The internal ribosome entry site (IRES) of hepatitis C virus visualized by electron microscopy

Abstract

Translation of hepatitis C virus (HCV) RNA is initiated via the internal ribosome entry site (IRES), located within the 59 untranslated region. Although the secondary structure of this element has been predicted, little information on the tertiary structure is available. Here we report the first structural characterization of the HCV IRES using electron microscopy. In vitro transcribed RNA appeared as particles with characteristic morphology and gold labeling using a specific oligonucleotide confirmed them to be HCV IRES. Dimerization of the IRES by hybridization with tandem repeat oligonucleotides allowed the identification of domain III and an assignment of domains II and IV to distinct regions within the molecule. Using immunogold labeling, the pyrimidine tract binding protein (PTB) was shown to bind to domain III. Structure-function relationships based on the flexible hinge between domains II and III are suggested. Finally, the architecture of the HCV IRES was seen to be markedly different from that of a picornavirus, foot-and-mouth disease virus (FMDV).

Structure of the full-length HCV IRES in solution

“…Domain II and domain III form independent long cylindrical stems that extend in different directions from the central III ef /IV pseudo-knot and constitute the dorsal spine of a structure from which subdomains III a , III c and III d and domain IV protrude on the sides. This structural architecture is in agreement with electron microscopy pictures of free HCV IRES 13 . In addition, the atomic model is in agreement with available biochemical data, in particular, with experimental accessibilities measured for chemical and enzymatic probes 16 .…”

“…S1) 13 . The molecule contains tertiary fold motifs, the IIIabc four-way junction and the IIIef/IV pseudoknot 4,14,15 , and adopts a defined ion-dependent fold under physiological conditions 16 , although it does not form a compact, globular structure 13,16,17 .…”

“…S1) 13 . The molecule contains tertiary fold motifs, the IIIabc four-way junction and the IIIef/IV pseudoknot 4,14,15 , and adopts a defined ion-dependent fold under physiological conditions 16 , although it does not form a compact, globular structure 13,16,17 . The structure at atomic resolution has been determined for several autonomous folding regions, including domain II, the III abc junction and, more recently, the III ef /IV pseudoknot [18][19][20][21][22][23][24] .…”

“…The structure at atomic resolution has been determined for several autonomous folding regions, including domain II, the III abc junction and, more recently, the III ef /IV pseudoknot [18][19][20][21][22][23][24] . The presence of several hairpin loops and regions predicted to be single-stranded likely confers dynamic flexibility to the entire IRES molecule 13,16,23 and has so far hampered structure determination of the entire molecule by X-ray crystallography or electron microscopy. As the IRES is essential for viral replication and its sequence is well conserved among all HCV genotypes, its structure represents a novel target for drug design 25 .…”

“…Little is known about the structure of the entire IRES in solution 13 and an atomic structure of this molecule in its free form would help to rationalize the conformational changes that occur during the assembly of the initiation complex. Characterizing the structure of large flexible macromolecules remains, however, a challenge.…”