©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Moloney Leukemia Retroviral Long Terminal Repeat trans-Activates AP-1-inducible Genes and AP-1 Transcription Factor Binding (*)

Haiqin Weng , Sang-Yun Choi , Douglas V. Faller (§)

From the (1) Cancer Research Center and the Departments of Medicine, Biochemistry, Pediatrics, Microbiology, and Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Moloney murine leukemia virus (Mo-MuLV) is a thymotropic and leukemogenic retrovirus which causes T lymphomas. The long terminal repeat (LTR) of Mo-MuLV affects the regulation of a number of cellular genes, including collagenase IV, monocyte chemoattractant protein-1, and c-jun genes, all of which contain 12-O-tetradecanoylphorbol-13-acetate-responsive ele-ment consensus sites within their promoters. We report here that Mo-MuLV stimulates the collagenase IV gene through transcription factor AP-1, and that the expression of a subgenomic portion of Mo-MuLV LTR alone is sufficient for this effect. Transient or stable expression of the viral LTR increases cellular AP-1 DNA binding activity. The collagenase IV 12-O-tetradecanoylphorbol-13-acetate-responsive element consensus sequence was shown to be required for this trans-activation. Deletions or mutations of this consensus site which abolished AP-1 binding also abolished trans-activation by the LTR. Transient or stable transfection of the viral LTR into cells stimulated c-jun gene expression, suggesting one mechanism whereby the viral LTR may induce cellular AP-1 activity. Thus, the Mo-MuLV LTR, through activation of the transcription factor AP-1, is capable of regulating cellular gene expression, including the induction of proto-oncogenes. This activity may be relevant to the mechanisms whereby retroviruses which do not contain oncogenes induce neoplasia.


INTRODUCTION

Moloney murine leukemia virus (Mo-MuLV)() is a type C leukemogenic retrovirus which causes T lymphocyte leukemia or lymphoma in mice over a period of months. The mechanism whereby Mo-MuLV causes T lymphocyte leukemia or lymphoma after this long latent period is not well understood. Because the leukemia viruses do not encode oncogenes which can directly transform cells, one potential mechanism of MuLV leukemogenesis may be through transcriptional activation of cellular genes, including proto-oncogenes. We have previously reported that infection of murine fibroblasts or human T lymphocyte lines with Mo-MuLV results in up-regulation of a number of genes encoding cell surface antigens and T cell activation antigens, including CD2, CD3, CD4, the T-cell receptor chain and MHC class I antigen expression (1-4). The ability of Mo-MuLV to trans-activate cellular genes is determined by a noncoding region of the genome, the long terminal repeat (LTR), and is localized to a region of the LTR which has been shown to be important for pathogenicity (5, 6, 7, 8, 9) . Stable transfection of cells with a single-copy retroviral LTR trans-activated specific endogenous cellular genes and specific transiently expressed reporter genes (3), eliminating the possibility that trans-activation is due to titration of a putative negative regulatory factor by multiple copies of the LTR DNA. The trans-activation of specific cellular genes by the Mo-MuLV LTR is independent of the physical location of either element, occurring regardless of whether the LTR is integrated (endogenous) or transiently expressed (exogenous), or whether the responsive cellular gene is integrated or transiently expressed.

The LTR sequences of murine leukemia viruses are not known to encode trans-acting viral functions, yet have been implicated in the etiology and progression of retroviral diseases (5, 6, 7, 8, 9) . The MCF-MuLV LTR, for example, is an important contributor to viral leukemogenicity in AKR mice (11) . The U3 region of BL/VL3 viral LTR has the ability to induce cellular suppressor factors, and generate intracellular immunity against itself and other MuLVs (9) . Deletions within the LTR alter disease specificity (7) , and analysis of the SL3-3 AKV recombinant provirus showed the determinant of leukemogenicity to lie within the SL3-3 LTR (12) .

We report here that infection of Balb or HeLa cells with Mo-MuLV, or transfection with vectors expressing regions of the viral LTR, results in induction of transcription factor AP-1 activity, as assayed on electrophoretic mobility shift assays (EMSA). This activation of AP-1 results in increases in transcription of a number of cellular genes, including collagenase IV, monocyte chemoattractant protein-1 (MCP-1, also called JE), and c-jun, all of which contain TRE (TPA responsive element) consensus sequences in their promoters. Subgenomic portions of the murine leukemia virus containing the LTR are sufficient to produce activation of transcription factor AP-1. The transient co-transfection of an LTR-expressing vector together with chimeric genes containing a collagenase promoter with an AP-1 binding consensus sequence driving the reporter gene CAT results in increases of CAT activity. Site-specific mutagenesis of this TRE binding site prevents binding of the LTR-induced complex and induction of the reporter gene. EMSAs performed in the presence of anti-Jun or anti-Fos Family antibodies demonstrate that the AP-1 complex induced by Mo-MuLV consists of Jun/Fos heterodimers.


