Article Figures & Data
Figures
- Fig. 1.
cis-acting elements in the U3 region of BLV. (A) Schematic map of the BLV LTR showing the locations of thecis-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.
Tables
- Table 1.
Sequences of primers and linkers used in this study
Name Location Sequence Gene-specific primers used for in vivo footprinting Primer set A (noncoding strand) A1 (bp −211 to −191) 5′-TGTATGAAAGATCATGCCGA-3′ A2 (bp −200 to −180) 5′-ATCATGCCGACCTAGGAGCCG-3′ A3 (bp −181 to −156) 5′-CCACCGCCCCGTAAACCAGACAGA-3′ Primer set B (coding strand) B1 (bp 8 to −16) 5′-AGCTCAATCGCCGTGGTCTTCGCAA-3′ B2 (bp −6 to −30) 5′-CCGCTAACTCGACAGGGCCGGCATT-3′ B3 (bp −30 to −57) 5′-TTATTAATTTATCAGCAGGTGAGGTCAG-3′ Primer set C (coding strand) C1 (bp 97 to 74) 5′-TACCTGACCGCTGCCGGATAGCCG-3′ C2 (bp 84 to 60) 5′-CCGGATAGCCGACCAGAAGGTCTCG-3′ C3 (bp 60 to 34) 5′-GGGAGCAAGAGAGCTCAGGACCGA-3′ Linkers 1 5′-GCGGTGACCCGGGAGATCTGAATTC-3′ 2 5′-GAATTCAGATC-3′ Primers used for mutagenesis Vector primers 1 5′-ACGGUUAUCCACAGAAUCA-3′ 2 5′-ACUGGAACAACACUCAACC-3′ Universal primers 1 5′-AUUCUGUGGAUAACCGUA-3′ 2 5′-AGUGUUGUUCCAGUTTGG-3′ Primers for CAT plasmid constructs 1 LTR 5′ primer 5′-TGTATGAAAGATCATGCCGAC-3′ 2 LTR 3′ primer 5′-ATTGTTTGCCGGTCTCTCCT-3′ - Table 2.
Sequence changes of 25 BLV LTR mutants made by site-directed mutagenesis
Namea Site of mutationb Original sequence Mutated sequence d-CRE1 −157 to −153 AGACG Deleted d-CRE2 −131 to −129 GAC Deleted d-CRE3 −57 to −54 TGAC Deleted s-CRE3 −56 G T d-CRE 1/2 −157 to −153 AGACG Deleted −131 to −129 GAC Deleted d-CRE 1/3 −157 to −153 AGACG Deleted −57 to −54 TGAC Deleted dCRE 1/s-GRE −157 to −153 AGACG Deleted −71 to −60 ACA---TG-CT TAT---AC--TA dCRE2/s-GRE −131 to −129 GAC Deleted −71 to −60 ACA---TG--CT TAT---AC--TA d-CRE2/3 −131 to −129 GAC Deleted −57 to −54 TGAC Deleted d-CRE3/a-GRE −57 to −54 TGAC Deleted −71 to −72 Wild type TATCC added d-CRE1/2/3 −157 to −153 AGACG Deleted −131 to −129 GAC Deleted −57 to −54 TGAC Deleted d-CRE1/2/s-GRE −157 to −153 AGACG Deleted −131 to −129 GAC Deleted −71 to −60 ACA---TG--CT TAT---AC--TA d-CRE2/3/a-GRE −131 to −129 GAC Deleted −57 to −54 TGAC Deleted −71 to −72 Wild type TATCC added d-CRE1/2/3/a-GRE −157 to −153 AGACG Deleted −131 to −129 GAC Deleted −57 to −54 TGAC Deleted −71 to −72 Wild type TATCC added s-GRE −71 to −60 ACA---TG--CT TAT---AC--TA s-167 −167 A G s-AP4-1 −151 G T s-142,3 −142 to −143 AG CT s-140 −140 A G s-AP4-2 −124 G T s-116 −116 C A s-107,8 −107 to −108 CC AT s-99 −99 C T s-83,4 −83 to −84 TC CT s,a-TATA −41 to −40 TA AT −37 to −38 Wild type T added
