An interferon regulatory factor binding site in the U5 region of the bovine leukemia virus long terminal repeat stimulates Tax-independent gene expression

Abstract

Bovine leukemia virus (BLV) replication is controlled by both cis- and trans-acting elements. The virus-encoded transactivator, Tax, is necessary for efficient transcription from the BLV promoter, although it is not present during the early stages of infection. Therefore, sequences that control Tax-independent transcription must play an important role in the initiation of viral gene expression. This study demonstrates that the R-U5 sequence of BLV stimulates Tax-independent reporter gene expression directed by the BLV promoter. R-U5 was also stimulatory when inserted immediately downstream from the transcription initiation site of a heterologous promoter. Progressive deletion analysis of this region revealed that a 46-bp element corresponding to the 5' half of U5 is principally responsible for the stimulation. This element exhibited enhancer activity when inserted upstream or downstream from the herpes simplex virus thymidine kinase promoter. This enhancer contains a binding site for the interferon regulatory factors IRF-1 and IRF-2. A 3-bp mutation that destroys the IRF recognition site caused a twofold decrease in Tax-independent BLV long terminal repeat-driven gene expression. These observations suggest that the IRF binding site in the U5 region of BLV plays a role in the initiation of virus replication.

FIG. 1

Stimulation of gene expression by the R-U5 region of BLV. (A) Schematic representation of the plasmid constructs. The numbering of the LTR sequence refers to the transcription initiation site (first nucleotide of R), considered +1. Solid and open arrows indicate transcription initiation sites in the BLV and HSV TK promoters, respectively. (B) Transient transfection of Raji cells with pLTR(−211/+47)-luc and pLTR-luc. These plasmids were cotransfected with the pRL-SV40 control plasmid, in which the SV40 early promoter is driving Renilla luc gene expression. Luc activities in the cellular extracts were measured 24 h after transfection. For each sample, the firefly Luc activity was corrected relative to the Renilla Luc value. Averages of triplicate samples from one of four experiments are shown. (C) Transient transfection of pTK-cat and pTK-(−22/+320)-cat in different cell lines. These two plasmids were transfected into Raji, Daudi, OVK, and MDBK cells. The Cat activities in the cell lysates were measured 48 h after transfection and are expressed as percentages of deoxychloramphenicol converted to the acetylated product. The average of three independent transfection experiments is shown for each cell line. (D) Transient transfection of pTK-cat, pTK-(−22/+320)-cat, pTK-(+26/+320)-cat, and pTK-(−22/+47)-cat in Raji cells. Cat assays were performed as described for panel C. Stimulation of HSV TK promoter-driven cat expression is defined as the ratio of the Cat activity obtained with an individual plasmid relative to the Cat activity yielded by the pTK-cat vector. The average values from four independent transfection experiments are shown.

FIG. 2

Progressive deletion analysis of the R-U5 stimulatory activity. A set of 3′ deletion mutants of the R-U5 region were obtained by PCR and cloned into pTK-cat, downstream from the HSV TK promoter. Cat activity was assayed after transient transfection of these plasmids in Raji cells. Stimulation of HSV TK promoter-driven cat expression was defined as for Fig. 1D. The stimulation by each deletion mutant is plotted against the boundary of the 3′ deletion in the R-U5 region. The designation of the corresponding plasmid is given below the x axis. Each point is the average from four independent transfection experiments. The R-U5 fragment is diagrammed at the top in scale with the x axis.

FIG. 3

U5 contains a transcriptional enhancer. (A) Schematic representation of the plasmids pLTR(−211/+275)-luc and pLTR(−211/+229)-luc. (B) Transient transfection of the pLTR-luc, pLTR(−211/+275)-luc, pLTR(−211/+229)-luc, and pLTR(−211/+47)-luc constructs in Raji cells. pRL-SV40 was cotransfected as an internal control for transfection efficiency. Luc activities (firefly and Renilla) were determined as described for Fig. 1B. Normalized Luc values from triplicate samples were averaged, and results from one representative experiment are shown. These results were reproduced three times by using two different plasmid preparations. (C) Transient transfection assay in Raji cells. The pTK-U5-I(s)cat plasmid contains the +230-to-+275 U5-I fragment downstream from the promoter, in the natural orientation. The pU5-I(s)-TK-cat and pU5-I(as)-TK-cat constructs contain the U5-I fragment upstream from the promoter, in the sense and antisense orientations, respectively. Cat assays were performed as described for Fig. 1C. Averages obtained from triplicate samples from one of four independent experiments are shown.

