Hepatitis C virus internal ribosome entry site (IRES) stem loop IIId contains a phylogenetically conserved GGG triplet essential for translation and IRES folding

The hepatitis C virus (HCV) internal ribosome entry site (IRES) is a highly structured RNA element that directs cap-independent translation of the vir al polyprotein. Morpholino antisense oligonucleotides directed towards stem loop md drastically reduced HCV IRES activity. Mutagenesis studies of this region showed that the GGG triplet (nucleotides 266 through 268) of the he xanucleotide apical loop of stem loop Ind is essential for IRES activity bo th in vitro and in vivo. Sequence comparison showed that apical loop nucleo tides (UUGGGU) were absolutely conserved across HCV genotypes and the GGG t riplet was strongly conserved among related Flavivirus and Pestivirus nontr anslated regions. Chimeric IRES elements with IIId derived from GB virus B (GBV-B) in the context of the HCV IRES possess translational activity. Muta tions 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.