Natural beta-phosphodiester 16mer and 15mer antisense oligonucleotides targeted against the HIV-1 and HIV-2 TAR RNAs respectively were previously described as sequence-specific inhibitors of in vitro retroviral reverse transcription. In this work, we tested chemically modified oligonucleotide analogues: alpha-phosphodiester, phosphorothioate, methylphosphonate, peptide nucleic acid or PNA, 2'- o -methyl and (N3'-P5') phosphoramidate versions of the 16mer anti-TAR oligonucleotide. PNA, 2'- O -methyl and (N3'-P5') phosphoramidate oligomers showed a strong inhibitory effect compared with the unmodified 16mer, with reverse transcription inhibition (IC50) values in the nanomolar range. The inhibition was sequence-specific, as scrambled and mismatched control oligonucleotides were not able to inhibit cDNA synthesis. No direct binding of the 2'- O -methyl, PNA or (N3'-P5') phosphoramidate anti-TAR oligonucleotides to the HIV-1 reverse transcriptase was observed. The higher T m obtained with 2'- O -methyl, (N3'-P5') phosphoramidate and PNA molecules concerning the annealing with the stem-loop structure of the TAR RNA, in comparison with the beta-phosphodiester oligonucleotides, is correlated with their high inhibitory effect on reverse transcription.

Human immunodeficiency virus type 2 (HIV-2) reverse transcription is initiated from cellular tRNA(Lys3) partially annealed to the RNA viral genome at the primer binding site (PBS). This annealing involves interactions between two highly structured RNA molecules. In contrast to HIV-1, in which the reverse transcription initiation complex has been thoroughly studied, there is still little information regarding a possible model to describe the secondary structure of the template-primer complex in HIV-2. To determine whether HIV-2 RNA sequences flanking the PBS may specifically interact with the natural primer tRNA, we performed site-directed mutagenesis and enzymatic footprinting. An RNA fragment corresponding to the HIV-2 U5 RNA domain and tRNA(Lys3) were probed either in their free form or in the binary complex. Important reactivity changes to nucleases were obtained upon complex formation. In addition to the canonical contacts between the viral PBS and the 3' end acceptor stem of tRNA(Lys3), we identified two additional interacting domains: (i) the U-rich region of the anticodon loop with the A-rich sequence of the internal loop within the U5-prePBS region; (ii) nucleotides 48-54 from the TPsiC domain of tRNA(Lys3) and the 240-247 region of viral U5-RNA. In view of these experimental data and sequence comparison between different HIV-2 isolates, we propose a model for the secondary structure of the HIV-2 template-primer initiation complex.

HIV‐2 reverse transcription is initiated by the retroviral DNA polymerase (reverse transcriptase) from a cellular tRNALys3 partially annealed to the primer binding site in the 5′‐region of viral RNA. The HIV‐2 genome has two A‐rich regions upstream of the primer binding site. In contrast to HIV‐1 RNA, no direct evidence of interactions with the U‐rich anticodon loop of tRNALys3 has been described to date. Here we address the question of the potential role of the interactions between these highly structured regions in the initiation of viral DNA synthesis. To evaluate this we used an antisense approach, first validated in our in vitro HIV‐1 reverse transcription system. Annealing of the antisense oligonucleotides to the pre‐primer binding site (the upstream region contiguous to the HIV‐2 primer binding site) was determined in the presence of native tRNALys3 or synthetic primers. Using natural and chemically modified antisense oligonucleotides we found that interactions between the anticodon of tRNALys3 and an A‐rich loop of viral RNA led to an important destabilization of the pre‐primer binding site; this region became accessible to anti‐pre‐primer binding site oligonucleotides in a cooperative manner. These studies allowed to identify an A‐rich region in HIV‐2ROD RNA capable of interacting with tRNALys3. Better knowledge of these interactions is very important for understanding the primer/template positioning in the early steps of HIV‐2 reverse transcription.

HIV reverse transcription is initiated from a cellular tRNA partially associated with the retroviral genome. Here we studied homologous HIV-2 cDNA synthesis using natural or synthetic primers. With natural tRNA vysQ , synthesis of early products comprising nucleotides +5 to +7 preceded the elongation step leading to synthesis of (3) strong-stop cDNA. In the presence of a poly(A)Woligo(dT) trap, no full-length product was observed while early products were still present, showing a transition between initiation and elongation. With DNA primers only an unspecific elongation was found. Our data show a similar mechanism of reverse transcription initiation by HIV-1 and HIV-2 reverse transcriptases. Furthermore, using a heterologous system we found that HIV-1 RNA, in contrast to data reported in the literature, was an excellent template for HIV-2 reverse transcriptase.z 1998 Federation of European Biochemical Societies.

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 tRNALys3 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 tRNALys3 primer for the PBS region. Modified 2'-O-methyl AS-ODN were able to inhibit in vitro HIV-1 reverse transcription and displace the tRNALys3 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 tRNALys3. 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 tRNALys3 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.