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Journal of Virology, July 2004, p. 7590-7601, Vol. 78, No. 14
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.14.7590-7601.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of cis-Acting Elements Residing in the Walleye Dermal Sarcoma Virus Promoter

Brett W. Hronek, Ashley Meagher, Joel Rovnak, and Sandra L. Quackenbush*

Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045

Received 5 November 2003/ Accepted 17 March 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Walleye dermal sarcoma virus (WDSV) is a complex retrovirus found associated with tumors that appear and regress on a seasonal basis. There are quantitative and qualitative differences in the amount of virus expression between developing and regressing tumors. To understand the role of host cell factors in WDSV expression, DNase I footprint analysis, electrophoretic mobility shift assays (EMSA), and reporter gene assays were employed. DNase I footprint analysis of the U3 region of the WDSV long terminal repeat with nuclear extract prepared from a walleye cell line revealed protection of an Oct1, AP1, Whn, and two E4BP4 sites. Additionally, three regions that contained no putative transcription factor binding sites were protected. EMSA confirmed the specific binding of the protected sites and revealed three additional sites, NF1, AP3, and LVa, not protected in DNase I footprint analysis. Site-directed mutagenesis of the individual sites, in the context of a luciferase reporter plasmid, revealed that the NF1, Oct1, AP1, E4BP4#2, AP3, and LVa sites contributed to transcription activation driven by the WDSV U3 region. Mutation of Novel#2 resulted in an increase in luciferase activity, suggesting the Novel#2 site may function to bind a negative regulator of transcription. Anti-Jun and anti-Fos antiserum specifically inhibited protein-DNA complex formation, indicating the presence of c-Jun and c-Fos in the walleye cell nuclear extracts and their participation in binding to the AP1 site. Interestingly, degenerative 15-bp repeats found in the U3 region are differentially protected in DNase I footprint analysis by the walleye cell line nuclear extract and regressing-tumor nuclear extract. EMSA utilizing the 15-bp repeat probe revealed that there are similarities of binding with W12 cell and developing-tumor nuclear extracts and that the binding differs from that observed with regressing-tumor nuclear extract.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Walleye dermal sarcoma virus (WDSV) is a complex retrovirus found etiologically associated with dermal sarcomas in walleye fish (Stizostedion vitreum) (14, 15, 31, 33, 34). There is a seasonal prevalence of this disease; tumors develop during the fall, regress in the spring, and are rarely seen in the summer (3). Dermal sarcoma can be experimentally transmitted to walleye fingerlings with cell-free tumor homogenates prepared from regressing spring tumors; however, homogenates prepared from developing fall tumors are unable to transmit the disease (4, 13). This result is due to the apparently low levels of only subgenomic transcripts during tumor growth, while regressing tumors contain 1,000-fold-greater levels of full-length viral RNA, subgenomic viral transcripts, and infectious virus (4, 22). Thus, control of WDSV gene expression is particularly important to tumor regression, because it correlates with a switch to high levels of virus expression.

Complex retroviruses encode viral accessory proteins that are necessary to upregulate transcription, while simple retroviruses have inherently active promoters. The protein product of WDSV open reading frame a, OrfA or retroviral cyclin, is involved in the regulation of transcription (26, 36). Transient-expression assays determined that OrfA decreased basal activity from the WDSV promoter in a walleye cell line (26, 36). OrfA was found to colocalize and copurify with RNA polymerase II and to interact with components of the mediator complex (26). These data suggest that OrfA may function to inhibit virus expression during tumor development, although the mechanism of transcription repression is not fully understood.

To elucidate the mechanisms responsible for the apparent shift in WDSV gene expression from a low level in growing tumors to overt virus expression in the spring, identification of cis-acting regulatory elements within the viral promoter is necessary. A number of putative cis-acting regulatory elements, including an AP1, AP3, LVa, and an NF1 motif, have been identified in the U3 region of the WDSV long terminal repeat (LTR) (11, 35). Other sequences previously identified include a pentanucleotide direct repeat, TRTGT, that occurs five times in the U3 region and a degenerate 15-bp repeat that occurs three times (11). Zhang et al. (35) used WDSV LTR deletion mutants to identify three regions within U3 that modulated LTR activity. Two of these regions positively regulated transcription from the WDSV LTR, and the third region was found to contain an apparent negative response element; however, individual sites were not conclusively identified.

In this report, we used DNase I footprint analysis and electrophoretic mobility shift assays (EMSA) to identify regions in the WDSV U3 bound by a walleye cell line nuclear extract. Site-directed mutagenesis of a WDSV U3 reporter gene construct was used to determine the contribution of individual sites to transcription activation. Further analysis of an AP1 site demonstrated specific binding of walleye cell nuclear extracts, and anti-Jun and anti-Fos antiserum disrupted this binding. In addition, the 15-bp repeats are differentially protected in DNase I footprint analysis by the walleye cell line nuclear extracts and regressing-tumor extracts. EMSA revealed similar patterns of binding to the 15-bp repeats with W12 cell and developing-tumor nuclear extracts, which differ from that observed with regressing-tumor nuclear extract.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reporter-gene constructs and luciferase assays. The pGL3-U3 reporter vector contains the WDSV U3 region cloned upstream of the luciferase gene (26). Deletion mutant versions of the U3 region were generated by PCR with a fixed 3' primer that incorporates a BglII restriction site (5'-GAAGATCTGAGACCCCGTTCTT-3') and the following series of 5' primers, which each incorporate an XbaI restriction site: U3{Delta}1 (5'-TGCTCTAGATTCTTAAATTGTTAGTAAGGT-3'), U3{Delta}2 (5'-TGCTCTAGATTTCTATGTTGTGTTAAACTA-3'), U3{Delta}3 (5'-TGCTCTAGATGTATACTGACTCATATGTAA-3'), U3{Delta}4 (5'-TGCTCTAGACCCAGATCAGCATGGTGCCAGA-3'), U3{Delta}5 (5'-TGCTCTAGATAAACCCATCTGTTTGTC-3'), U3{Delta}6 (5'-TGCTCTAGAGGCCTAAAGTAGAAATAACAA-3'), and U3{Delta}7 (5'-TGCTCTAGAGCATGTTGCCTTCAAACAGTGT-3'). The amplified products were digested with XbaI and BglII and ligated into the NheI and BglII sites in pGL3-Basic (Promega).

Mutations were made in the pGL3-U3 plasmid with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The resulting mutated pGL3-U3 plasmids were sequenced for confirmation.