MATERIALS AND METHODS

Cell Culture and Reagents

Balb/c-3T3 cells and HeLa cells were obtained from American Type Culture Collection. The mouse fibroblast Balb/c-3T3 cell line and the Moloney MuLV-infected Balb/c-3T3 cell line MoBalb (13) were carried in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated donor bovine calf serum (Sigma), 2 mML-glutamine, 100 units of penicillin/ml, 100 mg of streptomycin/ml. The human cervical carcinoma cell line HeLa was grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated newborn calf serum (Sigma). HeLa cells stably infected with the pZip-Neo-SV(X) vector were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated newborn calf serum, 0.5 mg of Geneticin/ml, and glutamine plus antibiotics as above. Geneticin and TPA were obtained from Sigma. Poly(dI-dC), acetyl-CoA, staurosporine, and okadaic acid were obtained from Sigma. [C]Chloramphenicol was obtained from DuPont NEN.

RNA Blot Analysis

Total cellular RNA was isolated by guanidine thiocyanate/phenol RNA extraction (13) , quantified, separated by electrophoresis on formaldehyde-agarose gels, and transferred to BioBlot-supported nitrocellulose (Costar, Cambridge, MA). Hybridizations were carried out at 42 °C in 50% formamide for 18 h. The filters were washed sequentially in 2 SSC, 0.5% SDS and in 0.2 SSC, 0.1% SDS, at room temperature and at 42 °C, respectively. Autoradiograms were scanned for quantitation, using an LKB laser densitometer (Pharmacia Biotech Inc.). [-P]-Labeled DNA Probes-Purified fragments of c-jun, c-fos, MCP-1, collagenase IV, and -actin cDNA were radiolabeled with [-P]dCTP using random primer synthesis (14) . Murine -actin, c-fos, and MCP-1 (JE) DNA probes have been described previously (15) . The c-jun probe was a 1.7-kb PstI fragment of murine c-jun, The c-fos fragment was a 1.1-kb EcoRI/SalI fragment of murine c-fos, and the MCP-1 probe was a 0.75-kb fragment of murine MCP-1 (JE). The collagenase IV probe was a 2.2-kb EcoRI fragment purchased from ATCC, and the actin probe was a 0.7-kb PstI fragment of murine -actin. The collagenase IV RNA probe was made from pGEM T7 promoter using T7 RNA polymerase (Promega).

Transfection and Infection of Cells

Transient transfections were performed using the DEAE-dextran method (16) . Cells were seeded in 60-mm dishes at 3 10 the day before transfection. Five µg of CAT-containing reporter plasmids were transfected together with 5 µg of LTR vector or control plasmid. 48 h after transfection, cells were stimulated with TPA at 25 ng/ml, and harvested for CAT assay 2 h later. Total cell extracts were made by the freeze-thaw method and the supernatants were used for CAT assay. The CAT activity was quantitated in extracts as described (17) . Co-transfection with the pRSV--galactosidase plasmid (0.5 µg/transfection) and cytofluorometric analysis of -galactosidase activity were performed to normalize for the transfection efficiency, as described (3) . pZip-Neo-SV(X) is a vector developed and provided by C. Cepko (Harvard Medical School, Boston, MA), which contains the 5` LTR of Mo-MuLV and 5` genomic sequences up to base 739 (using the nucleotide numbering system of Shinnick (18) ), the 3` region of Mo-MuLV from bases 7200 and including the 3` LTR, and also Mo-MuLV bases from 5407 to 5766. In addition, this vector also contains the prokaryotic gene from transposon Tn5, which confers resistance to neomycin, the pBR322 origin of replication, and the SV40 origin of replication. For stable transfections, the retroviral vector pZip-Neo-SV(X) was transfected into a canine cell amphotropic packaging line (DAMP) and filtered supernatants containing packaged vector were used to infect Balb/c3T3 and HeLa cells. Neomycin-resistant colonies were selected and cloned.

LTR Expression Vectors, CAT Reporter Genes, and PCR Mutagenesis

LTR-containing vectors pML4, pXF-MoA, and pGUXDP have been previously described and are shown in Fig. 1. pGUXDP has a deletion of 27 (nucleotide 96-122) and 75 base pairs (nucleotide 145-219) within the LTR U3 region (see Fig. 1). The vector pCollCAT-73 was the generous gift of P. Angel (19) . The MCP-1 promoter-CAT genes were the generous gift of A. J. van der Eb (20) . The mutation of the AP-1-binding site in pCollCAT-73 to create the plasmid pmutcollCAT-73 was performed using two synthetic oligonucleotides of 18 bases in length, encompassing the HindIII site on pCollCAT-73 to the XbaI site at +74. The 5` primer contained a complete HindIII site, and the 3` primer contained a complete XbaI site. The fragment containing the mutation was generated by synthesizing two overlapping fragments, denaturing and then annealing. Complementary strands were synthesized from the overhangs of the two annealed fragments, and ligation and transformation of bacteria were carried out. The mutation was confirmed by chain termination DNA sequencing (21) .


