Study of HIV-2 primer-template initiation complex using antisense oligonucleotides

HIV-2 reverse transcription is initiated by the retroviral DNA polymerase ( reverse transcriptase) from a cellular tRNA(Lys3) partially annealed to the primer binding site in the 5'-region of viral RNA. The HIV-2 genome has tw o 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 t RNA(Lys3) has been described to date. Here we address the question of the p otential role of the interactions between these highly structured regions i n the initiation of viral DNA synthesis. To evaluate this we used an antise nse approach, first validated in our in vitro HIV-1 reverse transcription s ystem. Annealing of the antisense oligonucleotides to the pre-primer bindin g site (the upstream region contiguous to the HIV-2 primer binding site) wa s determined in the presence of native tRNA(Lys3) Or synthetic primers. Usi ng natural and chemically modified antisense oligonucleotides we found that interactions between the anticodon of tRNA(Lys3) and an A-rich loop of vir al RNA led to an important destabilization of the pre-primer binding site; this region became accessible to anti-pre-primer binding site oligonucleoti des in a cooperative manner. These studies allowed to identify an A-rich re gion in HIV-2(ROD) RNA capable of interacting with tRNA(Lys3). Better knowl edge of these interactions is very important for understanding the primer/t emplate positioning in the early steps of HIV-2 reverse transcription.