FIG. 1.

Delivery of exogenous Tax leads to viral expression in transformed L267 B cells. (A) Diagram of the BLV provirus and major transcripts. The two LTRs and the genes gag, pro, pol, env, tax, and rex are represented. Vertical arrows indicate restriction sites SacI (S) and EcoRI (E) in the L267 provirus. The positions and directions of the PCR primers are indicated on the provirus map. Horizontal bars indicate regions that were used as probes. Double lines represent the sequenced regions. The genomic env and transcripts tax/rex 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 and C) Detection of BLV-specific transcriptional activity in total RNA isolated from L267, L267_LTaxSN, and YR2_LTaxSN cells. (B) RT-PCR analysis using two sets of splice-specific primers, LTR-R/EnvC and EnvA/Tax2, for detection of the singly and doubly spliced BLV transcripts, respectively. Amplified products (350 bp and 482 bp, respectively) were detected by Southern blot hybridization using a ³²P-labeled BLV full-length proviral DNA probe. G3PDH amplification was detected by ethidium bromide staining. (C) Northern blotting analysis of total RNA isolated from the same samples using a ³²P-labeled Tax probe. G3PDH hybridization was used as a control for RNA input. Sizes of mRNAs are indicated. (D) Southern blot hybridization of EcoRI-digested DNA extracted from PBMCs isolated from L267_LTaxSN-injected leukemic sheep (S2233 and S2311) 30 and 39 months postinoculation, respectively, from PBMCs from sheep injected with the silent L267 cells (S2013 and 2014) collected 24 months postinoculation, and from L267 and L267_LTaxSN cell cultures. The BLV full-length proviral DNA probe was used for hybridization. EcoRI-cleaved DNA generates two virus-host junction fragments for each integrated L267 provirus. Shown here in each lane is the fragment containing the unique 5′ flanking genomic region, a hallmark of monoclonal provirus integration characteristic of leukemic cells. The additional smaller band in L267_LTaxSN corresponds to the LTaxSN vector-derived EcoRI fragment detected with the BLV probe.