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Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit

Abstract

Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5′-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs1. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit2,3,4,5,6,7,8, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5, 6, 7, 9, 10, 11, 12), but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF313 and the HCV IRES8 revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components13. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S–IRES binary complex8, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.

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Figure 1: Cryo-electron microscopy structures of the CSFV ΔII-IRES–40S–DHX29 complex alone and bound to eIF3 compared to the structure of the DHX29-bound 43S preinitiation complex.
Figure 2: Different orientations of eIF3 and subdomain IIIb in the CSFV ΔII IRES–40S–DHX29 complex.
Figure 3: Structure and atomic model of the CSFV ΔII-IRES bound to the 40S subunit.
Figure 4: Binding of eIF3 to subdomain IIIb of the CSFV IRES and effects on translation of the eIF3–IRES interaction.

Accession codes

Accessions

Protein Data Bank

Data deposits

The electron microscopy map has been deposited in the EMBL-European Bioinformatics Institute Electron Microscopy Data Bank under accession codes EMD-2450 and EMD-2451. Coordinates of electron microscopy-based model of the CSFV IRES have been deposited in the RCSB Protein Data Bank under accession number 4c4q.

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Acknowledgements

We thank M. Thomas for assistance in the preparation of figures and A. Jobe for critical reading of the manuscript. This work was supported by the HHMI and National Institutes of Health (NIH) grants R01 GM29169 (to J.F.), NIH R01 AI51340 (to C.U.T.H.) and NIH R01 GM59660 (to T.V.P.).

Author information

Authors and Affiliations

Authors

Contributions

Y.H., A.d.G., V.D, T.V.P., C.U.T.H. and J.F. interpreted the data and wrote the manuscript. V.D. and T.V.P. prepared the sample. Y.H., A.d.G. and R.A.G. performed the cryo-electron microscopy experiments. H.Y.L. performed the three-dimensional variance estimation. Y.H. and R.L. performed the cryo-electron microscopy data processing. Y.H. modelled the CSFV IRES. T.V.P., C.U.T.H. and J.F. directed research.

Corresponding authors

Correspondence to Christopher U. T. Hellen or Joachim Frank.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Comparison of HCV and CSFV IRES-bound ribosomal complexes.

a, Secondary structures of (left) the HCV IRES and (right) the CSFV IRES. Domain II of each IRES is indicated by a red dashed oval; elements of the pseudoknot and subdomains IIIa–IIIe are colour-coded as in Extended Data Fig. 6. b, Cryo-electron microscopy reconstructions of the HCV IRES bound to the rabbit 40S subunit at 20 Å resolution8 (left), the HCV IRES bound to the 40S subunit of cycloheximide-stalled human 80S ribosomes at 15 Å resolution23 (middle) (accession code EMD-1138) and the CSFV ΔII-IRES bound to the rabbit 40S subunits at 8.5 Å resolution (right) (this study). In all panels, the IRES-40S subunit is viewed from the solvent side; the 40S subunit is displayed in yellow and the IRES in cyan. The red dashed circles in left and middle panels show a discontinuity in the density of domain II in the HCV IRES bound to the 40S subunit compared to the HCV IRES bound to 80S ribosomes. The dashed circle in the right hand panel highlights CSFV IRES subdomain IIId2, which has no counterpart in the HCV IRES.

Extended Data Figure 2 Analysis of 40S–ΔII-IRES–eIF3–DHX29 complexes.

40S–ΔII-IRES–eIF3–DHX29 complexes were assembled in vitro using CSFV ΔII-IRES mRNA, native eIF2, eIF3 and 40S subunits purified from rabbit reticulocyte lysate and recombinant DHX29, and assayed by toeprinting. Lanes C, T, A and G show the cDNA sequence corresponding to CSFV ΔII-IRES mRNA. The position of the initiation codon is indicated on the left. This analysis revealed (lane 2) that deletion of domain II of the IRES or the presence of DHX29 did not influence IRES’s contacts with either 40S subunit (the toeprint stops at UUU387–389, G345 and C334) or eIF3 (the toeprint stops at A250) that have been previously observed2,18. Moreover, upon addition of the eIF2-TC, 40S–ΔII-IRES–eIF3–DHX29 complexes were quantitatively converted into 48S complexes on the authentic initiation codon AUG373 (lane 3). The low-efficiency 48S complex formation on the preceding AUG366 was also observed before and was not related to the presence of DHX2918. The gel reported in the figure is representative of results obtained from three technical replicates.

Extended Data Figure 3 Unsupervised three-dimensional classification of IRES-bound ribosomal complexes.

Unsupervised three-dimensional classification of IRES-bound ribosomal complexes identified 423,000 particles inconsistent with the known structure of the 40S subunit (rejects) and six well-populated classes containing complexes of the 40S subunit in a binary complex with the ΔII-IRES (class 1), of the 40S subunit bound to the ΔII-IRES and DHX29 (class 2), of the 40S subunit bound to DHX29 and eIF3 (class 3) and of the 40S subunit bound to the ΔII-IRES, DHX29 and eIF3 in orientation 1 (class 4), in orientation 2 (class 5) and in orientation 3 (class 6), viewed from (left) the back, (centre) the intersubunit side and (right) the solvent side.

