Molecular Analysis of the Second Template Switch During Reverse Transcription of the HIV RNA Template

The molecular events leading to the second template switch during reverse transcription of the HIV genome were studied in a defined in-vitro system. In order to investigate displacement of the tRNAlys primer from the primer binding site (PBS) of the viral genomic RNA, following DNA synthesis, we produced an HIV RNA/DNA substrate that resembles the intermediate reverse transcription complex formed prior to the second template switch. Partial tRNAlys primer displacement was observed during plus (+) strand DNA synthesis and during minus (−) strand DNA elongation. We found two determinants that may serve as a stop signal for (+) DNA strong stop synthesis, the Am at position 19 of the natural tRNAlys and the secondary structure at the PBS sequence. The later signal appears to constitute a stronger terminator in-vitro. The 3‘ end of the nascent (−) DNA strand prior to the second template switch was also determined. It was mapped to the U5−PBS junction at the site for the first endonucleolytic cut introduced by ...