THE POLYPURINE TRACT, PPT, OF HIV AS TARGET FOR ANTISENSE AND TRIPLE-HELIX-FORMING OLIGONUCLEOTIDES

Replication of retroviral RNA into double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract, PPT, by the reverse transcriptase (RT). The PPT is highly conserved among the known HIV-1 retroviral isolates. It occurs twice, once within the coding region of the integrase and the other one adjacent to the 3' LTR. The data presented show that two antisense oligonucleotides, a 20-mer and a 40-mer, complementary to the PPT induce complete blocks of DNA synthesis whereas an antisense oligonucleotide outside the PPT is only slightly inhibitory. Previously polypurine sequences have been used by several groups for triplex-formation. During replication the HIV-polypurine tract, PPT, is present in a RNA-DNA hybrid. Therefore triple-helix formation consisting of RNA-DNA and a third DNA strand covering the PPT region was tested here by protection against RNase H cleavage in vitro. Incubation with a pyrimidine oligonucleotide in parallel orientation to the PPT-RNA shows some protection. GT-pyrimidine-purine mixed oligonucleotides (25-mer) led to protection against RNase H up to 50% independent of their orientation. The data suggest that triple-helix formation may have taken place with the PPT in vitro. Therefore, this highly conserved structure may prove useful in nucleic acid based anti-viral therapy with antisense or triple-helix approaches. Furthermore, the influence of HIV-1 nucleocapsid (NC) protein, NCp15, on reverse transcription is reported. The data show a two- to three-fold stimulatory effect of the NCp15 on RNA directed DNA synthesis.