EFFICIENT EXPRESSION BY AN ALPHAVIRUS REPLICON OF A FUNCTIONAL RIBOZYME TARGETED TO HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

Intracellular applications of ribozymes have been limited partly by th e availability of suitable high-expression systems. For RNA effecters, consideration of an RNA virus vector system for delivery and expressi on is reasonable. We show that alphavirus replicons can be highly effi cient nonintegrating ribozyme-expressing vectors. Using a hammerhead r ibozyme targeted to a highly conserved sequence in the U5 region of th e human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme ( SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged in to viral particles, and these particles transduce mammalian cells effi ciently. SFVRz-transduced BHK cells were found to produce large amount s of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay s hows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed in tracellularly from an RNA polymerase II promoter is quantitatively eli minated in SFVRz-transduced BHK cells.