Figure 1: Schematic drawings of the reporter genes and the LTR expression vectors. pCollCAT-73 contains an intact AP-1 site; pmutcollCAT-73 contains a mutated AP-1 site with a mutation of TGAGTCA to TGAGTAC; pCollCAT-71 (not shown) contains a deletion of the first two bases of the AP-1 binding site (TGAGTCA). Also depicted are the various vectors derived from the Mo-MuLV provirus, including pML4, pXFMoA, pMoV9, and pGUXDP (see Ref. 3 for description). The open bar represents the LTR of Mo-MuLV and its flanking proviral sequences, and the thin bar the adjacent cellular sequences.



Electrophoretic Mobility Shift Assay

HeLa cell nuclear extracts were isolated by the method of Andrews and Faller (22) . Binding reactions were performed for 20 min on ice with 10 µg of total nuclear protein in 20 µl of 10 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 10% glycerol, 3 µg of poly(dI-dC), 1 mM dithiothreitol, 1 nM phenylmethylsulfonyl fluoride, and 20,000 cpm of P-labeled oligonucleotide labeled with T4 kinase (Promega). The DNA-protein complexes were separated from the unbound probe on a native 5% polyacrylamide gel. The gels were vacuum-dried and exposed to Kodak X-AR film for 18 h. The sequence of the AP-1 consensus binding site oligonucleotide was 5`-CGCTTGATGAGTCAGCCGGAA-3` and 3`-GCGAACTACTCAGTCGGCCTT-5`. The transversion mutant oligonucleotide used for competition experiments was 5`-CGCTTGATGAGTACGCCGGAA-3` and 3`-GCGAACTACTCATGCGGCCTT-5`. The antibodies used in the EMSA, polyclonal rabbit antisera against all Jun family members, JunB and JunD, and polyclonal rabbit antibody against c-Fos, were purchased from Santa Cruz Biotech (Santa Cruz, CA) or were the generous gifts of Dr. Thomas Rothstein.


RESULTS

We have shown that stable infection of Balb/c-3T3 cells with Mo-MuLV (MoBalb cells) results in the induction of transcripts for specific cellular genes, including MCP-1 and collagenase IV, and an increase in their respective protein products (3).() MCP-1 is known to be regulated by the transcription factor AP-1. The expression of two other genes also inducible by AP-1, collagenase IV, and c-jun, were also examined in MoBalb cells. RNA blot analysis demonstrated that transcript levels for collagenase IV were elevated 13-fold and transcript levels for c-jun were elevated 15-fold in cells containing the Mo-MuLV genome (not shown in Fig. 2 ). We have previously shown that the entire leukemia virus is not required for trans-activation of cellular genes, including MCP-1 (3) . In order to examine whether the Mo-MuLV LTR alone could activate collagenase IV and MCP-1, the packaged defective retroviral vector pZip-Neo-SV(X), which contains two LTRs but no structural viral sequences, was used to infect Balb cells. After neomycin selection, the resulting cells had two copies of the Mo-MuLV LTR stably integrated in their genomes. The expression of collagenase IV mRNA was elevated 14-fold in Balb/c-3T3 cells with integrated Mo-MuLV LTRs, compared to the expression of the control gene -actin. Expression of MCP-1 mRNA was elevated 5-fold by the presence of the Mo-MuLV LTR (Fig. 2). Stable expression of the Mo-MuLV LTR into Balb cells resulted in over 60-fold increases in c-jun mRNA (Fig. 3). Induction of specific cellular gene transcripts by the Mo-MuLV LTR was not restricted to murine cells. Integration of the Mo-MuLV LTR into HeLa cells resulted in a 14.7-fold increase in c-jun mRNA levels (Fig. 3). In contrast, the levels of other gene transcripts, including c-fos, did not increase significantly in response to the Mo-MuLV LTR.


Figure 2: Analysis of MCP-1 and collagenase IV transcripts in Balb cells expressing Mo-MuLV or Mo-MuLV LTRs. A, total cellular RNA (20 µg) was obtained from Balb cells or Balb cells infected with Mo-MuLV. Lane 1, Balb cells; lane 2, Balb cells infected with Mo-MuLV. B, total cellular RNA (20 µg) was obtained from Balb cells or Balb cells stably infected with pZip-Neo-SV(X). RNA was separated on a formaldehyde-agarose gel, transferred to a nitrocellulose filter, and hybridized with labeled collagenase IV, MCP-1, or -actin probes. Lane 1, Balb cells; lane 2, Balb cells expressing pZip-Neo-SV(X). The figures are autoradiograms. The migration positions of the collagenase IV, -actin, and MCP-1 transcripts are indicated.




Figure 3: RNA blot analysis of c-jun and c-fos transcripts in Balb and HeLa cells expressing the Mo-MuLV LTR. Total cellular RNA (20 µg) from Balb cells or HeLa cells untreated or stably infected with pZip-Neo-SV(X) was separated on a formaldehyde-agarose gel, transferred to a nitrocellulose filter, and hybridized with labeled c-jun or c-fos probes, then stripped and reprobed for -actin. Lane 1, RNA from control Balb cells; lane 2, RNA from Balb cells containing an integrated Mo-MuLV LTR; lane 3, RNA from control HeLa cells; lane 4, RNA from HeLa cells containing an integrated Mo-MuLV LTR. The figure is an autoradiogram. The positions of the c-jun, c-fos, and -actin transcripts are indicated.



