FIG. 1.
Hairpin ribozyme Rz3′X in a GCV-reporter assay with HeLa 5′tk cells stably expressing a bicistronic hygromycin-HCV IRES-HSV TK transcript. (A) Schematic representation of Rz3′X binding to the antigenomic HCV RNA within the highly conserved 3′X tail. The substrate is cleaved 5′ of the GUC triplet required for hairpin ribozyme recognition (arrow). Conserved areas of the hairpin ribozyme are shaded. (B) Vector constructs for retrovirus vector production of the ribozyme (pLHPM) (including IRES-mediated translation of HCV core protein) and for expression of a bicistronic hygromycin-HCV 5′UTR-HSV TK transcript for negative selection in cell culture (14). The ribozyme is driven independently from the retrovirus transcript by a tRNAVal promoter expression cassette. (C) GCV-resistant HeLa 5′tk reporter colonies derived from transduction with Rz3′X retrovirus particles. Colonies were enumerated 21 days after GCV application (20 μM, 4 days), and numbers are fold enrichment values compared with that for control ribozyme RzBR1-transduced reporter cells.