W12 cells, a walleye fibroblast cell line, were seeded into 24-well plates in 1 ml of minimal essential medium supplemented with 10% fetal bovine serum (Gibco), 4 mM glutamine, 100 U of penicillin ml–1, and 100 µg of streptomycin ml–1. W12 cells were derived from walleye dermal sarcomas in the laboratory of Paul Bowser, Cornell University. They do not contain viral sequences (25); however, they are susceptible to infection with WDSV (S. L. Quackenbush, unpublished data). Cells were transfected with 0.2 µg of a luciferase reporter vector and 0.05 µg of pRL-TK (Promega) by using FuGENE6 (Roche) according to the manufacturer's suggestions. Experiments were performed using the dual-luciferase reporter assay system (Promega), which sequentially measures firefly and Renilla luciferase activities from a single sample. Cell lysates were harvested 72 h after transfection with passive lysis buffer and then centrifuged at 20,000 x g for 5 min at 4°C. Luciferase activities were obtained with a TD-20/20 luminometer (Turner Designs) according to the manufacturer's instructions. Luciferase activity from the reporter vector was normalized for transfection efficiency by using values obtained from the cotransfected pRL-TK vector. All transfections were performed in quadruplicate. Student's t test and 95% confidence intervals based on a t distribution were used for statistical analyses. A P value less than 0.05 was considered significant.

Nuclear extract preparation. Extracts were prepared by the method of Mayeda and Krainer (16), which is a modification of the method described by Dignam et al. (9). Briefly, W12 cells were grown in minimal essential medium supplemented with 10% fetal bovine serum (Gibco), 4 mM glutamine, 100 U of penicillin ml–1, and 100 µg of streptomycin ml–1 at 20°C to 80% confluence in 150-cm2 flasks. The cells were harvested and washed three times with cold Dulbecco's phosphate-buffered saline (D-PBS; 2.7 mM KCl, 1.5 mM KH2PO4, 0.137 M NaCl, 8.1 mM Na2HPO4 · 7H2O). The packed-cell volume (PCV) was measured, and the cell pellet was gently resuspended with 5 PCVs of buffer A (10 mM HEPES-KOH [pH 8.0], 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol [DTT], and EDTA-free protease inhibitor cocktail [Roche]). The cells were incubated on ice for 10 min and pelleted by centrifugation at 1,800 x g for 10 min. Buffer A was added to 2 PCVs, and the cell suspension was homogenized with 30 strokes of pestle B in a Dounce glass homogenizer (Kontes Glass Company) until the cells were ~90% lysed, as determined by microscopy. The lysate was centrifuged at 20,000 x g for 30 min at 4°C. The supernatant was removed, and the pellet, or packed nuclear volume (PNV), was measured. Then 0.4 ml of buffer C (20 mM HEPES-KOH [pH 8.0], 0.6 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% [vol/vol] glycerol, 1 mM DTT, EDTA-free protease inhibitor cocktail [Roche]) per ml of PNV was added. Cell nuclei were homogenized with 30 strokes of pestle A in a Dounce glass homogenizer. The suspension was rocked at 4°C for 45 min and centrifuged for 45 min at 20,000 x g. The supernatant (nuclear extract) was dialyzed against buffer D (20 mM HEPES-KOH [pH 8.0], 100 mM KCl, 0.2 mM EDTA, 20% [vol/vol] glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM DTT) overnight. The sample was centrifuged at 20,000 x g for 30 min, and the supernatant (nuclear extract) was aliquoted, frozen in liquid nitrogen, and stored at –80°C. Protein concentrations were determined by the Bradford method (Bio-Rad).

Regressing and developing tumors were provided by James W. Casey and Paul R. Bowser, Cornell University. Nuclear extracts from regressing and developing tumors were prepared from a modification of the method described by Braun et al. (5). Briefly, the solid tumors were processed with a Tissue Tearor (Biospec Products, Inc.) and washed in cold D-PBS. The pellet was resuspended in 0.5% NP-40 in PBS and rocked at 4°C for 30 min. The suspension was centrifuged briefly, and the supernatant was removed. The pellet was resuspended in a solution containing 20 mM Tris-HCl (pH 8.0), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 25% glycerol, and EDTA-free protease inhibitor cocktail (Roche) and rocked at 4°C for 1 h. The suspension was centrifuged at high speed for 30 min, and the supernatant (nuclear extract) was dialyzed against buffer D overnight. The nuclear extract was centrifuged for 30 min at 20,000 x g, and the supernatant was frozen and quantified as described above.

The W12 nuclear extract used to footprint the 15-bp repeats was processed identically to that from the regressing and developing tumors, except treatment with the Tissue Tearor was omitted.

DNase I footprinting. The DNA probe used in the DNase I footprinting was generated by PCR using PCR High Fidelity Supermix (Invitrogen). The primers used to amplify the U3 region of the WDSV promoter were RVprimer3 (5'-CTAGCAAAATAGGCTGTCCC-3') and GLprimer2 (5'-CTTTATGTTTTTGGCGTCTTCCA-3'). The RVprimer3 was labeled with [{gamma}-32P]ATP by using T4 polynucleotide kinase (PNK; New England Biolabs) according to the manufacturer's instructions. Briefly, the labeling reaction mixture contained 25 pmol of [{gamma}-32P]ATP (6,000 Ci/mmol, 10 mCi/ml), 20 pmol of RVprimer3, kinase reaction buffer (70 mM Tris-HCl [pH 7.6], 10 mM MgCl2, 5 mM DTT) (New England Biolabs), and 10 U of T4 PNK in a volume of 10 µl. The reaction mixture was incubated at 37°C for 1 h and then heated to 95°C for 2 min. The PCR mixture consisted of PCR High Fidelity Supermix (Invitrogen), 0.2 µM 32P-labeled RVprimer3, 0.2 µM GLprimer2, and 50 ng of pGL3-U3 plasmid, in a final volume of 50 µl. The labeled U3 probe was purified with the QIAquick PCR purification kit (QIAGEN) and resuspended in elution buffer (10 mM Tris-HCl, pH 8.5). The concentration of the labeled U3 was determined by measuring the absorbance at 260 nm.