To better study the effect of the Mo-MuLV LTR on trans-activation of cellular gene promoters, a number of plasmids containing the Mo-MuLV provirus (pMoV9), the Mo-MuLV LTR (pXF-MoA, pA5, pML4), or portions of the LTR (pGUXDP) (Fig. 1) were introduced into Balb/c-3T3 cells in combination with reporter gene constructs. Chimeric plasmids containing 5`-flanking (regulatory) regions of the human collagenase IV gene or the MCP-1 gene ligated the chloramphenicol transferase (CAT) gene were used as reporters for LTR trans-activation. Plasmid pCollCAT-73 contains the 7-base pair (-73 to -67) TPA-responsive element (TRE), which constitutes a binding site for transcription factor AP-1 (19, 23) . The plasmid pMCPCAT-2600 contains 2500 base pairs of 5`-regulatory sequence, including two putative AP-1 binding sites. The plasmid containing the provirus pMoV9 and the CAT reporter plasmids pCollCAT-73 or pMCPCAT-2600 were co-transfected into Balb/c-3T3 cells. The co-transfection of pMoV9 and pCollCAT-73 resulted in nearly 50-fold trans-activation of the CAT activity, and the co-transfection of pMoV9 and pMCPCAT-2600 resulted in 33-fold trans-activation of CAT activity (Fig. 4).


Figure 4: Induction of the collagenase IV and the MCP-1 gene promoters by pMoV9 in transient expression assays. Balb cells were transiently transfected with pCollCAT-73 together with a control plasmid pBR322 (lane 1), or with the plasmid pMoV9, containing the Mo-MuLV provirus (lane 2). Balb cells were transiently transfected with pMCPCAT-2600 together with a control plasmid pBR322 (lane 3), or with the plasmid pMoV9 (lane 4). Cells were harvested and extracts were assayed for CAT activity. The products were separated by thin layer chromatography, and an autoradiogram of the plate is shown here. The migration positions of chloramphenicol (Cam) and the acetylated products (AcCam) are indicated.



The viral LTR alone was as potent as the entire proviral genome in inducing trans-activation of the collagenase promoter in transient expression assays. In experiments using pCollCAT-73 as reporter gene, CAT activity was increased over 10-fold when the plasmid pML4, containing a single LTR, was co-transfected (Fig. 5A). Reporter genes with mutations in the AP-1 binding site were employed to determine if this binding site was required for trans-activation by the viral LTR. The vectors pCollCAT-71, in which the first two bases of the TRE consensus site are deleted, and pmutcollCAT-73, which contains a transversion of the last two bases of the binding site (CA to AC) were used as reporter genes in co-transfections with the vector pML4. Co-transfection of the viral LTR did not result in trans-activation of CAT activity from these TRE-mutant promoters (Fig. 5, A and B).


Figure 5: Regulation of collagenase IV promoter mutants by a co-transfected Mo-MuLV LTR. A, Balb cells were transiently transfected with pCollCAT-60 alone (lane 1), or pCollCAT-60 together with pML4 (lane 2), with pCollCAT-71 alone (lane 3), or pCollCAT-71 together with pML4 (lane 4), with pCollCAT-73 alone (lane 5), or pCollCAT-73 together with pML4 (lane 6) for 48 h prior to harvesting and assay for CAT activity. B, Balb cells were transiently transfected with pCollCAT-73 alone (lane 1), or pCollCAT-73 together with pXFMoA (lane 2), with pmutcollCAT73 alone (lane 3), or pmutcollCAT73 together with pXFMoA (lane 4) for 48 h prior to harvesting and assay for CAT activity. The products were separated by thin layer chromatography, and autoradiograms of the plates are shown here. The migration positions of chloramphenicol (Cam) and the acetylated products (AcCam) are indicated.



These experiments demonstrated that the TRE consensus site, which serves as binding site for transcription factor AP-1, was required for trans-activation by the LTR. To determine whether the presence of the LTR increased AP-1 binding activity in the cell, EMSAs for AP-1 DNA binding activity were performed using proteins from HeLa cell nuclear extracts incubated with a radiolabeled TRE consensus oligo. HeLa cell lines stably infected with a deleted retroviral vector containing two Mo-MuLV LTRs were also used for preparation of nuclear proteins. Cellular transcription factor AP-1 DNA binding activity was greatly augmented in the cells containing stably integrated Mo-MuLV LTRs (Fig. 6, lanes 1 and 2). To demonstrate that this DNA binding activity to the TRE consensus oligo was specific, a 100-fold excess of unlabeled TRE consensus or mutant oligo was incubated with the nuclear extracts. The wild-type AP-1 binding site-containing oligo competed efficiently for binding activity (lanes 3 and 6), while the same amount of unlabeled mutated TRE-site oligo (CA to AC transversion) could not compete away the binding (lanes 4 and 7). As a control, TPA, a known activator of AP-1 DNA binding activity, was used to stimulate the HeLa cells (lane 5). The DNA binding complexes stimulated by both TPA and Mo-MuLV LTR showed similar patterns on EMSA, both complexes could be competed away by unlabeled wild-type AP-1 oligos, and neither of the complexes were competed away by incubation with the cold TRE-mutant oligo containing the CA to AC transversion (lanes 4 and 7).


