The hepatitis C virus (HCV) internal ribosome entry site (IRES) is a highly structured RNA element that directs cap-independent translation of the viral polyprotein. Morpholino antisense oligonucleotides directed towards stem loop IIId drastically reduced HCV IRES activity. Mutagenesis studies of this region showed that the GGG triplet (nucleotides 266 through 268) of the hexanucleotide apical loop of stem loop IIId is essential for IRES activity both in vitro and in vivo. Sequence comparison showed that apical loop nucleotides (UUGGGU) were absolutely conserved across HCV genotypes and the GGG triplet was strongly conserved among related Flavivirus and Pestivirus nontranslated regions. Chimeric IRES elements with IIId derived from GB virus B (GBV-B) in the context of the HCV IRES possess translational activity. Mutations within the IIId stem loop that abolish IRES activity also affect the RNA structure in RNase T 1 -probing studies, demonstrating the importance of correct RNA folding to IRES function.Hepatitis C virus (HCV) is a member of the genus Hepacivirus in the Flaviviridae family (8). HCV infection is a global health problem, with chronically infected patients exhibiting an increased risk for the development of cirrhosis and hepatocellular carcinoma (19). HCV has a single-strand, positive-sense RNA genome known for its genetic heterogeneity, and HCV has been classified into six major genotypes and a series of subtypes (30, 31). However, the 5Ј nontranslated region (5ЈNTR) of the virus is relatively well conserved among all genotypes (5, 9).Located within the 5ЈNTR is the internal ribosome entry site (IRES) previously shown to contain sequence and structural elements responsible for directing cap-independent translation of the viral polyprotein (36, 38). Stem-loop I had been previously shown not to be essential for IRES activity (17,27,28). Based on a number of published reports, the minimal sequence required for IRES activity is believed to include nucleotide sequences spanning nucleotides (nt) 42 through 356 (12-14, 26-28). Within the minimal IRES sequence, three primary structured domains, known as stem-loops II, III, and IV, have been identified based on chemical and enzymatic probing as well as phylogenetic comparisons (4,13,14,20,40). Each primary domain of the IRES is further defined based on a combination of double-stranded (ds) helices and single-strand bulges or loops. A number of studies have been conducted to determine the importance of some of these structured elements in viral translation, including the pseudoknot and stemloops II, IIIb, IIIc, IIIe,17,25,[27][28][29]35,39,40). In general, mutations altering double-stranded helical regions can have deleterious effects on translation. However, when compensatory mutations are made to restore WatsonCrick base pairing, the translation efficiency can often be restored. In contrast, mutational changes in loop regions appear to be more amenable and may retain IRES activity (39). Recently, it has also been demonstrated that domains IIIb to IIIc are ...