DNase I footprinting reaction mixtures consisted of 0.5 µg of lambda DNA (New England Biolabs), 15 fmol of labeled U3, and nuclear extract in binding buffer (25 mM Tris-HCl [pH 8.0], 50 mM KCl, 6.25 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 10% [vol/vol] glycerol). Fifty microliters of a solution containing 5 mM CaCl2 and 10 mM MgCl2 was added to the reaction mixtures, and the mixtures were incubated at room temperature for 60 s. The reaction mixtures were treated with 0.01 U of DNase I (Roche) for 30 to 90 s, as determined for each probe, and the reaction was stopped by adding 90 µl of a stop solution (200 mM NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate, 100 µg of yeast tRNA/ml). The reaction mixtures were extracted with phenol-chloroform and precipitated with ethanol at –20°C. Samples were centrifuged at 20,000 x g for 10 min at room temperature. The supernatant was removed, and the DNA was washed once with 300 µl of 70% ethanol and allowed to air dry. The pellets were suspended in 11 µl of loading buffer (1:2 [vol/vol] 0.1 M NaOH-formamide, 0.1% xylene cyanol, 0.1% bromophenol blue). The samples were quantified by a Packard liquid scintillation counter, and equal counts (2 x 104 cpm) were loaded onto a 6% polyacrylamide sequencing gel (6% acrylamide-bisacrylamide [19:1], 7 M urea, 1x TBE [89 mM Tris base, 110 mM boric acid, 2 mM EDTA]). The gels were run at 1,500 V in 1x TBE, dried, and exposed to X-ray film at –80°C. Sequencing was performed with the Thermo Sequenase cycle sequencing kit (USB) according to manufacturer's instructions, using the 32P-labeled RVprimer3, and run adjacent to the DNase I footprinting lanes to identify the sequences of the protected regions.

Gel mobility shift assays. To prepare double-stranded DNA probes for gel mobility shifts, the DNA oligonucleotides (Invitrogen) were annealed by mixing 1 nmol of each primer in 1x TEN buffer (10 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl) and incubating the oligonucleotides at 95°C for 5 min and then slowly cooling them until reaching room temperature. The double-stranded oligonucleotides (probes) contained the specified binding site surrounded by irrelevant bases. The double-stranded DNA probes were end labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase (New England Biolabs). Ten micrograms of W12 nuclear extract and 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech) were added to EMSA reaction buffer (10 mM Tris [pH 7.5], 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 5% [vol/vol] glycerol) in a total volume of 20 µl and incubated for 10 min at room temperature. The labeled oligonucleotide probe (0.3 ng) was added, and the mixture was further incubated for 20 min at room temperature. Different concentrations of nuclear extracts ranging from 2.5 to 20 µg prepared from the W12 cell line were initially tested in EMSA. Ten micrograms of nuclear extracts was found to bind several different probes optimally and was subsequently used in all assays. When antibodies were included (c-Jun [Upstate Biotechnology] and c-Fos and sc-253X [Santa Cruz Biotechnology]), they were added following the 20-min incubation and the reaction mixture was incubated at 4°C overnight. The following day, the samples were placed at room temperature for 20 min and then separated on a 6% polyacrylamide gel (6% acrylamide, 37.5:1 acrylamide-bisacrylamide, 2.5% glycerol, 0.5x TBE [45 mM Tris base {pH 8.3}, 45 mM boric acid, 1 mM EDTA]) in 0.5 M TBE. The gels were run at 200 V for 45 min, dried, imaged and analyzed with the Cyclone storage phosphor system and OptiQuant image analysis software (Packard).

Western blotting. Fifty nanograms of recombinant human Jun (rhAP1; Promega), 20 µg of A431 cell lysate (Upstate Biotechnology), 10 µg of HeLa cell nuclear extract, and 10 µg of W12 cell nuclear extract were separated on a 12% NuPAGE gel (Invitrogen) in MOPS (3-[N-morpholino]propanesulfonic acid) running buffer (50 mM MOPS, 50 mM Tris base, 3.5 mM sodium dodecyl sulfate, 1 mM EDTA) at 200 V for 70 min. The proteins were then transferred to an Immobilon-P membrane (Millipore) at 150 mA for 2 h. The membrane was then blocked for 30 min in BLOTTO (5% nonfat dry milk and 0.5% Tween 20 in PBS) and incubated overnight with a 1:1,000 dilution of anti c-Jun rabbit antiserum (Upstate Biotechnology) in BLOTTO. The blot was then washed and incubated for 1 h with a 1:5,000 dilution of affinity-purified goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (Kirkegaard & Perry Laboratories) and developed with the substrate 3,3',5,5'-tetramethylbenzidine (Kirkegaard & Perry Laboratories).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of sequential 5' U3 deletion mutants. To identify the cis-acting regulatory sites that contribute to WDSV U3-driven transcription, luciferase reporter assays were employed. Progressive 50-bp deletions from the 5' end of the U3 region were constructed (Fig. 1), and luciferase activity was measured after transfection into the walleye cell line W12, which is susceptible to infection with WDSV (Fig. 1). Cells were cotransfected with reporter plasmid pRL-TK to control for transfection efficiency, and the activities of the deletions are expressed as percent activity relative to that of the full-length WDSV U3. Deletion of the first 50 bases of the U3 region (U3{Delta}1; nucleotides –440 to –391 based on the start of transcription at position +1) resulted in a significant decrease in luciferase activity to 50% of that expressed from the full-length U3 region (Fig. 1). Further deletion to nucleotide –341 (U3{Delta}2) did not result in significant changes in luciferase activity compared to that for U3{Delta}1. A significant increase in luciferase activity, above that of U3{Delta}2, was seen after removal of the next 50 bases (U3{Delta}3; P < 0.01), suggesting the presence of a negative regulator of transcription. There was a decrease in luciferase activity back to the levels of U3{Delta}1 and U3{Delta}2 when nucleotides –440 to –191 (U3{Delta}4 and U3{Delta}5) were deleted, and expression of luciferase activity from U3{Delta}6 and U3{Delta}7 (deletion of nucleotides –440 to –91) was 1 to 2% of wild type. These truncations of upstream elements of the U3 region of the WDSV promoter delineate the enhancer region (nucleotides –440 to –141) and the basal promoter element (–140 to +1).



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  		FIG. 1. Luciferase activity of WDSV U3 deletion mutants. WDSV wild-type and U3 deletion mutant reporter constructs were transfected into W12 cells. Relative luciferase values were corrected for transfection efficiency with pRL-Tk. The luciferase activity of each deletion mutant is expressed as percent activity relative to that for the full-length U3 region. Putative cis-acting sites previously identified by others are shown (11, 36). All experiments were performed in quadruplicate, and the means ± standard deviations of luciferase expression from three independent experiments are shown. *, statistically significant value (P < 0.05).

 
Walleye cell nuclear extracts protect sites within the WDSV U3 region. DNase I footprint experiments were conducted using nuclear extracts from the walleye cell line W12, in order to identify specific protein-DNA interactions in the WDSV LTR. The W12 walleye fibroblast cell line was originally derived from a dermal sarcoma but does not harbor WDSV (25). When W12 cells are infected with WDSV, only low levels of the accessory viral transcripts orf a and orf b are expressed, similar to the expression pattern seen in developing tumors (22; S. L. Quackenbush, unpublished data.). The U3 region of the WDSV LTR was 5' end labeled on the noncoding strand and incubated with increasing concentrations of nuclear extracts. The reaction products were treated with DNase I, separated on a 6% sequencing gel, and visualized by autoradiography.

