Abstract
Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by extension of the cellular tRNA3(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 tRNA3(Lys) and has been proposed to be essential for conferring specificity to tRNA3(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 tRNA3(Lys) priming on the viral RNA consequently blocking in vitro initiation of reverse transcription. Further, PNAAL can efficiently disrupt the preformed [tRNA3(Lys)--viral RNA] complex thereby rendering it non-functional for reverse transcription. The endogenous reverse transcription in disrupted HIV-1 virions containing packaged tRNA3(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 tRNA3(Lys) priming process. These findings suggest the potential of the A-loop region as a critical target for blocking HIV-1 replication.
Highlights
) upstream of the primerbinding site (PBS) interacts with the anticodon loop of tRNA3Lys and has been proposed to be essential for conferring specificity to tRNA3Lys for priming the initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription
The endogenous reverse transcription in disrupted HIV-1 virions containing packaged tRNA3Lys and its replicating enzyme reverse transcriptase (RT) was significantly inhibited by polyamide nucleotide analog (PNAAL), providing direct evidence of the involvement of the A-loop region of viral RNA genome in tRNA3Lys priming process
The initiation of HIV-1 reverse transcription occurs by extension of the cellular tRNA3Lys primer annealed near the 5′ nontranslated region of the viral RNA genome at a site called the PBS
Summary
) upstream of the PBS interacts with the anticodon loop of tRNA3Lys and has been proposed to be essential for conferring specificity to tRNA3Lys for priming the initiation of HIV-1 reverse transcription. The 495 base-long U5-PBS RNA transcript was incubated with the labeled tRNA3Lys (2 × 104 c.p.m.) at 37°C for 1 h in a buffer containing 50 mM Tris–HCl pH 7.8, 1 mM DTT and The initiation of HIV-1 reverse transcription occurs by extension of the cellular tRNA3Lys primer annealed near the 5′ nontranslated region of the viral RNA genome at a site called the PBS.
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