In vitro cleavage of HIV-1 vif RNA by a synthetic ribozyme

Abstract

Using the method of Haseloff and Gerlach, we constructed a ribozyme specifically targeted against the virion infectivity factor (vif) of HIV-1. Both, the vif gene and an oligonucleotide representing the catalytic RNA sequence were cloned into pSPT19 downstream of the T7 promoter and transcribed with T7 RNA polymerase. Efficient cleavage of vif RNA by the synthetic ribozyme occurred at pH 7.5 and 37 degrees C in the presence of magnesium ions in vitro. No measurable activity was observed with a vif antisense RNA. A deletion in the hybridizing region of the ribozyme decreased the cleavage rate, while a mutation in the consensus cleavage domain abolished its catalytic activity. Thus, we could demonstrate an in-vitro activity of a specifically designed ribozyme against HIV-1 vif RNA.