Inhibition of HIV-1 reverse transcription by triple-helix forming oligonucleotides with viral RNA

ABSRACT Reverse transcription of retroviral RNA into doublestranded DNA is catalyzed by reverse transcriptase (RT). A highly conserved polypurine tract (PPT) on the viral RNA serves as primer for plus-strand DNA synthesis and is a possible target for triple-helix formation. Triple-helix formation during reverse transcription involves either single-stranded RNA or an RNA·DNA hybrid. The effect of triple-helix formation on reverse transcription has been analyzed here in vitro using a three-strand-system consisting of an RNA·DNA hybrid and triplex-forming oligonucleotides (TFOs) consisting either of DNA or RNA. Three strand triple-helices inhibit RNase H cleavage of the PPT RNA·DNA hybrid and initiation of plus-strand DNA synthesis in vitro. Triple-helix formation on a single stranded RNA target has also been tested in a two-strand-system with TFOs comprising Watson-Crick and Hoogsteen base-pairing sequences, both targeted to the PPT-RNA, on a single strand connected by a linker (T)4 TFOs prevent RNase H cleavage of the PPT-RNA and initiation of plus-strand DNA synthesis in vitro. In cell culture experiments one TFO is an efficient inhibitor of retrovirus replication, leading to a block of p24 synthesis and inhibition of syncytia formation in newly infected cells.