Alterations to both the primary and predicted secondary structure of stem-loop IIIc of the hepatitis C virus ib 5 ' untranslated region (5 ' UTR) lead to mutants severely defective in translation which cannot be complementedin trans by the wild-type 5 ' UTR sequence

Cap-independent translation of the hepatitis C virus (HCV) genomic RNA is m ediated by an internal ribosome entry site (IRES) within the 5' untranslate d region (5'UTR) of the virus RNA. To investigate the effects of alteration s to the primary sequence of the 5'UTR on IRES activity, a series of HCV ge notype Ib (HCV-lb) variant IRES elements was generated and cloned into a bi cistronic reporter construct. Changes from the prototypic HCV-lb 5'UTR sequ ence were identified at various locations throughout the 5'UTR. The transla tion efficiencies of these IRES elements were examined by an in vivo transi ent expression assay in transfected BHK-21 cells and were found to range fr om 0.4 to 95.8% of the activity of the prototype I-ICV-lb IRES. Further mut ational analysis of the three single-point mutants most severely defective in activity, whose mutations were all located in or near stem loop IIIc, de monstrated that both the primary sequence and the maintenance of base pairi ng within this stem structure were critical for HCV IRES function. Compleme ntation studies indicated that defective mutants containing either point mu tations or major deletions within the IRES elements could not be complement ed in trans by a wild-type IRES.