites (22). Thereafter, we tested whether this region may be able to stimulate the transcriptional activity of a linked heterologous promoter after hormone administration. Initially, we inserted the DNA fragment spanning nucleotide 9134 to nucleotide 10439 (containing the putative GRE sequences) into the polylinker of piasmid pBLCAT.2 upstream of the Herpes Simplex virus thymidine kinase (HSV-TK) promoter. This promoter is commonly used for its responsiveness to glucocorticoid regulatory elements (12,23). This construct, called pC1.3CAT, contains both the coding region of the CAT gene and a viral ORF coding for the hypothetical BCRF1 protein in inverted orientation. Thus, the
Jh
GRE sequence was placed upstream of the TK-CAT gene and downstream of the putative promoter for an hypothetical ORF (Fig. 1). In Fig. 2, an example of the effect of DXM on Daudi cells transfected with pC1.3CAT is presented. A very low CAT activity was detected in cells transfected with pC1.3CAT without hormonal administration (lane 1). In hormonal treated cells, a 3-fold increase of CAT activity was observed (lane 2) as compared to the control (lane 1). The DXM effect is inhibited by a simultaneous equimolar addition of the potent antiglucocorticoid RU486 (lane 3); with RU486 alone the CAT activity remained at the basal level (lane 4). From these results we concluded that glucocorticoids may act on the EBV genome through a GRE region localized in the Aval digested C EBV fragment. Thus, it was interesting to investigate if this GRE region found in the EBV genome was also functional in non-EBV-positive cells and to define the minimal sequence required to detect a hormonal effect. Functional analysis of the EBV GRE sequence Transfection experiments were performed in HeLa cells with the pC1.3CAT construct co-transfected with an expression vector coding for the human glucocorticoid receptor (HG1). An 11 -fold induction of the CAT activity was observed in DXM-treated cells as compared to untreated cells (Fig. 3B). In the absence of glucocorticoid receptors no hormonal effect was observed (data not shown). Next, deletion mutants of pC1.3CAT were prepared, as described in Materials and Methods, creating plasmids pC1 .OCAT and pC0.25CAT (Fig. 3A). Each piasmid tested in HeLa cells were able to confer a strong DXM inducibility of CAT expression through
i
B OKFUCKFl
GRB' 10439
pC 1.3 CAT
c
Fig. 1. Linear representation of the EBV B95-8 SamHI digested genome and of the piasmid pC1.3CAT A, Sequence organization of the EBV B95-8 SamHI digested genome. B, Enlargement of the Ava\ restriction fragment. The location of the GRE sequence {black block) and of ORF BCRF1 are indicated. C, Detailed linear representation of the piasmid pC1.3CAT containing the Ava\ fragment inserted in front of the CAT coding region controlled by the TK promoter. Restriction sites abbreviations: S = Sail; A = Aval.
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269
Glucocorticoid Action on Epstein-Barr Virus Genome
CAT inductibility in the presence of DXM. Moreover, every induction observed required the presence of functional glucocorticoid receptors and was at least inhibited to 70% by simultaneous addition of the antiglucocorticoid RU486.
DISCUSSION
Fig. 2. Effect of DXM on the pC1.3CAT construct in Daudi
cells. CAT assays were performed essentially as described in Materials and Methods Lane 1, Extract of cells transfected with pC1.3CAT in absence of steroid; lane 2, extract of cells transfected with pC1.3CAT in presence of 3 IXM DXM; lane 3, extract of cells transfected with pC1.3CAT in presence of 3 ^M DXM and 3 ^M RU486; lane 4, extract of cells transfected with pC1.3CAT in presence of 3 HM RU486.
the TK promoter (Fig. 3B) and the induction measured was about three times over the induction rate obtained with the pC1.3CAT construct. One of the hexanucleotide —TGTYCT— found in the EBV genome was included in a DNA sequence forming an imperfect repeat showing a 85% homology with the GRE sequence. To define the contribution of this sequence —AGCACATTTTGTTCT— to the induction ratio observed with the total GRE zone, we carried out a construction pGREEBVCAT by inserting a 27 bp long oligonucleotide immediately upstream of the TK promoter. By transfecting HeLa cells with pGRE • EBVCAT, we measured also an equally 28-fold DXM induction. These experiments showed that the GRE region found in the EBV genome was able to confer DXM inducibility through the heterologous TK promoter in EBV negative nonlymphoma cells, such as HeLa cells. We also determined that the minimal GRE sequence —AGCACATTTTGTTCT— was able to confer maximal
In various systems, including viral models, glucocorticoids were found to regulate specific gene expression. The comparison of all the sequences involved in the glucocorticoid action led to the determination of a consensus GRE sequence —GNNACAANNTGTYCT— essential for the relaying of the hormonal message (18). A computer search in the EBV genome revealed a similar sequence in the C fragment of the BamH\ digested EBV B95-8 genome. This sequence had 85% homology with the consensus GRE. Moreover, we found three times the highly conserved motif — TGTTCT— in surrounding sequences. A restriction fragment spanning nucleotide 9104 to nucleotide 10439, including this putative GRE region, linked to the heterologous promoter TK (plasmid pC1.3CAT) conferred DXM responsiveness to the TK-CAT chimeric gene in Daudi cells. GRE are known to act as hormonal regulated enhancers and confer gene induction without regard to the orientation and the distance separating them from the target promoters (21, 24). In the pC1.3CAT, the GRE is located downstream of the ORF BCRF1 promoter and upstream of the TK promoter. A priory, one might think that this GRE would regulate these two independent promoters; however, glucocorticoids were found to act only through the TK promoter by inducing the transcription of the CAT gene in this construct (data not shown). With regard to the EBV cell specificity, restricted to B lymphoid cells and to poorly differentiated epithelial cells, it was interesting to test whether the glucocorticoid CAT induction observed in EBV positive lymphoma cells, such as Daudi cells, also occurs in other cell lines. The pC1.3CAT construct was therefore transfected in HeLa cells. These cells, known to be EBV negative, contain a low level of endogenous glucocorticoid receptors. In order to study the glucocorticoid responsiveness in these cells, it was necessary to transfect the CAT reporter genes together with a glucocorticoid receptor expressing vector, HG1. After DXM treatment, we measured a strong inducibility of the CAT expression in transfected HeLa cells. Maximal CAT induction was already obtained at a 5.10"8 M DXM concentration; this is in agreement with the saturation concentration obtained for other GREs (25, 26). However, in Daudi cells, the amount allowing a maximal effect was about 3.10~6 M. The difference may be relevant to the steroid metabolism occurring in lymphoma cells, which contain more hydroxylases than HeLa cells. Cotransfection with a glucocorticoid receptor expressing vector was essential to obtain hormonal induction; moreover, the CAT
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 November 2015. at 23:46 For personal use only. No other uses without permission. . All rights reserved.
Vol 5 No. 2
MOL ENDO-1991 270
pC 1J CAT
ORF BCRF1 "*1
GRE IB -1335
ITK [ V / ^ C J M ' V V / V
lp.l
A
TK r x ' x ' x O&T N ' N ' s ' N '*'
pC 1.0 CAT •364
•1335
pC
