Identification of novel alternatively spliced HTLV-1 mRNAs. a RT-PCR analysis of doubly spliced mRNAs produced in HLtat cells transfected with a plasmid containing the ACH molecular clone . RT-PCR was carried out with primers (Table 1) to detect transcripts joining exon 2 to the splice acceptor (SA) site of exon 3 (nucleotide 6950) or alternative to exon 3 (nucleotides 6875, 6878 and 6962). b Exon composition of the canonical (1-2-3) mRNA and of the three novel mRNAs (1-2-3a, 1-2-C and 1-2-Ca). The Hela-derived cell line HLtat  was maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (Invitrogen), 100 units/mL penicillin, and 20 units/mL streptomycin. HLtat cells were seeded into 35 mm cell culture plates at 1.5 × 105 cells/plate and transfected 1 day later with 1 µg of the ACH plasmid using GeneJuice Transfection Reagent (Novagene, Merck Millipore) and harvested 24 h after transfection for RNA extraction, DNAase treatment, reverse-transcription and PCR analysis (RT-PCR) as previously described .