Human T-cell leukemia virus type 1 uses a specific tRNA^Pro isodecoder to prime reverse transcription

Primer extension assays to probe the methylation state of A58 of tRNA^Pro in MT-2 cells. (A) Plus-strand strong-stop DNA ([+] ssDNA) synthesis terminates 1 nt before A58 due to N1 methylation (m¹A58); this ensures proper completion of plus-strand synthesis (left). In the absence of m¹A58, (+) ssDNA synthesis terminates downstream from A58, and the product contains sequences not present in the gRNA, resulting in failed plus-strand synthesis (right). (B) (Left) Sequence of unmodified human (isodecoder identified in this work as the HTLV-1 RT primer). (Right) Denaturing polyacrylamide gel showing migration of ³²P-labeled DNA primers and extension products. (Lanes 1 and 5) The 5′ ³²P-labeled 16 nt primers complementary to the 3′ end of tRNA^Pro or , respectively. (Lanes 2 and 6) Primer extension products with in vitro transcribed or . (Lanes 3 and 7) Primer extension products with cellular tRNA^Pro or . (Lanes 4 and 8) Primer extension products using cellular tRNA^Pro or following demethylation with AlkB.