Specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer tRNA₃^Lys

Initiation of RNA-dependent DNA synthesis by retroviral reverse transcriptases is generally considered as unspecific. In the case of human immunodeficiency virus type 1 (HIV-1), the natural primer is tRNA₃^Lys. We recently found evidence of complex interactions between tRNA₃^Lys and HIV-1 RNA that may be involved in the priming process. In this study, we compare the ability of natural and unmodified synthetic tRNA₃^Lys and 18mer oligoribo- and oligodeoxyribonucleotides complementary to the viral primer binding site to initiate replication of HIV-1 RNA using either homologous or heterologous reverse transcriptases. We show that HIV-1 RNA, HIV-1 reverse transcriptase and primer tRNA₃^Lys form a specific initiation complex that differs from the unspecific elongation complex formed when an oligodeoxyribo-nucleotide is used as primer. Modified nucleosides of tRNA₃^Lys are required for efficient initiation and transition to elongation. Transition from initiation to elongation, but not initiation of reverse transcrip-tion itself, is facilitated by extended primer-template interactions. Elongation, but not initiation of reverse transcription, is inhibited by Mn²⁺, which further differentiates these two different functional states of reverse transcriptase. These results define initiation of reverse transcription as a new target to block viral replication.