Figure 6: Induction of AP-1 activity by the Mo-MuLV LTR. A radiolabeled oligonucleotide containing a TRE consensus sequence was incubated with nuclear extracts made from: control HeLa cells (lane 1); HeLa cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)) (lane 2); HeLa cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)) with an excess of cold TRE oligo added during the binding reaction (lane 3); HeLa cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)) with an excess of cold mutant TRE oligo (CA to AC in the consensus sequence) added during the binding reaction (lane 4); HeLa cells treated with 20 nM TPA for 4 h (lane 5); HeLa cells treated with TPA, with an excess of cold TRE oligo added during the binding reaction (lane 6); HeLa cells treated with TPA, with an excess of cold mutant TRE oligo (CA to AC in the consensus sequence) added during the binding reaction (lane 7). The complexes were separated and analyzed as described. The figure is an autoradiogram. The positions of the AP-1 complex and the free probe are indicated.



Because c-jun transcription was strongly induced by the LTR in the absence of increases in c-fos transcription, the components of the AP-1 complex induced by the LTR was investigated. Antibodies against Fos and Jun were preincubated with the nuclear extracts to examine whether the binding of the AP-1 complex in the nuclear extracts to the TRE oligo could be interfered with by the presence of the antibodies. The LTR-induced AP-1 DNA binding to the TRE probe was almost completely inhibited by the anti-pan-Jun antibody, while preincubation of the nuclear extracts with anti-JunB or anti-JunD did not result in any interference with AP-1 binding to the TRE-specific probe, suggesting that the LTR-induced AP-1 DNA binding complex contained c-Jun protein (Fig. 7, A and B). The ability of the anti-JunB or anti-JunD antibodies to interfere with the ability of their respective ligands to participate in AP-1 complex formation was demonstrated by their inhibition of AP-1 complex formation induced by TPA treatment (Fig. 7B). Preincubation of a polyclonal antibody against c-Fos with nuclear extracts from both HeLa cells treated with TPA and HeLa cells containing the stably integrated Mo-MuLV LTR induced a supershift on EMSA, indicating that c-Fos was also a component of the LTR-induced DNA binding complex (Fig. 7C).


Figure 7: Identification of the components of the AP-1 complex induced by the LTR. A, a radiolabeled oligonucleotide containing a TRE consensus sequence was incubated with nuclear extracts made from: HeLa cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)) (lane 1); control HeLa cells (lane 2); HeLa cells treated with 20 nM TPA for 4 h (lane 3); HeLa cells treated with 20 nM TPA for 4 h, with 1 µg of anti-pan-Jun antibody added for 15 min prior to the binding reaction (lane 4); HeLa cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)), with 1 µg of anti-pan-Jun antibody added for 15 min prior to the binding reaction (lane 5). B, a radiolabeled oligonucleotide containing a TRE consensus sequence was incubated with nuclear extracts made from: HeLa cells treated with 20 nM TPA for 4 h (lane 1); HeLa cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)) (lane 2); HeLa cells treated with 20 nM TPA for 4 h with 1 µg of anti-JunB antibody added during the binding reaction (lane 3); HeLa cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)) with 1 µg of anti-JunB antibody added during the binding reaction (lane 4); HeLa cells treated with 20 nM TPA for 4 h with 1 µg of anti-JunD antibody added during the binding reaction (lane 5); HeLa cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)) with 1 µg of anti-JunD antibody added during the binding reaction (lane 6). Quantitation of AP-1 complexes by densitometer disclosed that whereas the presence of anti-JunB and anti-JunD antibodies decreased TPA-induced complex formation by 43%, there was no such inhibitory effect of the antibodies on the AP-1 complexes induced by the Mo-MuLV-LTR. C, a radiolabeled oligonucleotide containing a TRE consensus sequence was incubated with nuclear extracts made from: HeLa cells treated with 20 nM TPA for 4 h (lane 1); control HeLa cells (lane 2); HeLa cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)) (lane 3); HeLa cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)), with 1 µg of anti-c-Fos antibody added for 15 min prior to the binding reaction (lane 4); HeLa cells treated with 20 nM TPA for 4 h, with 1 µg of anti-c-Fos antibody added for 15 min prior to the binding reaction (lane 5). The complexes were separated and analyzed as described. The figures are autoradiograms. The positions of the AP-1 complex and the free probe are indicated.