The presence of a putative Oct1 binding site in the U3 region (position –366 to –359) was predicted by analysis with the MatInspector database (www.genomatix.de) (23). This site was protected with nuclear extracts from W12 cells (Fig. 2) and was accompanied by a DNase hypersensitive site as the amount of nuclear extracts increased.



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  		FIG. 2. DNase I footprint of the WDSV U3 region with W12 nuclear extracts. DNA probes were 5' end labeled on the noncoding strand and incubated with increasing amounts of W12 nuclear extracts. Dideoxy sequence reactions were run in adjacent lanes for reference. *, hypersensitive site. The locations of the protected sequences are indicated on the right.

 
Two footprints which do not contain consensus binding sites present in the MatInspector database were identified in the U3 region of the WDSV LTR. One of these regions, Novel #1, maps to –350 to –345 in the U3 region (Fig. 2), and the other protected region, referred to as Novel #2, maps to –147 to –141 in the U3 region (Fig. 2).

Holzschu et al. (11) previously identified a 5-bp direct repeat, TRTGT, that is present in the WDSV U3 region five times. No known transcription factors are predicted to bind to this sequence. However, two of these 5-bp repeats are part of two E4BP4 sites. All five repeats were protected by DNase I footprinting, and four of the five 5-bp repeats are shown in Fig. 2. One region of protection overlaps the first two 5-bp repeats and maps to –335 to –328. The second region of protection, maps to –319 to –311 in U3 (Fig. 2). This second region contains the third and fourth 5-bp repeats, and the first of two E4BP4 sites, E4BP4 #1, that are present in the U3 region.

Using nuclear extracts prepared from W12 cells, a large footprint was found to map to –293 to –270 in the U3 region (Fig. 2). The footprint overlaps a predicted Winged helix nude (Whn) binding site, an AP1 transcription factor binding site, and the second E4BP4 binding site, E4BP4 #2. The fifth 5-bp direct repeat, is contained within E4BP4 #2. The AP1 and E4BP4 #2 binding sites are directly adjacent to each other and overlap by 1 bp (Fig. 2).

These results demonstrate that proteins present in walleye cell nuclear extracts bind to specific sequences in the U3 region of the WDSV promoter. These proteins may include homologs of Oct1, Whn, E4BP4, and components of the AP1 binding complex. Also included in the walleye cell nuclear extracts are proteins capable of binding DNA sequences that do not contain known transcription factor binding sites.

Protected sites are specifically bound by nuclear proteins. EMSA were performed to confirm the specific binding of a cellular factor(s) to sites protected by DNase I footprinting and also to identify additional sites that were not protected.

A probe containing the predicted Oct1 binding site from the WDSV U3 region was incubated with W12 cell nuclear extracts and separated on a nondenaturing polyacrylamide gel. The formation of a protein-DNA complex was observed as a single shifted band (Fig. 3A, lane 2). The specificity of protein binding to the Oct1 probe was demonstrated by competition with a 100-fold molar excess of the unlabeled Oct1 probe (Fig. 3A, lane 3). Competition of nuclear extract binding was not possible with a 100-fold molar excess of an unlabeled mutated Oct1 probe or with a nonspecific competitor probe, further demonstrating specific binding (Fig. 3A, lanes 4 and 5).



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  		FIG. 3. W12 nuclear extracts specifically bind to sites found protected by DNase I footprint analysis. Oligonucleotides containing individual binding sites (Oct1, Novel #1, 5-bp repeats, E4BP4 #1, Whn, AP1, E4BP4 #2, and Novel #2) were end-labeled with 32P and incubated with 10 µg of W12 nuclear extract. Lane 1, oligonucleotide alone; lane 2, oligonucleotide and 10 µg of W12 nuclear extract; lane 3, oligonucleotide, 10 µg of W12 nuclear extract, and unlabeled oligonucleotide; lane 4, oligonucleotide, 10 µg of W12 nuclear extract, and unlabeled mutated oligonucleotide; lane 5, oligonucleotide, 10 µg of W12 nuclear extract, and unlabeled nonspecific competitor oligonucleotide. In panels D and G the three bands are indicated.

 
Three regions that were protected in DNase I footprint analysis but that do not contain known transcription factor binding sites were subjected to EMSA with walleye cell nuclear extracts. Two shifted bands were observed when the Novel #1 probe was used in EMSA (Fig. 3B, lane 2). The upper band was competed with excess unlabeled probe, but not with the nonspecific competitor probe (Fig. 3B). There appears to be a little competition with the mutated Novel #1 probe for binding to nuclear extracts (Fig. 3B, lane 4). The lower band was not competed with any of the probes (Fig. 3B) and likely represents a nonspecific band. When a probe containing the first two 5-bp repeats was incubated with W12 nuclear extracts, a predominant shifted band was observed (Fig. 3C, lane 2), the majority of which could be competed with excess unlabeled probe (Fig. 3C, lane 3). A nonspecific competitor did not compete for binding of nuclear extracts; however, the mutated 5-bp-repeat probe seemed to compete for some binding (Fig. 3C, lanes 4 and 5). A minor, slower-migrating band was also present; however, it was not competed with excess unlabeled probe, suggesting that this is a nonspecific band. Incubation of nuclear extracts with the Novel #2 probe resulted in the formation of a protein-DNA complex that appears to contain two bands (Fig. 3H, lane 2). A 100-fold molar excess of unlabeled probe competed for binding of the nuclear extracts, as did the mutated Novel #2 probe (Fig. 3H, lanes 3 and 4), whereas a nonspecific competitor was unable to compete for binding (Fig. 3H, lane 5).

EMSA revealed the formation of three protein-DNA complexes when W12 nuclear extracts were incubated with a probe that contained the E4BP4 #1 site (Fig. 3D, lane 2). Band 1 was successfully competed by 100-fold molar excess of unlabeled probe (Fig. 3D, lane 3), and the majorities of bands 2 and 3 were competed. The nonspecific competitor did not compete for binding of nuclear extracts (Fig. 3D, lane 5). The mutated E4BP4 #1 probe competed for binding to nuclear extracts (Fig. 3D, lane 4). The mutation made in the E4BP4 probe did not change the 5-bp repeat sequence, which may still bind nuclear extract.

