Structure of the full-length HCV IRES in solution

Abstract

The 5 0 -untranslated region of the hepatitis C virus genome contains an internal ribosome entry site (IRES) that initiates cap-independent translation of the viral RNA. Until now, the structural characterization of the entire (IRES) remained limited to cryo-electron microscopy reconstructions of the (IRES) bound to different cellular partners. Here we report an atomic model of free full-length hepatitis C virus (IRES) refined by selection against small-angle X-ray scattering data that incorporates the known structures of different fragments. We found that an ensemble of conformers reproduces small-angle X-ray scattering data better than a single structure suggesting in combination with molecular dynamics simulations that the hepatitis C virus (IRES) is an articulated molecule made of rigid parts that move relative to each other. Principal component analysis on an ensemble of physically accessible conformers of hepatitis C virus (IRES) revealed dominant collective motions in the molecule, which may underlie the conformational changes occurring in the (IRES) molecule upon formation of the initiation complex.

Fingerprinting the junctions of RNA structure by an open-paddlewheel diruthenium compound

“…In agreement with the results found for the FMDV IRES, OPW-Ru reactive positions tend to be located close to junctions, within flexible regions of the three-dimensional HCV IRES structure (Fig. 4B, inset) as defined by Perard et al (2013).…”

“…The potential correlation between structural features of both RNAs and reactivity toward OPWRu was also analyzed (Supplemental Tables 2, 3). We could not conclude that there is a direct correlation among the OPW-Ru reactivity and any of the structural features (solvent accessibility measured by OH-radical reactivity in both the HCV and FMDV IRES elements [Kieft et al 1999;Lozano et al 2014]; and interacting edges, sugar conformation, or backbone turn in the case of the HCV IRES element [Kieft et al 2002;Lukavsky et al 2003;Berry et al 2010;Perard et al 2013]). Concerning the type of secondary structure motif, a preferential attack to positions present in four-way junctions was observed (Figs.…”

“…To reinforce these results and provide further support to the validity of this compound for RNA structural analysis, we investigated the reactivity of a different RNA molecule for which three-dimensional structure has been determined using different approaches alone or in complex with the 80S ribosome (Lukavsky et al 2000(Lukavsky et al , 2003Kieft et al 2002;Melcher et al 2003;Easton et al 2009;Berry et al 2010;Perard et al 2013;Quade et al 2015). To this end, the reactivity of the hepatitis C virus (HCV) IRES toward OPW-Ru was analyzed in parallel to NMIA (Fig.…”

Dynamics of IRES-mediated translation

“…Early structural work applied a 'divide and conquer' approach to determine three-dimensional structures of isolated IRES domains using NMR and X-ray crystallography [14,81]. By incorporating structures of known fragments, a recent study using SAXS and molecular dynamics simulations found that the free IRES is best fit by an ensemble of at least five conformations as a result of rigid domains moving with respect to one another by flexible linkers [95]. Many of the HCV IRES structural elements are conserved in related flaviviruses, as well as certain picornavirus genera, with variability especially in domain II and apical domain III regions [16].…”

“…These observed positions of domain II are probably only a subset sampled during initiation, as a complete 48S complex of the 40S : IRES : eIF3 : eIF2-TC has yet to be solved. It has been proposed that the IRES must undergo a multi-step rearrangement during binary-complex formation to position domain II [95]; and clearly, for 80S ribosomes to transition into elongation, domain II must be entirely vacated from its E site position to make room for deacyclated initiator tRNA moving from the P site. The structural observations help explain the multipurpose role of domain II during tRNA recruitment and positioning, where it participates in both the formation of productive 48S complexes and the progression of 80S initiation complexes into elongation [82,129,131].…”