Destabilization of tRNA(3)(Lys) from the primer-binding site of HIV-1 genome by anti-A loop polyamide nucleotide analog

Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by extension of the cellular tRNA(3)(Lys) which anneals to the primer-binding site (PBS) on the 5′ non-translated region of the viral RNA genome. The A-rich sequence (A-loop) upstream of the PBS interacts with the anticodon loop of tRNA(3)(Lys) and has been proposed to be essential for conferring specificity to tRNA(3)(Lys) for priming the initiation of HIV-1 reverse transcription. We observed that polyamide nucleic acid targeted to the A-loop sequence (PNAal) exhibits high binding specificity for its target sequence. The PNAal pre-bound to the A-loop sequence prevents tRNA(3)(Lys) priming on the viral RNA consequently blocking in vitro initiation of reverse transcription. Further, PNAal can efficiently disrupt the preformed [tRNA(3)(Lys)–viral RNA] complex thereby rendering it non-functional for reverse transcription. The endogenous reverse transcription in disrupted HIV-1 virions containing packaged tRNA(3)(Lys) and its replicating enzyme RT was significantly inhibited by PNAal, thus providing direct evidence of the involvement of the A-loop region of viral RNA genome in tRNA(3)(Lys) priming process. These findings suggest the potential of the A-loop region as a critical target for blocking HIV-1 replication.