Figure 1.

Schematic representation of the reporter construct and the randomized hairpin ribozyme library for the discovery of HCV IRES cofactors. (A) A bicistronic construct is transcribed from the SV 40 promoter, allowing for cap-dependent translation of hygromycin B phosphotransferase (hygro) and cap-independent HCV IRES-mediated translation of HSV-tk. (B) The hairpin ribozyme library (RzLib) was constructed such that 12 positions of the substrate binding domains were randomized (8 nucleotides in helix 1, 4 nucleotides in helix 2). The ribozyme preferentially recognizes a GUC triplet within the substrate RNA and cleaves 5′ of the G residue as indicated. A hairpin ribozyme can be disabled by changing the bases AAA to CGU (as shown), without changing the substrate binding ability of the ribozyme. A ribozyme library cassette under control of the tRNA^val promoter was cloned into a murine retrovirus vector (pLHPM) to generate pLHPM-RzLib. The LTR transcript expresses a neomycin-resistance gene (neo) to allow for selection of stable transductants, and the 5′-UTR of HCV, which directs the translation of the authentic HCV core (nucleotides 342–874). Arrows depict transcriptional start sites. Polyadenylation signal is indicated as pA.