To examine the large protected region from position –293 to –270, which contains the Whn, AP1, and E4BP4 #2 sites, individual probes for each of the sites were tested by EMSA. A predominant protein-DNA complex formed with the Whn probe (Fig. 3E, lane 2). A 100-fold molar excess of unlabeled probe efficiently competed for binding of the complex (Fig. 3E, lane 3). The mutated Whn and the nonspecific competitor probes did not compete for binding to nuclear extracts (Fig. 3E, lanes 4 and 5). A probe containing only the AP1 site also yielded a predominant shifted band (Fig. 3F, lane 2), and specificity of binding was demonstrated by competition with excess unlabeled probe and lack of competition with the mutated AP1 and nonspecific competitor probes (Fig. 3F, lanes 3 to 5). Mobility shift assays of the E4BP4 #2 site revealed the formation of three protein-DNA complexes, similar to that observed with the E4BP4 #1 probe (Fig. 3G and D). Again, the majorities of bands 2 and 3 were competed with excess unlabeled probe, whereas band 1 was completely eliminated (Fig. 3G, lane 3). Interestingly, analysis by densitometry determined that bands 2 and 3 consistently bound 40% more of the labeled E4BP4 #2 probe than of the E4BP4 #1 probe (Fig. 3G and D). The two sites differ by 1 bp (Table 1), although the contribution of this base in binding E4BP4 has not been previously determined. The nonspecific competitor probe did not compete for binding of nuclear extracts (Fig. 3G, lane 5). The mutated E4BP4 #2 probe competed somewhat for binding (Fig. 3G, lane 4) and again, like the E4BP4 #1 probe, contained the 5-bp repeat sequence, suggesting that a protein(s) other than E4BP4 may bind to this probe.


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  		TABLE 1. cis-acting sites in the WDSV U3 region

 
Three sites not protected from DNase I in footprinting assays were evaluated with EMSA. When a probe containing a predicted NF1 site was incubated with W12 nuclear extract, a shifted band was observed, which was competed by an excess of unlabeled probe and not by mutated NF1 or nonspecific competitor probes (Fig. 4A). Two sites identified by Holzschu et al. (11), LVa and AP3, were also investigated. A large, predominant protein-DNA complex was observed with both probes (Fig. 4B and C, respectively), and both complexes could be competed by a 100-fold molar excess of unlabeled probe (Fig. 4B and C). Neither a mutated AP3 probe nor a nonspecific competitor probe competed for binding of nuclear extract, indicating the specificity of binding (Fig. 4C). The mutated LVa probe was not successful at eliminating binding of nuclear extracts (Fig. 4B, lane 4).



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  		FIG. 4. W12 nuclear extracts specifically bind to sites not found protected by DNase I footprint analysis. Oligonucleotides containing individual binding sites (NF1, LVa, and AP3) were end labeled with 32P and incubated with 10 µg of W12 nuclear extract. Lanes 1, oligonucleotide alone; lanes 2, oligonucleotide and 10 µg of W12 nuclear extract; lanes 3, oligonucleotide, 10 µg of W12 nuclear extract, and unlabeled oligonucleotide; lanes 4, oligonucleotide, 10 µg of W12 nuclear extract, and unlabeled mutated oligonucleotide; lanes 5, oligonucleotide, 10 µg of W12 nuclear extract, and unlabeled nonspecific competitor oligonucleotide.

 
These results confirm specific binding of proteins present in W12 nuclear extracts to sites found protected by DNase I footprinting and two sites that were not protected by footprint analysis.

AP1 binds to the WDSV LTR. Characterization of the proteins that bind to the AP1 site in the WDSV U3 region required an antibody that recognizes components of AP1 from walleye cells. An amino acid alignment was performed with c-Jun from humans (Homo sapiens), mice (Mus musculus), chickens (Gallus domesticus), carp (Cyprinus carpio), and fugu (Fugu rubripes) (data not shown). Based on the conservation of the DNA-binding domain of Jun among the species examined, an antibody that recognized the DNA-binding domain was selected for the detection of c-Jun from walleye. Nuclear extracts from W12 cells were subjected to Western blot analysis with an antibody that was generated against the avian DNA-binding domain of c-Jun (Fig. 5). The anti-Jun antibody recognizes recombinant human Jun (rhAP1), Jun from two human cell lines, A431 and HeLa, and a protein that migrates at the correct molecular mass from walleye cell (W12) nuclear extracts (Fig. 5), which suggests that this protein is the Jun equivalent in W12 nuclear extracts.



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  		FIG. 5. Detection of Jun in walleye cell nuclear extracts. Recombinant human Jun (rhAP1), A431 cell lysate, HeLa cell nuclear extract (NE), and W12 cell nuclear extract were separated on a NuPage 12% Bis-Tris gel and transferred to an Immobilon membrane. The blot was probed with rabbit anti-Jun antiserum that recognizes the DNA-binding domain of avian Jun.

 
EMSA were used to further investigate the binding of W12 nuclear extracts to the AP1 site alone and also to the region of WDSV U3 that encompasses the Whn, AP1, and E4BP4 #2 sites. Anti-Jun antiserum could specifically disrupt the protein-DNA complex formed with W12 nuclear extracts and a probe containing only the AP1 site (Fig. 6A, lane 3), whereas normal rabbit serum did not affect complex formation (Fig. 6A, lane 4). A distinct protein-DNA complex was observed when an oligonucleotide containing the Whn, AP1, and E4BP4 #2 sites was incubated with W12 nuclear extracts (Fig. 6B, lane 2). Inclusion of anti-Jun antiserum in the reaction inhibited the formation of the complex (Fig. 6B, lane 3). The anti-Jun antiserum was generated against the DNA-binding domain of Jun, suggesting that the anti-Jun antibody prevents the binding of walleye Jun to DNA, which results in disruption of the complex, instead of formation of a supershift. As illustrated in Fig. 6B, lane 4, normal rabbit serum was unable to disrupt complex formation, indicating that inhibition of complex formation with anti-Jun antiserum was specific and that walleye Jun was present in the complex. To further assess the composition of the AP1 complex, EMSA was performed with an antibody that recognizes all of the Fos family members (c-Fos, FosB, Fra1, and Fra2). The anti-Fos antibody was also able to disrupt complex formation (Fig. 6A), suggesting that the AP1 complex is a c-Jun/Fos heterodimer.



