HTLV-2 antisense transcripts are detected and are spliced. (A) The HTLV-2 molecular clone pH6neo provirus is depicted with all known genes and is adapted from Feuer and Green.²  The positioning of the putative APH-2 ORF from the antisense strand is indicated below the proviral DNA. A 5′ deleted version of this vector termed pHTLV-2 ΔEcoRI is also presented and encompasses nt 4683 to 8952. The primers used for the RT-PCR experiments are also represented with their positioning. The dashed line represents the hypothetical antisense transcript. (B-C) The 293T cells were transfected either with pHTLV-2 ΔEcoRI and/or (C) with pH6neo. RNA was extracted and analyzed by RT-PCR for the presence of antisense transcripts using the primer combinations 19-1/21-2 and 22-1/21-2. (C) The 22-1/21-2 primer set that allows the amplification of the spliced transcript was used. When indicated, RT was omitted during the RT step. Control indicates no cDNA; M, 100-bp marker. (D) The position of exon 1 and exon 2 of the APH-2 transcript (■) is presented. The size of the RT-PCR products is also indicated. (E) The splice donor and acceptor sequences of the APH-2 transcript are conserved. DNA sequences containing the APH-2 splice donor and splice acceptor sites were compared between various HTLV-2 and STLV-2 isolates retrieved from GenBank. The modified sequence of the MO proviral DNA was used as a reference. Bold letters represent exonic sequence; underlined, SD and SA sequences. The HTLV-2 subtype is indicated on the left of the panel. Predicted amino acid sequence of the spliced APH-2 transcript at the splice junction are also presented above the exonic nucleotide sequence.