Extended Data Figure 4 Measured resolution and reference-free two-dimensional classification of IRES-bound ribosomal complexes.

a, Gold-standard Fourier shell correlation (FSC) curves of the cryo-electron microscopy reconstruction of classes 2 (red line) and 4 (blue line) (also see Extended Data Fig. 3) indicating their estimated resolution. b, Right column on each side, two-dimensional classes obtained by reference-free classification of particles corresponding to 40S–eIF3–DHX29–ΔII-IRES complexes (class 4 in Extended Data Fig. 3). Middle column on each side, projection views of the class 4 cryo-electron microscopy map corresponding to the two-dimensional classes. Right column on each side, corresponding views of the segmented three-dimensional map coloured as in Fig. 1.

Extended Data Figure 5 Correspondence between individual subunits and anthropomorphic features of the eIF3 core complex and three-dimensional variance of the 40S–DHX29–ΔII-IRES–eIF3 map.

a, b, Front (upper panels) and back views (lower panels) of cryo-electron microscopy reconstructions of eIF3 as it appears in class 4 of the CSFV ΔII-IRES–40S–DHX29–eIF3 complex bound to the CSFV ΔII-IRES (a) and alone13 (b), labelled to show anthropomorphic terms12 and the localization of individual subunits in the core complex11,24. c, three-dimensional variance of the class 4 cryo-electron microscopy map, filtered to 20 Å, and coloured according to the computed three-dimensional variance (see Methods), from dark blue for the lowest variance to red for the highest variance. The map is filtered to the resolution at which the three-dimensional variance was estimated (20 Å).

Extended Data Figure 6 Comparison between the CSFV and the HCV pseudoknots.

Views of the structures of the HCV pseudoknot, from the 3.6 Å resolution crystal structure, with an additional crystallization module extending from helix III127 (a) (PDB: 3T4B) and the CSFV pseudoknot in the context of the 40S-subunit-bound ΔII-IRES (b) (this study) are shown in ribbon representation and coloured according to the scheme of the respective secondary structure diagrams (Extended Data Fig. 1a). c, d, Close-up views of HCV and CSFV pseudoknots, showing the ‘main’ helix, formed by helix III1 and pseudoknot (pk) stem 1A (in HCV) or helix III1, pk stem 1a and pk stem 1b (in CSFV), and the ‘sidecar’ helix, which contains subdomain IIIe, pk stem 2 and the two-base-pair helical segment of subdomain IIIf (see Extended Data Fig. 5a, b).

Extended Data Figure 7 Molecular interactions of the CSFV ΔII-IRES with the 40S subunit and interactions of eIF3 with the HCV and CSFV IRESs.

a, Secondary structure diagram of the CSFV ΔII-IRES, with nucleotides shown in different degrees of bold to show qualitatively their flexibility in the cryo-electron microscopy map (the more flexible, the bolder). Circled nucleotides interact with the indicated components of the 40S subunit. Ribosomal protein names and residue numbers are indicated according to the Tetrahymena thermophila 40S subunit42. bd, Secondary structure diagram of the apical region of domain III of the CSFV IRES (b, c) and the HCV IRES (d). b, Contacts of eIF3 with the IRES in the cryoEM map of the 40S–ΔII-IRES–eIF3 complex. c, d, Sites of strong protection of CSFV and HCV IRESs by native eIF3 from enzymatic cleavage and chemical modification, of protection of the HCV IRES by a 10-subunit form of eIF3 from 1M7 modification, or of interference with binding of eIF3 to the IRES by modification, as indicated in the keys5,9,11. Abbreviations: dimethyl sulphate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulphonate (CMCT), diethylpyrocarbonate (DEPC), 1-methyl-7-nitroisatoic anhydride (1M7). The inset panels show CSFV(c) and HCV IRESs (d), with helix III4 and subdomains IIIa, IIIb and IIIc in bold.

Extended Data Figure 8 Formation of elongation-competent 80S ribosomes on the HCV IRES depending on the presence of eIF3.

Toe-printing analysis of 48S initiation and 80S pre-termination complexes (pre-TC) assembled on the wild-type and ΔIIIb HCV (MSTN-STOP) mRNAs with translation components as indicated. The positions of the initiation and stop codons are shown on the left. Lanes C, T, A and G depict the cDNA sequence corresponding to the wild-type HCV (MSTN-STOP) mRNA. The gel reported in the figure is representative of results obtained from three technical replicates.

Extended Data Figure 9 Unsupervised three-dimensional classification protocol.

Details of the unsupervised three-dimensional classification. The classification included 6 rounds. For each round, the number of the particles included is indicated, as well as their percentages calculated over the full data set. The classes of rejected particles are crossed out in red and their percentages are indicated, also in red, as calculated over the full data set. Lines and brackets are drawn in different colours for clarity. Classes generated in rounds 3 to 6 are displayed and coloured by radial distance in Chimera UCSF43 in order to help in the visual discrimination of differences in features among the classes.

Supplementary information

Range of different orientations of eIF3 within the 40S•DHX29•∆II-IRES•eIF3 complex

This video displaying a morphing of density maps from classes 4 through 6 (Fig. S3), displaying eIF3 in three distinct orientations when bound on the IRES•40S complex. The complex is viewed from the top and the morphing illustrates the continuum of orientations sampled by eIF3 due to the flexibility of domain IIIb of the CSFV IRES. The video was made using Chimera UCSF47. (MP4 974 kb)

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Hashem, Y., des Georges, A., Dhote, V. et al. Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit. Nature 503, 539–543 (2013). https://doi.org/10.1038/nature12658

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