Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Sicotte, Saint-Hyacinthe, PQ J2S 7C5, Canada1
Author for correspondence: D. Martineau.Fax +1 450 778 8113. e-mail martinea{at}ere.umontreal.ca
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Abstract |
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Introduction |
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WDSV is a complex retrovirus which has three putative non-structural/accessory genes in the viral genome, designated orfA, orfB and orfC, in addition to the obligatory retroviral structural genes, gag, pol and env (Holzschu et al.,1995 ). Like all complex retroviruses identified to date, WDSV contains two non-overlapping short open reading frames downstream of the env gene, one of which, orfA, is similar to cyclins at the amino acid sequence level. Most strikingly, an open reading frame, orfC (119 aa), is located in the leader region preceding the gag gene, a unique feature among members of the family Retroviridae.
To understand the mechanisms behind the seasonal tumour development and regression induced by WDSV, we have functionally characterized the long terminal repeat (LTR) of WDSV by constructing a WDSV LTRluciferase reporter construct and 5' deletion mutants. Based on data from transient transfection studies, we identified several regions within the U3 region of the WDSV LTR that have positive or negative effects on viral promoter transcriptional activity.
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Methods |
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NIH 3T3 cells obtained from ATCC were cultured in DMEM, supplemented with 10% FBS and 50 µg/ml gentamycin, and were maintained at 37 °C with 5% CO2.
Identification of transcription factor elements by DNA sequence analysis.
The University of Wisconsin Genetic Computer Group Find program was used to identify putative transcription factor elements (TFEs). The locations and sequences of potential TFEs identified by this analysis are presented in Fig. 1. The consensus sequences on which these TFEs are based are presented in Table 1
, where the coordinates of each TFE are denoted as the first base of the sequence relative to the viral transcription start site in WDSV 5' LTR.
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Cell transfection and luciferase assay.
Plasmids used for DNA transfection were prepared using Qiagen columns following the suppliers protocol. Transfection was carried out with the LipofectAMINE reagent (2 mg/ml) (GIBCO BRL). To achieve the highest transfection efficiency, several parameters including the amounts of DNA and LipofectAMINE reagent, cell density, transfection time and post-transfection expression time were optimized. Briefly, 5x105 cells/cm2 were seeded in 6-well plates the day before transfection. Plasmid DNA (1 µg) and LipofectAMINE reagent (6 µl) in each well were complexed in twelve 75 mm sterile tubes for 45 min at room temperature in 0·2 ml (for each well) of MEM without serum and antibiotics. During lipid/DNA complex formation, the cells were washed once with 2 ml serum-free MEM medium. For each transfection, 0·8 ml serum-free MEM was added to the tubes containing the lipid/DNA complexes. The transfection medium containing lipid/DNA complexes was then overlaid on the cells. The cells were incubated at 25 °C for 9 h. Following incubation, 1 ml growth medium containing 20% FBS was introduced into each well without removing the transfection mixture and incubation was carried out for 12 h. The transfection medium was then replaced with fresh, complete medium containing 10% FBS. The transfected cells were allowed to grow for 24 h and were harvested for luciferase assays.
For the functional assays at various temperatures, cells were transfected at 25 °C, kept for 6 h without serum, and were allowed to grow at the selected temperatures (25, 20, 15 and 7 °C). For the luciferase assay, the medium was removed from the transfected cells followed by a single wash with PBS. The cells were trypsinized and pelleted by centrifugation at 800 g for 5 min. The pellets were then washed once with 1 ml PBS. Following the washing, the cells were lysed with 100 µl reporter gene lysis buffer (Promega) at room temperature for 30 min. Cell lysis solution (50 µl) was used to perform the luciferase assay in a LUMAT LB 9501/16 (Berthold). Each assay was carried out with 100 µl luciferin substrate solution using 10 s for light counting time.
Three separate experiments were carried out in fish cells (W12 cells) to assay WDSV LTR and deletion mutants at various temperatures; two experiments were carried out in triplicate and one experiment in duplicate. Four separate experiments were carried out in fish cells in triplicate and three separate experiments were carried out in mammalian cells (NIH 3T3 cells) in triplicate to assay chimeric promoters composed of the WDSV LTR 5' end and the SV-40 minimal early promoter.