We have previously demonstrated that mutations of the LTR which prevent the generation of a polymerase III transcript from the LTR render it incapable of trans-activation of cellular genes (3). To determine the specificity of the induction of AP-1 activity by the Mo-MuLV LTR, wild-type and mutant LTRs were co-transfected into HeLa cells. Nuclear extracts were made from these transient transfectants and then tested for their ability to enhance AP-1 DNA binding activity. pXF-MoA, which contains the wild-type LTR, has been shown to strongly activate a co-transfected pCollCAT-73 vector (3). Plasmid pGUXDP, which contains an LTR with a mutation within the U3 region and does not generate a transcript, has been shown to have no trans-activating ability for MHC or MCP-1 genes (3). Nuclear extract made from pXF-MoA-transfected HeLa cells showed a 5-fold induction of AP-1 DNA binding activity, in comparison to extracts from control HeLa cells (Fig. 8). Nuclear extracts made from pGUXDP transfectants, however, demonstrated no trans-activation of AP-1 DNA binding activity. Thus, the trans-activation ability of the LTR correlates with the ability to activate AP-1 DNA binding activity.


Figure 8: Induction of AP-1 activity by vectors containing deleted LTRs. A radiolabeled oligonucleotide containing a TRE consensus sequence was incubated with nuclear extracts made from: control HeLa cells (lane 1); HeLa cells transiently transfected with pGUXDP (lane 2); HeLa cells transiently transfected with pXFMoA (lane 3); HeLa cells treated with 20 nM TPA for 4 h (lane 4). The complexes were separated and analyzed as described. The figures are autoradiograms. The positions of the AP-1 complex and the free probe are indicated.



The expression of integrated, cellular AP-1 inducible genes could be induced by even transient expression of the Mo-MuLV LTR. Collagenase IV mRNA was elevated 8-fold in Balb cells transiently transfected with pMoV9, compared to cells transfected with a control plasmid not containing an LTR. The expression of the -actin gene was unaffected (Fig. 9, lanes 1 and 2). As a control, treatment with TPA was also used to stimulate collagenase IV mRNA expression (Fig. 9, lane 3). These findings demonstrate again that induction of the expression of specific cellular genes by the MoMuLV LTR is independent of the location of either the LTR or the responsive gene.


Figure 9: RNA blot analysis of collagenase IV transcripts in Balb cells transiently transfected with pMoV9. Total cellular RNA (20 µg) from Balb cells or Balb cells transiently transfected pMoV9 were separated on a agarose gel, transferred to a nitrocellulose filter, and hybridized with a radiolabeled collagenase RNA probe and subsequently with a probe for -actin. Lane 1, Balb cells; lane 2, Balb cells transiently transfected with pMoV9; lane 3, Balb cells stimulated with TPA for 2 h. The figures are autoradiograms. The migration positions of the collagenase IV, -actin, and MCP-1 transcripts are indicated.



The protein kinase C inhibitor staurosporine and the protein serine/threonine phosphatase inhibitor okadaic acid have been reported to affect TPA induction of AP-1 DNA binding activity. To study the effect of staurosporine or okadaic acid on LTR induction of AP-1 activity, electrophoretic mobility shift assays for AP-1 binding activity were performed using HeLa cell nuclear extract incubated with a radiolabeled AP-1 consensus oligo. HeLa cell lines containing a stably integrated Mo-MuLV LTR (pZip-Neo-SV(X)) were also used for preparation of nuclear proteins. As expected, AP-1 DNA binding activity was greatly augmented in the cells containing an integrated Mo-MuLV LTR. After treatment with staurosporine for 6 h, however, the induction of AP-1 DNA binding activity by the LTR was reduced by 50% (Fig. 10A, lanes 2 and 3). Basal expression of the AP-1 complex in control cells not expressing the LTR was not affected by staurosporine (not shown). The TPA induction of AP-1 DNA binding activity was also reduced by 50% after the same treatment with staurosporine (Fig. 10A, lanes 4 and 5). In contrast, the protein phosphatase inhibitor okadaic acid increased basal expression and the enhanced induction of AP-1 DNA binding activity by the Mo-MuLV LTR (Fig. 10B, lanes 1 and 2).


Figure 10: The effect of protein kinase C inhibitors and serine/threonine phosphatase inhibitors on Mo-MuLV LTR induction of AP-1 activity. A, a radiolabeled oligonucleotide containing a TRE consensus sequence was incubated with nuclear extracts made from: control HeLa cells (lane 1); HeLa cells with a stably integrated Mo-MuLV LTR (pZip-Neo-SV(X)) (lane 2); HeLa cells with a stably integrated Mo-MuLV LTR treated with staurosporine at 0.1 µM for 6 h (lane 3); HeLa cells stimulated with TPA for 6 h (lane 4); HeLa cells stimulated with TPA and treated with staurosporine at 0.1 µM for 6 h (lane 5). B, HeLa cells (lane 1); HeLa cells were treated with okadaic acid at 0.1 µM for 6 h (lane 2); HeLa cells with a stably integrated Mo-MuLV LTR (pZip-Neo-SV(X)) (lane 3); HeLa cells with stably integrated Mo-MuLV LTR treated with okadaic acid at 0.1 µM for 6 h (lane 4). The complexes were separated and analyzed as described. The figures are autoradiograms. The positions of the AP-1 complex and the free probe are indicated.




