A NOVEL RIBOZYME TARGET SITE LOCATED IN THE HIV-1 NEF OPEN READING FRAME

We have tested the sequence UUC CAG UCA GAC CU, at position 9016-9029 within the HIV-1(SF2) nef open reading frame, for accessibility to ant isense and hammerhead ribozyme attack. The accessibility was first stu died using a phosphorothioate-modified 14-nt DNA oligo (complementary to the nef(9016-9029) site). A dose-dependent repression of HIV-1(SF2) growth was observed in human peripheral blood mononuclear cells after exogenous administration of the oligo to the cell culture medium. A h ammerhead ribozyme with a 6+7-nt antisense specificity for the nef(901 6-9029) site (hhRz.nef(9016-9029)) was constructed and transfected int o the human T-cell line HUT78. Again, a dose-dependent repression of v irus growth was observed when different individual clones expressing h hRz.nef(9016-9029) were infected with HIV-1(SF2). A complete abrogatio n of virus production was observed after infection with a low (0.5 TCI D50) HIV-1 titer. Increasing doses (2.5 and 12.5 TCID50) of HIV-1 viru s yielded a low production (10(3)-fold reduced) of virus particles in most cases: but a complete, or close to complete, abrogation was obser ved even in individual cultures infected with the highest dose. Presen ce of proviral pol and gag sequences in hhRz.nef(9016-9029)-expressing HUT78 clones was assayed, using PCR. Interestingly, since no pol and gag PCR products could be detected, the results strongly indicated tha t the hammerhead ribozyme was already acting on the infecting HIV RNA before its reverse transcription and integration as proviral DNA. In s ummary, the results obtained in this study support the nef(9016-9029) site as a strong new candidate for ribozymal gene therapy against HIV- 1 infection. (C) 1996 Academic Press, Inc.