The poliovirus CRE functions when located in the 5′ NCR of the genome. Complementary oligonucleotides for the CRE were directionally cloned between the Bam HI and Sac I sites located, respectively, in the 5′ NCR and region encoding the amino-terminus of VP4. The CRE sequence was modified by substitution of U15A/A49U (Fig. 1A) to replace the AUG49–51 to generate pT7/SL3HVCRE-AUG. The same region of pT7/SL3 was also replaced with oligonucleotides encoding the Synth2 CRE, and a Synth2-derived CRE in which one of the conserved A triplet was deleted (generating pT7/SL3-HVSynth2 and pT7/SL3-HVSynth2ΔA, respectively). Normalized levels of RNA generated in vitro, and serial 10-fold dilutions, was transfected into HeLa cells and overlaid with agar. Plaques were visualized after 3 d by staining with crystal violet.