DISCUSSION

We have previously demonstrated that infection of human or murine cells with murine leukemia viruses rapidly increases the transcription and expression of a number of genes belonging to the immunoglobulin superfamily and involved in T-lymphocyte activation, including the Class I MHC antigens (1, 2, 3, 4) . More recently, we have reported that the LTR of Moloney murine leukemia virus encodes a trans-activator which induces transcription and expression of MHC Class I genes, T cell activation antigens, and certain cytokine genes. The portion of the LTR responsible for trans-activation was mapped by deletions to lie within the U3 region (3) . We have also demonstrated that a transcript is initiated by RNA polymerase III within the U3 region, and its presence correlates with the trans-activating activity. The studies reported herein confirm and extend these findings to another group of cellular genes, and define a transcription complex and cis-acting promoter element required for this trans-activation. Trans-activation of the distinct set of 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible genes by the Mo-MuLV LTR reported here is independent of the physical location of the genes or of the LTR, occurring whether the LTR is integrated in the genome or transiently expressed, and whether the responsive cellular genes or gene promoters are integrated or transiently expressed.

The transcriptional response to TPA is mediated in many cases by the AP-1 transcription factor, which recognizes and binds to a specific DNA sequence motif that functions as cis-acting promoter element (23). Induction of AP-1 activity by the viral LTR was demonstrated during transient or stable expression of the LTR. We have also shown that the ability of a mutated LTR to trans-activate AP-1-dependent CAT activity correlates both with the ability of that LTR to generate a transcript, and with the ability to activate cellular AP-1 DNA binding activity. The mutant LTR contained in the vector pGUXDP is incapable of generating the polymerase III-directed transcript previously demonstrated to be necessary for trans-activation of a number of cellular genes. When pGUXDP was co-transfected with a collagenase promoter-CAT vector, both the trans-activation of CAT activity and the activation of cellular AP-1 DNA binding activity were minimal compared to the effects of the wild-type LTR (e.g. pXFMoA).

The transcriptional activation of endogenous cellular genes by the LTR of Mo-MuLV represents a novel way in which retroviruses can control the expression of genes in the cells they infect. The mechanisms through which this activation occurs are beginning to be elucidated. Our finding that a number of TPA-inducible genes, including MCP-1, collagenase IV, and c-jun,are induced in response to the viral LTR led us to determine which cis-acting elements of these gene promoters are required for induction by the viral LTR. The promoter region of MCP-1 harbors the sequence TGAGTCC, which resembles the consensus site for AP-1 binding (TPA-response element, TRE), and TGAGTCA, which is also present in the collagenase IV gene promoter. The c-jun gene promoter also contains the sequence TGAGTCA, raising the possibility that the c-jun gene can be positively autoregulated by AP-1 through its TRE site (19) . Trans-activation of the collagenase IV gene by TPA and by the LTR both required a TRE site in the promoter. The Mo-MuLV LTR trans-activated a collagenase IV promoter-CAT construction which contained an intact TRE site, but the level of trans-activation by the LTR was significantly lower for the same promoter-CAT vector with either a 2-base pair deletion or 2-base pair transversion of the AP-1 binding site. These same mutations were shown in EMSAs to be unable to bind TPA- or LTR-induced AP-1 complexes. Therefore, an intact TRE site apparently is required for the trans-activation of certain genes by the viral LTR. Gel-shift assays using a TRE consensus oligo supported the hypothesis that the viral LTR could activate an AP-1 complex which bound to the TRE site. The DNA binding activity induced by the integrated or transiently expressed LTR in cells also appeared identical in its binding site sequence requirements and mobility to the AP-1 complexes induced by TPA treatment.

The activity of the transcription factor AP-1 is modulated at least four levels (24, 25, 26) : 1) the regulation of the levels or location of c-Fos or c-Jun (27) ; 2) the regulation of the DNA binding activity of the transcription factor (28, 29) ; 3) the modulation of the trans-activating potential of c-Jun (30, 31, 32, 33) , through post-translational modulation of the protein; and 4) through regulation of dimerization (34) .

Expression of the viral LTR elevated the level of AP-1 DNA binding activity coincidentally with a marked increase in the expression of c-jun transcripts. In contrast to c-jun mRNA, increases in c-fos transcripts were minimal. c-jun is also an AP-1-inducible gene, so its transcriptional up-regulation may be the result, rather than the cause, of AP-1 activation by the LTR (35, 36) . It was therefore conceivable that the AP-1 activity induced by the LTR might consist predominantly of Jun/Jun homodimers rather than the Fos family/Jun family heterodimers induced by TPA. The use of antibodies specific for the components of AP-1 in EMSA demonstrated, however, that both Jun and Fos family members were present in the AP-1 complexes induced by the LTR. It is possible that the basal cellular level of pre-existing c-Fos proteins is greater than c-Jun proteins, so that the sudden increase in c-jun driven by the LTR enables the formation of more c-Jun/c-Fos heterodimers, but this remains to be determined. Changes in intracellular redox potentials may regulate AP-1 activity (37, 38, 39) . Reducing agents like pyrrolidine dithiocarbamate and N-acetylcysteine activate AP-1 activity in HeLa and Balb cells, and the presence of the LTR does not result in a further increase in AP-1 activity,() but we have no further evidence that redox mechanisms regulate transcription of AP-1 inducible genes or the formation of AP-1 binding complexes by the viral LTR.

