Inhibitor-induced structural change in the HCV IRES domain IIa RNA

Abstract

Translation of the hepatitis C virus (HCV) RNA is initiated from a highly structured internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) of the RNA genome. An important structural feature of the native RNA is an approximately 90 degrees helical bend localized to domain IIa that positions the apical loop of domain IIb of the IRES near the 40S ribosomal E-site to promote eIF2-GDP release, facilitating 80S ribosome assembly. We report here the NMR structure of a domain IIa construct in complex with a potent small-molecule inhibitor of HCV replication. Molecular dynamics refinement in explicit solvent and subsequent energetic analysis indicated that each inhibitor stereoisomer bound with comparable affinity and in an equivalent binding mode. The in silico analysis was substantiated by fluorescence-based assays showing that the relative binding free energies differed by only 0.7 kcal/mol. Binding of the inhibitor displaces key nucleotide residues within the bulge region, effecting a major conformational change that eliminates the bent RNA helical trajectory, providing a mechanism for the antiviral activity of this inhibitor class.

Fig. 1.

IRES region of HCV genomic RNA. (A) Consensus secondary structure of the HCV IRES RNA (8). Domain II is outlined in the large dashed box, and subdomain IIa in the smaller dashed box. (B) Sequence and secondary structure of the RNA construct used for NMR. A UUCG tetraloop sequence was used to promote folding. The bulge nucleosides from A6–A10 (corresponding to residues 53–57 of the full length IRES) of domain IIa result in an approximately 90° bend in the helix trajectory. (C) The small-molecule inhibitor, Isis-11.

Fig. 2.

Chemical shift perturbation of Isis-11 binding. (A) The free domain IIa RNA model built from the structure of Lukavsky et al. (12) was refined with CH RDC restraints. Residues C8, U9, C11, C34, and C35 are highlighted in orange and correspond to the pyrimidines with the greatest TOCSY cross-peak changes. Residues A6, A7, and A10 are magenta, green, and cyan, respectively. (B) 2D TOCSY NMR spectrum showing pyrimidine base H5–H6 cross-peaks in the absence (blue) and in the presence (red) of a 7-fold excess of Isis-11. Pyrimidine residues experiencing large chemical shift changes are predominantly localized to the bulge region, whereas residues in the stems and the tetraloop (C18, C19, U20, U21, C22) are largely unaffected.

Fig. 3.

NMR structures and RDC data. (A) Superposition of 10 S-isomer structures from 15 nsec AMBER trajectories. RMSD = 2.3 Å. (B) The free RNA model (black) is shown superimposed (residues 12∶19, 24∶32) on the complex structure (orange backbone) illustrating the change in the helical axis. The inhibitor is shown in yellow. (C) RDC fit. CH RDCs were calculated from a representative final complex structure and from the free RNA starting structure; each set of calculated RDCs is plotted against the experimental RDC set for the complex. Filled squares are for the complex, and open circles for the free RNA.

Fig. 4.

Base stacking changes in the bulge region of domain IIa. (A) Molecular graphics of the heavy atoms of the free RNA using the first three models from PDB 1P5M. (B) Isis-11 bound RNA structures (the two lowest energy water-refined NMR structures from each stereoisomer). Residues whose environment changes upon binding are labeled and colored (C11, C34, C35: red; C8, U9: yellow; G5, A6, A7: magenta; A10: cyan; G33: green; Isis-11: gray). The structures were fit using residues 5–11, 33–34 for the free structure and residues 5, 10, 11, 33, 34 for the bound structure. (C) electrostatic potential surface calculated for the RNA only using the structure of the complex. Areas of highest negative charge are indicated by the more red colors and are localized near the dimethylamino groups. (D) Fluorescence intensity increase for 2AP-6 substituted RNA (1 μM) as a function of inhibitor concentration (see Fig. S5). Emission at 370 nm was fit to a two-state binding model providing a calculated K_D = 2.4 μM, comparable to the value previously determined by mass spectrometry.

Fig. 5.

Model for domain IIa function in 80S ribosomal assembly. (Center) Domain IIa of the HCV IRES has a 90° bend that positions the apical loop of domain II in contact the 40S subunit near the E-site, and along with eIF5, promotes hydrolysis of eIF2-GTP and subsequent release of eIF2-GDP. (Right) The release of eIF2-GDP allows recruitment of the 60S subunit and eIF3 release to assemble a translation competent 80S ribosome. (Left) The inhibitor (X) would straighten IRES domain II, reduce the eIF2-GTP hydrolysis rate, and slow 80S assembly.