Figure 1

Follow-up of sheep S2531: silencing occurssimultaneously with the onset of leukemia. (A) Blood samples were collected from S2531 at regular time intervals over a 18-month period from the time of inoculation to the leukemic stage and examined for several parameters. WBC counts per mm³ are indicated. Provirus load and integration were examined by Southern blot hybridization of SacI- and EcoRI-digests respectively, showing increasing provirus load and the progression from polyclonal to monoclonal integration as leukemia develops. The nucleotide sequence of the 3' end of the proviral tax DNA is illustrated by a polyacrylamide gel autoradiography of dideoxynucleotide sequenced PCR-amplified DNA. Boxes highlight nucleotides at positions 8149, 8150 and 8151 of the BLV sequence [29]. Arrows indicate the nucleotide identified at position 8149: a G at pre-leukemic stages (yellow arrow); a G to A transition at the time of the first documented WBC increase (17-month post-infection, red arrow). The resulting amino acid at position 303 of the corresponding Tax proteins is shown below. The transactivation potential of the putative S2531 proviral Tax proteins were examined in a luciferase reporter assay following co-transfection of HeLa cells with the pSGTax₂₅₃₁ expression vectors containing tax sequences cloned from S2531 PBMCs collected at different times post-infection and the reporter plasmid pLTR-Luc as detailed in B. "+" indicates a luciferase activity equivalent to that resulting from transfection with the wild-type pSGTax; "-" indicates the background level activity similar to that obtained when the empty expression vector pSG5 is co-transfected with pLTR-Luc. (B) Luciferase assay reflecting the transactivation potential of a selection of four S2531-derived tax sequences. Each pSGTax₂₅₃₁ construct containing the different S2531-derived tax sequences downstream of the CMV promoter was used in HeLa co-transfection with pLTR-Luc which expresses the firefly luciferase under the control of the BLV-LTR promoter. Luciferase activities were measured in cell lysates 42 h posttransfection and were normalized to protein concentrations as previously described [19]. Results are represented as histograms indicating basal luciferase activities (arbitrary units). pSGTax_2531–6 and pSGTax_2531–14 contain sequences amplified from PBMCs isolated during the aleukemic stage, 6 and 14 months post-inoculation respectively; pSGTax_2531–18 contains tax sequences from leukemic PBMC isolated 18 months post-inoculation, and the pSGTax_2531-tum construct resulted from the insertion of lymphoma-derived tax sequences collected 18 months post-infection. pSGc is the empty control vector. Values represent the means of the results of triplicate samples. The results from a representative experiment of four independent experiments are shown.