Figure 2
Sheep S267: non-transformed blood-derived B-cells carry a potentially active provirus while virus and Tax expression are completely suppressed in the the co-existing malignant lymphoma B-cells. (A) Diagram of the BLV L267 provirus and major transcripts. The two LTRs and the gag, pro, pol, env, tax, and rex genes are represented. Vertical arrows indicate restriction sites in the L267 provirus: S, SacI; E, EcoRI. The position and direction of the PCR primers are indicated on the provirus map. The horizontal bar indicates the 8.4 kb-long region that was used as probe. Double lines represent the sequenced regions. The genomic, env, and tax/rex transcripts are represented below. Alternatively spliced RNAs are not shown. The translation products of the singly- and doubly-spliced transcripts and the positions of the RT-PCR primers are indicated. (B) Southern blot analysis following hybridization with a full-length BLV probe of SacI-digested DNA isolated from blood (BL267) and lymphoma (L267-1, -2 and -3) cells collected from S267 twenty nine months post-infection. SacI is indicative of the proviral load (upper row). Southern blot analysis of EcoRI-digested DNA indicates the presence of a single monoclonally-integrated provirus for all three lymphoma (L267) whereas the blood-derived BL267 cells display a polyclonal integration pattern (middle and lower panels). EcoRI-cleaved DNA generates two virus-host junction fragments for each integrated L267 provirus as illustrated in the diagram. Shown here in each lane are the fragments containing the 5' flanking genomic region. (C) Southern blot analysis of EcoRI-digested DNA isolated from the lymphoma (L267-1, -2, -3) and the cell lines derived from each of these lymphoma (CL267-1, -2, -3) cultured for four weeks. (D) RT-PCR analysis of RNA isolated from lymphoma-derived cell lines (CL267), 24 h-cultured blood-derived lymphocytes (BL267-24 h), fresh lymphoma (L267) and freshly isolated blood-derived lymphocytes (BL267). EnvA/Tax2 primers for the detection of the doubly-spliced tax/rex RNA were used. In the controls YR2 and YR2LTaxSN, provirus is silent and active respectively. (E) PCR analysis using BLV tax-specific primer pair Tax1/Tax2 of DNA isolated from sheep inoculated with the various S267-isolated B-cell populations: six sheep were inoculated using either cultured (CL267) or fresh (L267) transformed B-cells, two sheep were injected with nontransformed PBMCs (BL267).