In vivo protein binding and functional analysis of cis-acting elements in the U3 region of the bovine leukemia virus long terminal repeat

Abstract

Bovine leukemia virus (BLV) is a member of the human T-cell leukemia virus (HTLV)/BLV group of retroviruses. These viruses regulate their own transcription by producing Tax, a protein which activates the virus promoter region, the long terminal repeat (LTR). To explore the molecular mechanisms involved in the transactivation, we identified protein binding elements by in vivo footprinting and analyzed their function by site- directed mutagenesis. We used in vivo dimethyl sulfate footprinting by ligation-mediated PCR to detect constitutive in vivo protein-DNA interactions in a BLV-producing cell line, Bat2Cl6. The U3 region and part of the R region of the LTR were footprinted. In addition to the cis-acting elements (three cyclic AMP-responsive elements [CREs] and two AP4 sites) reported by others to be important for Tax-mediated activation of the BLV LTR, we found footprints in regions flanking these elements and in the core promoter region. The importance of these sites for transcriptional activation was studied by site-directed mutagenesis followed by promoter function analysis of the mutants with a chloramphenicol acetyltransferase reporter system. Our data corroborate those of others showing that the CREs are necessary for transactivation of the LTR, and they identify two new functional sites not previously reported by others. We show that the middle region of the BLV U3 contains multiple dual-functioning cis-acting elements which act as either positive or negative regulatory elements depending on the cell type tested. This is the first report of a functional mapping of the cis-acting elements of a virus of the HTLV/BLV group.

FIG. 1

cis-acting elements in the U3 region of BLV. (A) Schematic map of the BLV LTR showing the locations of the cis-acting elements in the U3 region and the binding sites for the primers used for in vivo footprinting. (B) Nucleotide sequence comparison of response elements in the BLV U3 region: the TREs, consensus CRE and CRE-like sequences including the AP4 sites, and the consensus GRE and GRE-like sequence. Bases different from the consensus sequences are shown in boldface type.

FIG. 2

In vivo DMS footprinting of the BLV U3 region in the presence of Tax. BLV-infected Bat₂Cl₆ cells were used as the source of DNA. Lanes: 1 and 2, noncoding strand with primer set A; 3 and 4, coding strand with primer sets B and C. In vitro (purified DNA) DMS-treated samples are in lanes 1 and 3. In vivo (living cells) DMS-treated samples are in lanes 2 and 4. The locations of the bands relative to the RNA start site are labeled at the left of each gel (negative numbers for sequences upstream and positive numbers for the downstream of the start site). Increased sensitivity to DMS (band in lane 2 darker than in lane 1, and band in lane 4 darker than in lane 3) is indicated by arrows pointing away from the gel, and decreased sensitivity to DMS (lighter or no bands in lanes 2 and 4) is indicated by arrows pointing toward the gel. cis-acting elements reported in the literature are indicated by open boxes on the right of the gel, the consensus sequences of the AP4 sites are indicated by solid boxes, the core sequences of three CRE-like elements are indicated by the hatched boxes, and the GREs are indicated by dotted boxes.

FIG. 3

Gel shift assay to detect in vitro protein binding to wild-type and some mutant cis-acting elements in the BLV LTR. Synthetic double-stranded oligonucleotides were end labeled with [γ-³²P]ATP and used as probes. Nuclear protein extracts were prepared from Bat₂Cl₆ (BLV-producing cell line) and Tb₁Lu (BLV-free parental cell line of Bat₂Cl₆). Labeled probes were incubated with nuclear extracts in the presence (+) or absence (−) of unlabeled probe (100 times the concentration of the labeled probe) and separated on a 5% nondenaturing polyacrylamide gel. Protein-bound and free probes are indicated by the arrowheads. Tb₁Lu cell extracts were used for lanes 1, 4, 7, and 10 and Bat₂Cl₆ cell extracts were used for the remaining lanes.

FIG. 4

Basal promoter function of the wild-type BLV U3 and 25 LTR mutants in the absence (A) and presence (B) of p34Tax (solid bars) with values in the absence of p34Tax (open bars) shown for comparison. LTRs were inserted into a CAT reporter construct and transfected into BLV-negative Tb₁Lu cells with or without cotransfection with a tax-containing plasmid. Mutants are arranged according to their location on the U3 map (Fig. 1). Promoter activity was measured as nanograms of CAT protein per 10 μg of total protein, and values were standardized by scaling the activity of the wild-type LTR to 1.0. Each value is the mean of five (A) or two (B) trials in separate experiments. Asterisks indicate that the CAT activity of the mutant is significantly different from the wild-type LTR (P ≤ 0.05 [Dunnett t test]). Abbreviations: a, addition; d, deletion; s, substitution; WT, wild type. Acronyms in capital letters refer to sites illustrated in Fig. 1.

FIG. 5

Promoter function of the wild-type BLV LTR and 11 LTR mutants in six different cell lines: BL3 (bovine B lymphocytes) (A), Raji (human B lymphocytes) (B), FLK (sheep fibroblasts) (C), GR (mouse mammary epithelial cells) (D), MCF-7 cells (human mammary epithelial cells) (E), and Tb₁Lu (bat lung epithelium) (F). For BL3 and FLK cells, only the plasmid with the wild-type or mutant LTR sequences was transfected, since these cell lines contain endogenous p34Tax. For the other four lines, wild-type or mutant LTR was cotransfected into cells with the pSGtax plasmid. Bars represent the mean values (nanograms of CAT protein per 10 μg of total protein) from two to four independent experiments. Values were standardized by scaling the activity of the wild-type LTR to 1.0. Asterisks represent statistically significant differences (P ≤ 0.05 [Dunnett t test]) relative to the wild-type LTR.

FIG. 6

Functional regions of the BLV U3. The genome structure of the whole BLV provirus is shown at the top. A schematic map of the U3 region is shown in the middle of the figure. The locations of three DRs are indicated by brackets. Possible protein binding sites are marked with boxes of different shapes (indicating different types of proteins). The locations of sequence changes in 15 mutants are indicated by asterisks (14 of the sites had altered in vivo footprints; no footprints were detected at bp −167). Mapping of the U3 region was based on the promoter activity of the mutants. Mutants with increased promoter activity were used to define an NRE, and the ones with decreased activity were used to define a PRE. The core promoter region was defined as reported in the literature (6, 9, 10). Regulatory regions were defined according to the reactions represented in Fig. 4B. Elements within the dually functional region function as NREs in some cell lines and as PREs in other cell lines.