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Results |
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Effects of 5' progressive deletions on WDSV LTR-directed luciferase activity
There were no statistically significant differences between protein concentrations of W12 cells transfected with different constructs (pGL2-Basic, pLTR and pActin) or incubated at various temperatures (7, 10, 15, 20 and 25 °C). To investigate the cis-acting regulatory elements present in U3, the effect of sequential, progressive deletions on the LTR-directed luciferase activity was examined. Each mutant was assayed for its ability to direct the synthesis of a reporter gene product, firefly luciferase, following transfection of constructs into W12 cells cultured at various temperatures. The full-length LTR was active in W12 cells and in NIH 3T3 cells (Figs 2 and 3
). In W12 cells kept at 25 °C, LTR-directed luciferase activity was 3·4- to 6·9-fold higher than that of the carp
-actin promoter. Both WDSV-LTR and the carp
-actin promoter were most active at 25 °C and showed the same pattern of temperature-dependent activity.
In cells kept at 25 °C, deletion of the 5' 60 bp (p-380) of the LTR (containing four potential transcription regulatory elements; Table 1, Fig. 1
), decreased luciferase activity levels 1·2- to 2·3-fold compared to the full-length LTR. In cells kept at lower temperatures, luciferase activity levels remained unchanged. A similar temperature-specific pattern was shown by mutants p-380 to p-336.
Removal of 8, 3 and 22 additional bp (p-372, p-369 and p-347, respectively), which deleted potential AP-1-SV-40, CRE and the first E-box elements, did not affect luciferase activity. Removal of a further 4 bp (p-343), resulting in the disruption of a putative TEF1 binding site, increased luciferase activity 1·4- to 2·7-fold at all temperatures. Removal of an additional 7 bp (p-336) decreased luciferase activity (1·3- to 7·2-fold) at all temperatures. Deletion of the first two of five 5 bp direct repeats (p-325) increased luciferase activity 3- to 7-fold at 25 °C, 1·9- to 4-fold at 20 °C, 2- to 8-fold at 15 °C and 2·2- to 4·8-fold at 7 °C.
Deletion of two additional 5 bp direct repeats (p-288) decreased the luciferase activity 1·1- to 2·2-fold. Deletion of a potential AP-1 element (p-275) and most of the second E-box sequence decreased the luciferase activity 1·7- to 3·3-fold at 25 °C, 2·3- to 3·1-fold at 20 °C, 1·5- to 2·9-fold at 15 °C and 2- to 3·1-fold at 7 °C. Further deletions (p-223 to p-89) produced a gradual decrease in luciferase activity, with p-89 showing the lowest level. Removal of the adjacent three 15 bp direct repeats (p-43) slightly increased luciferase activity compared to that of p-89.
Effects of U3 5' half on SV-40 promoter activity
Based on the presence of temperature-specific promoter elements in the 5' half of WDSV U3, experiments were conducted to determine whether this region can confer temperature responsiveness to a heterologous promoter. Two chimeric promoters were constructed by inserting the 5' half of WDSV U3 (nt -440 to -218), upstream of the SV-40 early promoter in both orientations. The resulting constructs were introduced into W12 cells and transfected cells were allowed to grow at different temperatures. Surprisingly, the 5' half of U3 inserted in the forward orientation slightly (1·3- to 2-fold) increased the SV-40 early promoter activity at 20 °C in fish cells when compared to the SV-40 early promoter alone (Fig. 3) while in the reverse orientation, the 5' half of U3 slightly decreased (1050%) luciferase activity at all temperatures (Fig. 3
).
To determine whether this LTR region is functional in mammalian cells, these chimeric promoters were transfected into NIH 3T3 cells allowed to grow either at 37 °C or at 25 °C after transfection. Compared to the luciferase activity directed by the SV-40 early promoter alone, the insertion of this region in a forward orientation resulted in a 3·7- to 5·1-fold increase at 37 °C and in a lesser increase (1·4- to 4·2-fold) at 25 °C (Fig. 3), whereas the same fragment in the reverse orientation had no effect (0·65- to 1·6-fold).
Functional analysis of a putative GRE in the U3 of WDSV LTR
As sequence analysis of the LTR revealed the presence of a putative GRE in the U3 region of WDSV LTR, this putative element was functionally examined in NIH 3T3 cells, which express the glucocorticoid receptor (GR) (Hatzoglou et al.,1991 ). The luciferase reporter gene under the control of the MMTV promoter was used as a positive control. The plasmid pLTR containing the entire WDSV LTR was transfected into NIH 3T3 cells. Dexamethasone increased the MMTV promoter activity 10-fold, whereas treatment with progesterone did not affect it (Fig. 4
). In contrast, dexamethasone and progesterone had no effect on WDSV LTR-directed luciferase activity in NIH 3T3 cells.