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  		FIG. 6. c-Jun in W12 nuclear extracts binds to the AP1 consensus site in the WDSV LTR. (A) W12 nuclear extracts were incubated with a 5'-end-labeled oligonucleotide containing the AP1 binding site. (B) W12 nuclear extracts were incubated with a 5'-end-labeled oligonucleotide containing the Whn, AP1, and E4BP4 #2 binding sites. Anti-Jun antiserum (Jun), normal rabbit serum (N), anti-Fos antiserum (F), or irrelevant antiserum (I) was added to the specified reaction mixture. The reaction products were separated on a 6% nondenaturing polyacrylamide gel and visualized with a phosphorimager.

 
Effect of individual cis-acting sites on WDSV U3-driven transcription. To evaluate the functional role of the cis-acting elements protected in DNase I footprint analysis and identified with EMSA, transcriptional activity of the WDSV U3 was analyzed with a luciferase reporter gene assay. Protein-binding regions identified in both footprint analysis and EMSA were subjected to site-directed mutation (Table 1). Mutations of Novel #1 and Novel #2 were analyzed to confirm that mutations did not result in the generation of known sites. Walleye W12 cells were transfected with each luciferase reporter construct and pRL-TK, a Renilla reporter plasmid, to control for transfection efficiency, and the ratio of relative luciferase activity to Renilla activity for the wild-type WDSV U3 was set at 100%.

The Oct1 site that was protected by DNase I footprint analysis (Fig. 2A) was mutated, and expression levels were compared to wild-type U3 expression. Expression from the WDSV Oct1 mutant reporter construct resulted in significantly less activity than that observed with the wild-type U3 construct (69.2% ± 9.3%; Fig. 7B; P < 0.03).



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  		FIG. 7. Activity of mutant WDSV U3 reporter constructs. (A) Diagram of the transcription factor binding sites characterized by DNase I footprint analysis, EMSA, and luciferase assays. (B) Relative luciferase activity of the pGL3-U3 luciferase plasmid with mutations in the indicated sites (Table 1) found protected by DNase I footprint analysis. (C) Relative luciferase activity of the pGL3-U3 luciferase plasmid with mutations in the indicated sites (Table 1) that were not found protected by DNase I footprint analysis. The luciferase activity of the mutants is expressed as percent activity relative to that for the full-length U3 region. All experiments were performed in quadruplicate, and the means ± standard deviations of luciferase expression from three independent experiments are shown. *, statistically significant (P < 0.05).

 
The Novel #1 site protected in DNase I footprint analysis was subjected to mutation, and luciferase activity was measured (Fig. 7B). Three bases were mutated in the Novel #1 site (Table 1), and mutation of this site did not significantly affect luciferase expression (104.5% ± 5.6%; Fig. 7A).

The first two of the 5-bp repeats present in the U3 region mapping to –336 to –326 (Fig. 2D) were mutated, and luciferase expression was measured. Mutation of six of the protected bases (Table 1) did not result in a measurable change in luciferase activity compared to that for the wild type (99.9% ± 10.3%; Fig. 7B). Mutation of the predicted E4BP4 binding site, E4BP4 #1, which overlaps the fourth 5-bp repeat at position –317 to –310, did not result in a significant change in luciferase activity compared to that for the wild type, (94.4% ± 4.7%; Fig. 7B).

Mutation of the AP1 site in the WDSV U3 reporter construct resulted in a significant decrease in luciferase expression compared to that of the wild-type U3 construct (73.0% ± 4.4%; Fig. 7B; P < 0.009). The predicted E4BP4 site adjacent to the AP1 binding site, E4BP4 #2, was mutated and luciferase activity was measured. In contrast to the activity from the mutated E4BP4 #1 site, luciferase activity resulting from mutation of the second E4BP4 site was significantly reduced compared to wild type (58.5% ± 8.9%; Fig. 7B; P < 0.02). Also, mutation of the Whn site resulted in significantly reduced luciferase activity (70.2% ± 3.0%; Fig. 7B, P < 0.003).

A third site, Novel #2, found to be protected by DNase I footprinting (Fig. 2C), was mutated, and luciferase activity was measured. Transfection of this mutated U3 reporter construct into W12 cells resulted in a significant increase in luciferase activity (137.8% ± 1.7%; Fig. 7B; P < 0.001). These data suggest that the Novel #2 region may bind a transcriptional repressor.

Zhang et al. (35) conducted a functional analysis of the WDSV LTR using a luciferase reporter construct by introducing progressive deletions from the 5' end of the WDSV LTR and measuring the luciferase activity of these deletion mutants. When a region of the WDSV U3 containing an NF1 site was deleted, there was a decrease in luciferase activity compared to that for the wild-type LTR (35). This site was not found to be protected by DNase I footprinting but did bind W12 nuclear extract in EMSA (Fig. 4A). Mutation of the NF1 site resulted in a significant decrease in expression compared to that for the wild-type WDSV U3 (86.2% ± 1.8%; Fig. 7C; P < 0.006).

Two putative transcription factor binding sites in the WDSV U3, AP3, and LVa, were previously identified by Holzschu et al. (11). Again, these sites were not protected by DNase I footprint analysis (data not shown); however, they were found to bind nuclear extracts in mobility shift assays (Fig. 4B and C). Transfection of the WDSV U3 reporter constructs containing either an AP3 or LVa mutation into W12 cells resulted in significantly lower levels of luciferase activity: 76.1% ± 9.7% (Fig. 7C; P < 0.05) or 56.5% ± 8.7% (Fig. 7C, P < 0.01), respectively.

These data demonstrate that several cis-acting sites present in the U3 region of the WDSV LTR functionally contribute to transcription activation.

Regressing-tumor nuclear extracts protect the 15-bp repeats. Nuclear extracts prepared from regressing tumors obtained from naturally infected walleye fish were used in DNase I footprint analysis. The sites found protected with W12 cell nuclear extracts (Fig. 2) were also protected with the tumor cell nuclear extracts (data not shown). In addition, a very strong footprint was observed overlapping the degenerative 15-bp repeats that map to –82 to –36 in the U3 region (Fig. 8A, C, and D). Immediately apparent is a very predominant DNase I hypersensitive site (Fig. 8A and C), which corresponds to the A in the middle of the first 15-bp repeat. The corresponding A in the middle of the second 15-bp repeat is also present as a hypersensitive site, although it is not as intense as that in the first 15-bp repeat (Fig. 8B and C). There are four regions of protection which are interspersed between four additional hypersensitive sites (Fig. 8A, C, and D).



