Fig. 5.

Functional competence of chimera HCV IRES elements. (A) Effect on in vitro translation of domain II replacement in the HCV IRES by corresponding motifs from other viral IRES elements and additional deletion of two base pairs between the hairpin loop IIb and the loop E motif (Δ2bp), or deletion of the internal loop IIa (ΔIIa). See SI Appendix, Fig. S3 for chimera structure and sequences. (B) Construction of a chimera X (orange) by an exact structural motif swap based on superimposition of the subdomain IIa crystal structures from HCV (red/white) and SVV (orange/white). (C) Effect on in vitro translation of subdomain IIa replacement in the HCV IRES by the corresponding motif from SVV (chimera X, outlined in B). The chimera P (brown) is a control construct that contains a subdomain IIa swap with a one base pair offset relative to the X construct (SI Appendix, Fig. S8). HCV IRES wild-type and subdomain IIa deletion (ΔIIa, 0) are shown as controls. Translation efficiencies were normalized to the cap-driven expression in bicistronic dual reporter constructs. (D) Function of the HCV/SVV IRES chimera X in replicon-transfected human cells. Reporter expression was measured 4 and 24 h after transfection, with the subgenomic replicon RNA carrying the IRES subdomain IIa replaced by the corresponding motif from SVV. Error bars represent ±1 SD calculated from triplicate experiments, except for in D, where triplicates of three biological replicates (nine values) were used.