Inhibition of HIV-1 replication in vitro and in human infected cells by modified antisense oligonucleotides targeting the tRNA(Lys3) /RNA initiation complex

摘要

The untranslated 5' leader region of the human immunodeficiency virus type 1 (HIV-1) RNA plays an essential role in retroviral replication. It is the first retrotranscribed RNA region, primed from a cellular tRNA(Lys3) partially annealed to the HIV-1 primer binding site (PBS). The structural and functional features of the HIV-1 reverse transcription initiation complex have been thoroughly studied. In this work, we used chemically modified antisense oligonucleotides (AS-ODN) as competitors of the natural tRNA(Lys3) primer for the PBS region. Modified 2'-O-methyl AS-ODN were able to inhibit in vitro HIV-1 reverse transcription and displace the tRNA(Lys3) previously annealed to the PBS. The destabilization of the initiation complex by 2'-O-methyl ODN was a sequence-specific process. We further demonstrated the importance of an anchor region contiguous to the PBS in the annealing of the antisense molecule, allowing the displacement of tRNA(Lys3). The 20-mer 2'-O-methyl molecules were also able to inhibit viral replication in HIV-1-human infected cells, either by blocking cDNA synthesis during the early phase or by interfering with the annealing of the tRNA(Lys3) primer to the PBS during the late phase of the viral cycle. Thus, the highly conserved retroviral initiation complex was shown to be a promising target when using the antisense strategy.