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  		FIG. 8. Differential protection of the degenerate 15-bp repeats in WDSV U3. DNase I footprint analysis of the degenerate 15-bp repeats after incubation with regressing-tumor nuclear extracts (NE) (A), W12 nuclear extracts (B), and both W12 and regressing-tumor nuclear extracts (C). DNA probes were 5' end labeled on the noncoding strand and incubated with the indicated quantities of nuclear extracts. Dideoxy sequence reactions were run in adjacent lanes for reference. *, hypersensitive sites. (D) Representative diagram showing the sequence of the 15-bp repeats (boxed). The regions of protection (black bars) and hypersensitive sites (*) found with the W12 nuclear extracts and the regressing-tumor nuclear extracts are shown on the top and bottom of the sequence, respectively.

 
DNase I footprint analysis of the 15-bp repeats with W12 nuclear extracts revealed a striking difference from that observed with the tumor nuclear extract (Fig. 8B to D). Two regions of protection are found with W12 nuclear extracts, as well as five faint hypersensitive sites (Fig. 8B to D). Two of the hypersensitive sites are located at the A in the middle of the first two 15-bp repeats, similar to that found with the regressing-tumor extracts (Fig. 8). The additional three hypersensitive sites do not correspond to the hypersensitive sites found by using regressing-tumor nuclear extract.

EMSA was used to confirm binding of nuclear extracts to the 15-bp repeats. An oligonucleotide containing the 15-bp repeat region was incubated with W12 cell and regressing- and developing-tumor extracts and separated on a nondenaturing polyacrylamide gel (Fig. 9). Two predominant protein-DNA complexes were observed with all three of the nuclear extracts. A slower-migrating protein-DNA complex was detected with the W12 cell and developing-tumor nuclear extracts but was not present with the regressing-tumor extracts. An additional protein-DNA complex formed with the developing-tumor and W12 cell nuclear extracts; however, the intensity of the band was stronger with the developing-tumor extracts.



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  		FIG. 9. Binding of W12 and developing- and regressing-tumor nuclear extract to the 15-bp repeats. An oligonucleotide containing the 15-bp repeats was end labeled with 32P and incubated with the indicated nuclear extract. Lane 1, oligonucleotide alone; lane 2, oligonucleotide and 10 µg of W12 nuclear extract (W12); lane 3, oligonucleotide and 10 µg of developing-tumor nuclear extract (Dev); lane 4, oligonucleotide and 10 µg of regressing-tumor nuclear extract (Reg).

 
These data demonstrate a significant difference in the footprinting patterns of the 15-bp repeats between the W12 nuclear extracts and regressing-tumor nuclear extracts. Also, a striking similarity in binding of nuclear extracts from W12 cells and developing tumors to the 15-bp repeat probe was demonstrated by EMSA.

The 15-bp repeat region contributes to negative regulation of WDSV U3 transcription. Analysis of the 15-bp repeat region with the MatInspector database identified two cell cycle gene homology region sites (CHR) and one each of forkhead-related activator 4 (FREAC4), hepatocyte nuclear factor 3ß (HNF-3ß), STAT5, CCAAT displacement protein (CDP), and PAX 2/5/8 sites (Fig. 10B). These sites were mutated by site-directed mutagenesis in the context of the pGL3-U3 luciferase plasmid and assayed for luciferase expression as described above. The CDP and Pax sites overlap; therefore, it was not possible to mutate one without disrupting the other site. As a result, both sites were disrupted in the luciferase reporter construct. Mutation of STAT5, CHR#1, and FREAC4 did not result in a significant difference in luciferase activity (86.7% ± 7.5%, 79.1% ± 12.7%, and 105.1% ± 14.3%, respectively) compared to that of the wild type (100%) (Fig. 10A). However, mutation of the HNF-3ß, CHR#2, and CDP/Pax sites resulted in an increase in luciferase activity: 163.8% ± 20.8% (P < 0.04), 151.4% ± 10.3% (P < 0.02), and 148.5% ± 17.5% (P < 0.05), respectively (Fig. 10A). These data demonstrate that three sites within the 15-bp repeat region of the WDSV promoter contribute to negatively regulate transcription in W12 cells.



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  		FIG. 10. Activity of mutant WDSV U3 reporter constructs. (A) Relative luciferase activity of the pGL3-U3 luciferase plasmid with mutations in the indicated sites found in the 15-bp repeats. The luciferase activity of the mutants is expressed as percent activity relative to that for the full-length U3 region. All experiments were performed in quadruplicate, and the means ± standard deviations of luciferase expression from three independent experiments are shown. *, statistically significant (P < 0.05). (B) Representative diagram showing the sequence of the 15-bp repeats (boxed). The transcription factor-binding consensus sites are underlined.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There are large differences in WDSV gene expression between developing and regressing tumors collected from naturally infected walleye (4, 22). The WDSV protein OrfA functions to inhibit expression from the WDSV promoter in transient-transfection experiments conducted with a walleye cell line. However, very little is known regarding what host cell factors are necessary for transcription from the WDSV promoter or whether OrfA functions through a DNA-binding protein. In this study, specific host cell protein-DNA interactions in the WDSV U3 region were identified and the functional contribution of each site was evaluated.

Initially, mutants with 5' progressive 50-bp deletions of the U3 region were constructed and luciferase activity was measured. Based on these data, the enhancer region of the WDSV U3 was mapped to nucleotides –440 to –141 and the basal promoter was mapped to –141 to +1. DNase I footprint analysis was performed using walleye cell nuclear extracts, and six regions of protection were found. Individual sites included in these protected regions were an Oct1 site, an AP1 site, a Whn site, two E4BP4 sites, a series of 5-bp repeats, and two novel sites. Specific binding of nuclear extracts to these sites was confirmed by EMSA. There was a little competition with the mutated Novel #1 and the 5-bp repeat probes. Currently we do not know what proteins bind to these sites; therefore, it is possible that the mutations did not completely disrupt binding. A region of protection, including a prominent hypersensitivity band, of the Novel #2 site was clearly identified in DNase I footprint analysis. EMSA confirmed binding of nuclear extracts to the Novel #2 probe. The unlabeled probe competed for binding, as did the mutated probe, indicating that the mutation did not disrupt binding. Interestingly, the mutation of the Novel #2 site resulted in an increase in luciferase activity, suggesting the presence of a negative element; however, this site does not contain consensus binding sites for any known transcription factors. To further address the importance of the Novel #2 site in transcription will require identification of the host proteins that bind to this region.

We also examined three additional putative binding sites identified by others (11, 35) for their contribution to transcription of WDSV. Two sites, AP3 and LVa, were previously identified in the U3 region (11). Mobility shift assays were performed with these sites using walleye cell nuclear extract, and specific binding was observed. Mutation of both the AP3 and LVa sites resulted in decreased luciferase activities, suggesting the importance of these two sites in the transcription activation driven by the WDSV U3 region. The reason we did not observe binding in the DNase I footprinting experiments is not known; however, difficulties in DNase I footprinting using crude nuclear extracts have been previously reported (19).