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Discussion |
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We have used a transient expression assay to study the effects of sequential deletions of the U3 region on the transcriptional activity of WDSV LTR. In fish cells, deletion of the first 60 bp significantly decreased luciferase activity levels only at 25 °C, that is in a temperature-specific manner. Thus, this region, which contains four putative TFEs (Table 1, Fig. 1
) and a potential EcRE (1 bp mismatch) in tandem repeat, modulates the levels of viral gene expression in a temperature-specific manner. A putative EcRE is also present as an overlapping repeat at the same position in SnRV, the only other fish retrovirus whose nucleotide sequence has been determined (Table 1
; Fig. 1
) (Hart et al., 1996
), and at the 5' end of the regulatory region (-770 to -760) of the temperature-induced carp myosin-heavy-chain gene. In the latter gene, disruption of the putative EcRE abolishes temperature response (Gauvry et al., 1996
). Considered together, the conservation of this putative element in the same orientation and in the same position in two viruses whose hosts are widely different, and in the regulatory region of a fish cellular gene, along with the involvement of EcRE in the regulation of heat-shock protein expression in insect cells (Dobens et al.,1991
) suggest that this element could be functional and could be involved in temperature response in both viruses. Additional mutagenesis studies specifically targeting the potential EcRE will be necessary to confirm this hypothesis.
Several reasons could explain the lack of response to dexamethasone or progesterone treatments in NIH 3T3 cells. Firstly, only the consensus core of the GRE is present in the WDSV LTR and, thus, this putative GRE could be non-functional. Secondly, the GRE consensus present in the WDSV LTR could be the core of a fish-specific GRE that is not recognized by the activated mammalian GR. Lastly, GREs, often present in retroviral enhancers, are not all activated by dexamethasone, probably because contextual sequences impair GRE recognition by the activated GR (Golemis et al.,1989 ). Whether the putative eukaryotic transcription binding element CTF/NF-1 is functional in fish cells remains to be determined. In addition, considering the unique physiological characteristics of fish, we cannot rule out the possibility that other unidentified transcriptional regulatory elements are present in this region.
Further removal of 44 bp did not significantly alter WDSV LTR activity (p-380 to p-344). This suggests that the motifs present in this region (CRE, E-box) do not play a major role in transcription under the conditions used in this study. Seven base pairs located between nt -343 and -336 positively modulated luciferase activity at all temperatures since deletion of this region decreased luciferase activity. Although no putative transcriptional motifs were found within this region, as yet unidentified transcriptional motifs specific to fish might be present.
Short direct repeats are often associated with enhancing the transcriptional activity of retroviral LTRs (Barnhart et al.,1997 ). For instance, Tax activates human T-lymphotropic virus type 1 transcription through DNA sequences present in U3 known as the Tax-responsive elements which consist of three copies of an imperfect 21 bp repeat (Brady et al., 1987
). In WDSV, however, deletion of the first two 5 bp repeats present between nt -336 and -325 increased luciferase activity at all temperatures, implying that these repeats have a negative effect on viral gene expression.
Removal of 13 bp between nt -288 and -275 markedly decreased luciferase activity, suggesting that this region is important to maintain basal levels of transcription at all temperatures. The deleted region encompasses a perfectly matched AP-1 element overlapping an E-box, the fifth 5 bp repeat and the second 10 bp repeat. AP-1 elements have been involved in positively regulating many eukaryotic genes. The U3 region of many retroviruses contains AP-1 elements which positively modulate viral gene expression (Weng et al.,1995 ). In SnRV, a bona fide AP-1 element is also present in an almost identical position, also in proximity of a putative TEF1 element (Table 1
; Fig. 1
) (Hart et al.,1996
). These similarities support the proposal that the putative AP-1 element present in WDSV LTR is functional and plays a crucial role in WDSV expression. Site-specific mutagenesis experiments will be necessary, however, to confirm that AP-1, alone or with an overlapping element, plays a major role in maintaining basal transcriptional activity.
Insertion of a U3 fragment (-440 to -275) containing several potential regulatory elements upstream of the SV-40 early promoter increased the activity of that promoter in an orientation-dependent manner. This finding implies that promoter elements located within this region act positively on the transcription machinery of both poikilotherm and homeotherm animals.
The results presented here represent the first functional analysis of a fish retrovirus LTR and provide insight into the temperature/hormone-dependence of WDSV transcripts. These studies also provide the basis for more detailed study of transcriptional regulation from this interesting and complex retrovirus.
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Acknowledgments |
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Footnotes |
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References |
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Received 19 May 1999;
accepted 30 August 1999.