The dichotomy between the strong induction of c-jun transcription and lack of c-fos induction by the viral LTR may provide some information regarding mechanisms of action of the LTR. Both the c-fos and c-jun genes are induced by exposure to phorbol ester, but the cis-acting elements controlling this response are distinct. The AP-1 complex appears to bind directly to c-jun promoter, but phorbol esters may act indirectly on the c-fos promoter, through the serum response element. While c-jun transcription is rapidly induced by many stimuli, its pattern of expression is not identical to c-fos. For example, ultraviolet (UV) light is a very potent inducer of c-jun transcription (mediated by a divergent AP-1 binding site in the c-jun promoter), but UV light only modestly induces expression of c-fos(40) .

The AP-1 complex, through its cognate DNA-binding site, may serve to integrate signaling from a number of diverse stimuli, by transcriptionally activating genes resulting in proliferation, activation, tumor promotion, transformation, or protection from apoptosis. Changes in phosphorylation state may regulate AP-1 activity. The activation of AP-1 by growth factors and tumor promoters is in many cases through activation of protein kinase C (41) . The growth factor receptor- or phorbol ester-stimulated protein kinase C-mediated pathway may activate AP-1 indirectly by altering the phosphorylation status of the Jun family of proteins (28, 29, 42, 43, 44, 45, 46, 47) , allowing dimerization and DNA binding by the complex to occur. More recently, another protein kinase which is activated by phorbol esters and phosphorylates c-Jun has been described (48) . We have shown that the protein kinase C inhibitor staurosporine and the protein serine/threonine phosphatase inhibitor okadaic acid exert opposite effects on Mo-MuLV LTR induction of AP-1 DNA binding activity. These findings suggest cellular serine/threonine kinase activity may play a role in the Mo-MuLV LTR stimulation of AP-1 activity.

We and others have reported the activation by the retroviral LTR of cellular genes which are not directly dependent on AP-1 activity, and which do not contain consensus TRE binding sites in their promoters, including MHC antigens and T cell activation antigens. We have identified by deletion and footprinting studies another cis-acting element which appears responsive to activation by the retroviral LTR and which may be responsible for the trans-activation of some of these genes (3).() Alternatively, the activation of c-jun and AP-1 may indirectly affect the expression of these genes. Overexpression of c-jun in malignant cells has been reported to affect MHC class II expression, and transfection of c-jun and c-fos genes into B-16 melanoma cells resulted in the activation of H-2 class I gene expression, suggesting that the activation of other genes which are not directly regulated by AP-1 may be affected indirectly (49) .

Murine leukemia viruses cause lymphoid neoplasia after an extended latent period by an unknown mechanism. The U3 region of the LTR identified here as important in trans-activation of the proto-oncogene c-jun and the transcription factor complex AP-1 has been recently shown to be a critical determinant of leukemogenicity and latency of Mo-MuLV (5, 6, 7, 8, 9) . The ability of this portion of the Mo-MuLV to regulate genes and transcription factors associated with growth control and transformation may be relevant to the oncogenic properties of the virus. These findings suggest a novel mechanism of retrovirus-induced activation of cellular gene expression, potentially contributing to leukemogenesis. Furthermore, the ability of the retroviral LTR to regulate cellular genes should be taken into consideration in the use of retroviral vectors for genetic therapies.


FOOTNOTES

*
This work was supported by Grants R01-CA50459 and R01-CA65420 from the National Cancer Institute and Grant 1RG-97S from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be sent: Cancer Research Center, Boston University School of Medicine, 80 E. Concord St., Rm. E124, Boston, MA 02118. Tel.: 617-638-4173; Fax: 617-638-4176.

The abbreviations used are: Mo-MuLV, Moloney murine leukemia virus; TRE, TPA-responsive element; TPA, 12-O-tetradecanoylphorbol-13-acetate; CAT, chloramphenicol acetyltransferase; LTR, long terminal repeat; MHC, major histocompatibility complex; MCP-1, monocyte chemoattractant protein-1; EMSA, electrophoretic mobility shift assay; kb, kilobase(s).

H. Weng and D. V. Faller, manuscript submitted for publication.

H. Weng and D. V. Faller, unpublished observations.

S.-Y. Choi and D. V. Faller, manuscript in preparation.

S.-Y. Choi and D. V. Faller, manuscript submitted for publication.


ACKNOWLEDGEMENTS

We thank Drs. C. Cepko, R. Bravo, and T. Rothstein for generously providing reagents.


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