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HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation

Nature Structural & Molecular Biology volume 20, pages 150–158 (2013)Cite this article

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Abstract

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have focused on 80S ribosome formation but have not explored its role after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause only a local perturbation in IRES structure and result in conformational changes in the IRES–rabbit 40S subunit complex. Functionally, the mutations decrease IRES activity by inhibiting the first ribosomal translocation event, and modeling results suggest that this effect occurs through an interaction with a single ribosomal protein. The ability of the HCV IRES to manipulate the ribosome provides insight into how the ribosome's structure and function can be altered by bound RNAs, including those derived from cellular invaders.

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Figure 1: In vitro translation analysis of dIIb mutations.
Figure 2: EM of HCV IRES mutant ΔGCC in complex with the 40S subunit.
Figure 3: WT and mutant IRES ribosome assembly assays and position of domain IIb.
Figure 4: Characterization of the structural changes induced by dIIb mutation.
Figure 5: Biochemical analysis of AUG docking and potential frameshift.
Figure 6: Puromycin and toeprinting assays with antibiotic.
Figure 7: Model of the role of dIIb in HCV IRES translation initiation.

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Acknowledgements

The authors thank the members of J.S.K.'s lab and R. Davis, D. Bentley, D. Barton and T. Evans for useful suggestions and discussions, and M. Ruehle, T. Blumenthal, T. Cech and M. Johnston for critical reading of this manuscript. We also thank C. Spahn (Institut für Medizinische Physik und Biophysik, Charite–Universitätsmedizin Berlin) for data files and advice with structural modeling; P. Lukavsky (Central European Institute of Technology, Masaryk University) for NMR resonance assignments and the pUC18 plasmid for toeprinting experiments; A.Willis (Medical Research Council Toxicology Unit) for the pRL plasmid for LUC experiments and G. Armstrong and E. Eisenmesser for assistance in NMR data collection and processing. This work was supported by US National Institutes of Health grant GM081346 to J.S.K. M.E.F. was supported as an American Heart Association predoctoral fellow (grant no. 0815655G). J.S.K. is an Early Career Scientist of the Howard Hughes Medical Institute. T.G.'s laboratory is supported by the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, Colorado, USA

    Megan E Filbin & Jeffrey S Kieft

  2. Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    Breanna S Vollmar, Dan Shi & Tamir Gonen

  3. Howard Hughes Medical Institute, University of Colorado Denver School of Medicine, Aurora, Colorado, USA

    Jeffrey S Kieft

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Contributions

M.E.F. conducted all biochemical experiments. J.S.K. and M.E.F. conducted and analyzed the NMR experiments. B.S.V., D.S., T.G. and J.S.K. conducted the cryo-EM experiments, with structure calculation by B.S.V. Results were interpreted by M.E.F., B.S.V., J.S.K. and T.G. M.E.F. and J.S.K. designed the overall study and wrote the manuscript. All authors contributed to figure construction.

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Correspondence to Tamir Gonen or Jeffrey S Kieft.

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Filbin, M., Vollmar, B., Shi, D. et al. HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation. Nat Struct Mol Biol 20, 150–158 (2013). https://doi.org/10.1038/nsmb.2465

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