Inhibition of hepatitis C virus-directed gene expression by a DNA ribonuclease

Background/Aims: The aim of this study was to determine whether DNA analogs of ribozymes could be prepared to inhibit hepatitis C virus (HCV) gene exp ression. Methods: Two DNA ribonucleases, Dz2 and Dz4, were designed with varying arm lengths, to cleave at the 5'-noncoding region (NCR) just upstream from the translation start site, and core region of HCV genome, respectively. A rep orter vector was prepared to contain target HCV regulatory sequences contro lling a downstream luciferase gene. DNA ribonucleases with normal phosphodi ester, as well as with terminal phosphorothioate linkages, were administere d to Huh7 cells, and luciferase activity was measured. Results: DNA ribonucleases were highly active in cleaving HCV RNA targets. Enzymes with longer arms had consistently higher cleavage activity compared to enzymes with shorter arms under cell-free conditions. Furthermore, in H uh7 cells, terminal phosphorothioate derivatives, Dz2 and Dz4, significantl y suppressed HCV-luciferase fusion gene expression up to 45% and 67% of con trols, respectively. Interestingly, phosphorothioate-modified DNA ribonucle ases had greater inhibitory effects on target gene expression than their un modified counterparts. In contrast, DNA ribonucleases with point mutations in the catalytic domain had significantly lower inhibitory effects compared to wild-type DNA ribonucleases. However, activity was not eliminated, sugg esting that some antisense contribution was present. Conclusions: DNA ribonucleases directed against the HCV genome can specific ally cleave target HCV RNA. Modifications of the extreme 3'- and 5'-termini protect against nuclease degradation without appreciable reduction in inhi bitory activity against viral gene expression under intracellular condition s.