Figure 4

RT-associated mutations frequently occur during BLV minus strand synthesis. A Generation of U5 substitution during minus strand synthesis. RNA is represented as a thin line whereas DNA is represented as thick lines. The first 6 horizontal lines represent the synthesis of the provirus. Line 7 represents the integrated provirus flanked with its two integration sites represented as black boxes. NlaIII restriction sites are represented on both the provirus and the 3' cellular flanking sequence. Lines 8 and 9 represent the first two mitoses of the infected cells harboring the integrated provirus. For each cell, the RU5 sequence and the 3' flanking sequence encompassed by the 2 NlaIII restriction sites, i.e., the sequences obtained after inverse PCR, are represented. Line 10 represents the sequences obtained after inverse PCR, cloning and sequencing. The open circle represents a RT-associated mutation that has occurred during the synthesis of the 5' RU5 minus strand. As shown in lines 1 to 6, this substitution appears to be harbored by both strands of the two LTRs of the integrated provirus. Accordingly, all infected cells from the corresponding clone (identified by their common integration site) harbor a provirus with the same substitution (see lines 8 and 9). As a consequence, all sequences from this clone obtained after inverse PCR harbor the same mutation at the same position. B PCR detection of the G511A U5 substitution along the 5' LTR. Top: 5' BLV LTR from the wild-type (left) and from clone M36m3-1 (right) carrying the putative G511A substitution having occurred during minus strand synthesis, thereby generating the G8696A substitution identified by sequencing IPCR product derived from the sample harvested in animal 4536 three days before seroconversion. In the absence of digestion, specific 5' LTR PCR amplifies a fragment of 632 bp. EaeI digestion of the wild-type sequence abolishes this signal while incubation with EaeI has no effect on the sequence carrying the G511A mutation, leading to the detection of the 632 bp fragment. Samples studied in the presence (+) or in the absence (-) of EaeI digestion derived from BLV infected animals 4536 and 4538, both harvested 3 days before seroconversion, and from the uninfected control animal 4533. C Detection of the T8617C and T8651C substitutions in their corresponding positions along the 5' LTR. Each LTR was specifically amplified by PCR and substitutions were detected by direct sequencing of PCR product, as detailed in the experimental procedures.