Cleavage of hepatitis B virus RNA by three ribozymes transcribed from a single DNA template

Hepatitis B virus is a DNA virus which replicates asymmetrically through reverse transcription of an RNA intermediate (pregenomic RNA). As a first step toward an antiviral strategy, a single synthetic oligodeoxynucleotide coding for 3 ribozyme motifs directed against three adjacent sites within the pregenomic RNA was synthesized, cloned and transcribed in vitro. Experiments utilizing 5' and 3' end labeling of RNA demonstrated that the three ribozymes accurately cleaved hepatitis B virus substrate RNA. Cleavage efficiency was similar to that of single ribozyme constructs. These results demonstrate that hepatitis B virus RNA is susceptible to ribozyme induced cleavage and illustrate that three ribozymes were simultaneously active when transcribed from a single DNA template. The expression of multiple ribozyme motifs may offer an advantage if there is high viral target sequence variability.