The hepatitis C virus internal ribosome entry site adopts an ion-dependenttertiary fold

Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) loc ated in the 5' untranslated region of the genomic RNA that drives cap-indep endent initiation of translation of the viral message. The approximate seco ndary structure and minimum functional length of the HCV IRES are known, an d extensive mutagenesis has established that nearly all secondary structura l domains are critical for activity. However, the presence of an IRES RNA t ertiary fold and its functional relevance have not been established. Using chemical and enzymatic probes of the HCV IRES RNA in solution, we show that the IRES adopts a unique three-dimensional structure at physiological salt concentrations in the absence of additional cofactors or the translation a pparatus. Folding of the IRES involves cooperative uptake of magnesium and is driven primarily by charge neutralization. This tertiary structure conta ins at least two independently folded regions which closely correspond to p utative binding sites for the 40 S ribosomal subunit and initiation factor 3 (eIF3). Point mutations that inhibit IRES folding also inhibit its functi on, suggesting that the IRES tertiary structure is essential for translatio n initiation activity. Chemical and enzymatic probing data and small-angle X-ray scattering (SAXS) experiments in solution show that upon folding, the IRES forms an extended structure in which functionally important loops are exposed. These results suggest that the 40 S ribosomal subunit and eIF3 bi nd an HCV IRES that is prefolded to spatially organize recognition domains. (C) 1999 Academic Press.