FIG. 4

Characterization of factors that bind to the U5-I element. (A) Oligonucleotides used in the EMSA. The ISG15 ISRE and the ISRE-like motif in U5 are underlined. Boldface characters represent mutated residues. The sequences presented are the positive strand of the BLV LTR and the antisense strand of the ISG15 promoter. (B) Competition EMSAs. Oligonucleotides containing the well-characterized ISRE from the ISG15 gene (ISRE_ISG15wt) or the BLV ISRE-like motif (IRF_BLVwt) were tested in competition experiments. The labelled U5-I oligonucleotide was incubated with Raji cell nuclear extracts and 5 μg of poly(dI)-poly(dC), either in the absence of a specific competitor (control lanes, indicated by minus signs) or in the presence of increasing concentrations (4-, 20-, 100-, and 500-fold molar excesses) of the homologous oligonucleotide (U5-I), IRF_BLVwt, an oligonucleotide containing three substitutions in the ISRE-like motif (IRF_BLVmut), ISRE_ISG15wt, or a mutated oligonucleotide (ISRE_ISG15mut) in which the ISRE was destroyed. (C) Competition EMSAs. The labelled IRF_BLVwt oligonucleotide was incubated with nuclear extracts of Raji cells (lane 1) or Daudi cells (lanes 2 to 17) in the absence of specific competitor (controls, lanes 1 and 2) or in the presence of increasing concentrations (4-, 20-, and 100-fold molar excesses) of the homologous competitor (IRF_BLVwt), IRF_BLVmut, ISRE_ISG15wt, or a nonspecific competitor (Sp1 consensus). Poly(dI)-poly(dC) was 1.5 μg per binding reaction. (D) Supershift assay. The ISRE_ISG15wt (lanes 1 to 14) and IRF_BLVwt (lanes 15 to 28) oligonucleotide probes were incubated with nuclear extracts from untreated (−IFNα) or treated (+IFNα; 500 U/ml for 6 h) Daudi cells. Supershift assays were performed in the absence of antibodies (lanes 1, 8, 15, and 22) or in the presence of anti-IRF-1 antibody (lanes 3, 10, 17, and 24), anti-IRF-2 antibody (lanes 4, 11, 18, and 25), purified rabbit IgG as a control (lanes 2, 9, 16, and 23), anti-Stat-1 antibody (lanes 6, 13, 20, and 27), anti-Stat-2 antibody (lanes 7, 14, 21, and 28) or preimmune serum for the control (lanes 5, 12, 19, and 26). Poly(dI)-poly(dC) was 0.5 μg per binding reaction. A 48-h exposure of the autoradiograms is shown for nuclear extracts of untreated cells (lanes 1 to 7 and 15 to 21), and a 16-h exposure is shown for the extracts of IFN-treated cells (lanes 8 to 14 and 22 to 28). Lanes 22′ to 25′ are a longer exposure (48 h) of the gel portion from lanes 22 to 25. (E) Competition EMSAs. The ISRE_ISG15wt probe was incubated with nuclear extract from IFN-α-treated Daudi cells and 0.5 μg of poly(dI)-poly(dC), either in the absence of a specific competitor or in the presence of a 50-fold molar excess of the ISRE_ISG15wt, ISRE_ISG15mut, IRF_BLVwt, or IRF_BLVmut competitor.

FIG. 5

Mutation of the IRF binding site in U5 decreases Tax-independent LTR-driven gene expression. Plasmids containing the firefly luc gene under the transcriptional control of either the BLV U3 region (−211 to +47), U3-R region (−211 to +229), wild-type LTR, or mutated LTR (where the IRF binding site was destroyed) were cotransfected in Raji cells with the pRL-SV40 control plasmid. Luc activity was measured and corrected as described for Fig. 1B. Averages obtained from triplicate samples in a representative experiment are shown. Similar results have been obtained in five independent transfection experiments using two different plasmid preparations.