An additional putative site, NF1, was first identified in database analysis performed by Zhang et al. (35). In their deletion analysis study, removal of the first 60 bp of the U3 region, including the putative NF1 site mapping to –403 to –397, resulted in decreased luciferase activity (35). In our deletion analysis, we also observe a decrease in luciferase activity after removal of the first 50 bp (U3{Delta}1), thus supporting Zhang et al. (35). Based on this information, we further investigated the NF1 site by performing mobility shift analysis and site-directed mutagenesis on the NF1 site in the context of the luciferase reporter plasmid. Specific binding was observed in mobility shift analysis, and mutation of NF1 resulted in a significant decrease in luciferase activity.

Previous work by Zhang et al. (35) using deletion mutants demonstrated that deletion of nucleotides –440 to –325, which removed the first four 5-bp repeats, resulted in an increase in luciferase activity, suggesting that a negative regulator of transcription functions through these repeats. In this report, deletion of –440 to –291 also resulted in an increase of luciferase activity. However, site-directed mutation of the 5-bp repeats did not result in a significant change in luciferase activity. Therefore, we cannot unequivocally define this region as binding a negative regulator of transcription.

The Whn, AP1, and E4BP4 #2 sites reside in close proximity to each other (nucleotides –294 to –269). When Zhang et al. deleted the portion of the U3 region from –440 to –275, which removes the Whn binding site, the entire AP1 site, and the E4BP4 #2 site, a reduction in luciferase activity was observed (35). In our deletion analysis, removal of the Whn, AP1, and E4BP4 #2 sites (U3{Delta}4) resulted in a decrease in luciferase activity (Fig. 1, compare U3{Delta}3 to U3{Delta}4), confirming the observation by Zhang et al. (35). We further extend these studies with site-directed mutations of the Whn, AP1, and E4BP4 #2 sites, which all resulted in significant decreases in activity compared to that observed with the wild-type U3 construct.

The AP1 site present in the U3 region was further analyzed with antiserum generated against the DNA-binding domain of avian Jun. Western blot analysis demonstrated the presence of walleye Jun in W12 nuclear extracts, and this antiserum could specifically disrupt the protein-DNA complex formed in mobility shift assays. This demonstrates that Jun is present in walleye cell nuclear extracts and participates in binding to the AP1 site. Antiserum that recognizes all of the Fos family members was also able to disrupt complex formation, suggesting that a c-Jun/Fos heterodimer binds to the AP1 site. The identification of the Fos family member will require antibodies that recognize individual Fos proteins. Interestingly, the anti-Jun antiserum disrupted the majority of the protein-DNA complexes formed with W12 nuclear extracts and a probe containing the Whn, AP1, and E4BP4 #2 sites. This suggests that AP1 is the predominant factor binding to this region or that AP1 binding is required for Whn and E4BP4 to bind. However, footprint analysis revealed protection over all three sites, and site-directed mutagenesis of all three sites resulted in decreased levels of luciferase activity. Therefore, further analysis of this region using additional antibodies and/or purified proteins is necessary.

Experiments addressing the functional role of the two E4BP4 sites revealed that mutation of only E4BP4 #2 resulted in a decrease in luciferase activity. Mobility shift analysis of each site revealed similar patterns of binding, although bands 2 and 3 of E4BP4 #2 consistently bound more nuclear extract than those of E4BP4 #1. The binding pattern observed is consistent with a previously published report investigating the role of E4BP4 regulation by interleukin-3 in Baf-3 pro-B lymphocytes (12). E4BP4 #1 matches the E4BP4 consensus sequence determined by Cowell et al. (7) exactly, whereas E4BP4 #2 differs from the consensus sequence by one base. The base that differs from the consensus in E4BP4 #2 is the last base of the AP1 site. We currently don't know why mutation of E4BP4 #2 results in decreased activity from the WDSV reporter construct, while no difference is apparent with mutation of E4BP4 #1. However, the difference could be attributed to the sequence difference between the two sites, which leads to a weaker binding of E4BP4 to E4BP4 #1, as suggested by the mobility shift assays, or perhaps E4BP4 needs to interact with other transcription factors, such as AP1. With respect to this idea, a recent report has indicated that the purified dimerization domains of Jun and Fos family members do not interact with the purified dimerization domain of E4BP4; however, these experiments do not rule out an interaction of the full-length proteins (18).

Perhaps the most interesting observation resulting from these experiments concerns the 15-bp repeats in the U3 region. The footprint patterns of the 15-bp repeats for the W12 and regressing-tumor nuclear extracts are clearly distinct. This difference could be attributed to different cellular factors binding to the 15-bp repeats, which is supported by the distinct footprint patterns, and to the presence of different hypersensitive sites. Another possibility would be different levels of the same cellular factors, which is supported by two similar regions of protection and by the presence of two identical hypersensitive sites. This leads to the hypothesis that the 15-bp repeats contribute to the differential expression of WDSV. EMSA revealed a different pattern of protein-DNA complex formation when nuclear extracts from regressing tumors were compared to those formed on the 15-bp repeat probe with W12 cell and extracts from developing tumors. These results suggest that the transcription factor compositions of the developing tumors and W12 cells are similar. Interestingly, when W12 cells are infected with WDSV, only low levels of accessory viral transcripts, like those seen with developing tumors, are detected (Quackenbush, unpublished). Six different transcription factor-binding sites were identified within the 15-bp repeat region. Mutation of three of these sites, HNF3ß, CHR2, and CDP/Pax, resulted in an increase in luciferase activity when activity in W12 cells was assayed, suggesting that these proteins may contribute to the low level of viral gene expression observed early in infection with WDSV. CDP is a repressor of mouse mammary tumor virus (MMTV) expression (37, 38). Expression of CDP decreases as the mammary gland differentiates, which correlates with increased MMTV expression. Further studies identifying the factor(s), either host or viral or a combination of both, that binds to the 15-bp repeats will provide insight into the regulation of WDSV gene expression.


   ACKNOWLEDGMENTS
 
We thank Paul R. Bowser and James W. Casey for providing tumor samples.

This research was supported by research project grant RPG-00-313-01-MBC from the American Cancer Society. J.R. was supported by National Research Service Award F32 CA88572-01.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Biosciences, 7047 Haworth Hall, 1200 Sunnyside Ave., The University of Kansas, Lawrence, KS 66045. Phone: (785) 864-4022. Fax: (785) 864-5294. E-mail: squack{at}ku.edu. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Virology, July 2004, p. 7590-7601, Vol. 78, No. 14
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.14.7590-7601.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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