Nothing Special   »   [go: up one dir, main page]
Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Subjects

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.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Accession codes

Primary accessions

Electron Microscopy Data Bank

Referenced accessions

Electron Microscopy Data Bank

References

  1. Tsukiyama-Kohara, K., Iizuka, N., Kohara, M. & Nomoto, A. Internal ribosome entry site within hepatitis C virus RNA. J. Virol. 66, 1476–1483 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Bukh, J., Purcell, R.H. & Miller, R.H. Sequence analysis of the 5′ noncoding region of hepatitis C virus. Proc. Natl. Acad. Sci. USA 89, 4942–4946 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Simmonds, P. et al. Sequence variability in the 5′ non-coding region of hepatitis C virus: identification of a new virus type and restrictions on sequence diversity. J. Gen. Virol. 74, 661–668 (1993).

    Article  CAS  PubMed  Google Scholar 

  4. Fraser, C.S. & Doudna, J.A. Structural and mechanistic insights into hepatitis C viral translation initiation. Nat. Rev. Microbiol. 5, 29–38 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Jackson, R.J., Hellen, C.U. & Pestova, T.V. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat. Rev. Mol. Cell Biol. 11, 113–127 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kieft, J.S., Zhou, K., Jubin, R. & Doudna, J.A. Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA 7, 194–206 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kolupaeva, V.G., Pestova, T.V. & Hellen, C.U. An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus. J. Virol. 74, 6242–6250 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lytle, J.R., Wu, L. & Robertson, H.D. The ribosome binding site of hepatitis C virus mRNA. J. Virol. 75, 7629–7636 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lytle, J.R., Wu, L. & Robertson, H.D. Domains on the hepatitis C virus internal ribosome entry site for 40s subunit binding. RNA 8, 1045–1055 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pestova, T.V., Shatsky, I.N., Fletcher, S.P., Jackson, R.J. & Hellen, C.U. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev. 12, 67–83 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Otto, G.A. & Puglisi, J.D. The pathway of HCV IRES-mediated translation initiation. Cell 119, 369–380 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Ji, H., Fraser, C.S., Yu, Y., Leary, J. & Doudna, J.A. Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA. Proc. Natl. Acad. Sci. USA 101, 16990–16995 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Fraser, C.S., Hershey, J.W. & Doudna, J.A. The pathway of hepatitis C virus mRNA recruitment to the human ribosome. Nat. Struct. Mol. Biol. 16, 397–404 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Locker, N., Easton, L.E. & Lukavsky, P.J. HCV and CSFV IRES domain II mediate eIF2 release during 80S ribosome assembly. EMBO J. 26, 795–805 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Terenin, I.M., Dmitriev, S.E., Andreev, D.E. & Shatsky, I.N. Eukaryotic translation initiation machinery can operate in a bacterial-like mode without eIF2. Nat. Struct. Mol. Biol. 15, 836–841 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. Kim, J.H., Park, S.M., Park, J.H., Keum, S.J. & Jang, S.K. eIF2A mediates translation of hepatitis C viral mRNA under stress conditions. EMBO J. 30, 2454–2464 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kieft, J.S. et al. The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold. J. Mol. Biol. 292, 513–529 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Lukavsky, P.J. Structure and function of HCV IRES domains. Virus Res. 139, 166–171 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sizova, D.V., Kolupaeva, V.G., Pestova, T.V., Shatsky, I.N. & Hellen, C.U. Specific interaction of eukaryotic translation initiation factor 3 with the 5′ nontranslated regions of hepatitis C virus and classical swine fever virus RNAs. J. Virol. 72, 4775–4782 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Honda, M., Brown, E.A. & Lemon, S.M. Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA. RNA 2, 955–968 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Berry, K.E., Waghray, S. & Doudna, J.A. The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit. RNA 16, 1559–1569 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Filbin, M.E. & Kieft, J.S. HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove. RNA 17, 1258–1273 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Spahn, C.M. et al. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit. Science 291, 1959–1962 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Boehringer, D., Thermann, R., Ostareck-Lederer, A., Lewis, J.D. & Stark, H. Structure of the hepatitis C virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES. Structure 13, 1695–1706 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Fukushi, S. et al. Ribosomal protein S5 interacts with the internal ribosomal entry site of hepatitis C virus. J. Biol. Chem. 276, 20824–20826 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Wower, J., Scheffer, P., Sylvers, L.A., Wintermeyer, W. & Zimmermann, R.A. Topography of the E site on the Escherichia coli ribosome. EMBO J. 12, 617–623 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yusupov, M.M. et al. Crystal structure of the ribosome at 5.5-Å resolution. Science 292, 883–896 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Döring, T., Mitchell, P., Osswald, M., Bochkariov, D. & Brimacombe, R. The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop. EMBO J. 13, 2677–2685 (1994).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Odreman-Macchioli, F., Baralle, F.E. & Buratti, E. Mutational analysis of the different bulge regions of hepatitis C virus domain II and their influence on internal ribosome entry site translational ability. J. Biol. Chem. 276, 41648–41655 (2001).

    Article  CAS  PubMed  Google Scholar 

  30. Kalliampakou, K.I., Psaridi-Linardaki, L. & Mavromara, P. Mutational analysis of the apical region of domain II of the HCV IRES. FEBS Lett. 511, 79–84 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Passmore, L.A. et al. The eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome. Mol. Cell 26, 41–50 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Rabl, J., Leibundgut, M., Ataide, S.F., Haag, A. & Ban, N. Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1. Science 331, 730–736 (2011).

    Article  CAS  PubMed  Google Scholar 

  33. Lukavsky, P.J., Kim, I., Otto, G.A. & Puglisi, J.D. Structure of HCV IRES domain II determined by NMR. Nat. Struct. Biol. 10, 1033–1038 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Pestova, T.V., Hellen, C.U. & Shatsky, I.N. Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol. Cell Biol. 16, 6859–6869 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wilson, J.E., Pestova, T.V., Hellen, C.U. & Sarnow, P. Initiation of protein synthesis from the A site of the ribosome. Cell 102, 511–520 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Hartz, D., McPheeters, D.S., Traut, R. & Gold, L. Extension inhibition analysis of translation initiation complexes. Methods Enzymol. 164, 419–425 (1988).

    Article  CAS  PubMed  Google Scholar 

  37. Devaraj, A., Shoji, S., Holbrook, E.D. & Fredrick, K. A role for the 30S subunit E site in maintenance of the translational reading frame. RNA 15, 255–265 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Monro, R.E. & Marcker, K.A. Ribosome-catalysed reaction of puromycin with a formylmethionine-containing oligonucleotide. J. Mol. Biol. 25, 347–350 (1967).

    Article  CAS  PubMed  Google Scholar 

  39. Peske, F., Savelsbergh, A., Katunin, V.I., Rodnina, M.V. & Wintermeyer, W. Conformational changes of the small ribosomal subunit during elongation factor G-dependent tRNA-mRNA translocation. J. Mol. Biol. 343, 1183–1194 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Dibrov, S.M. et al. Structure of a hepatitis C virus RNA domain in complex with a translation inhibitor reveals a binding mode reminiscent of riboswitches. Proc. Natl. Acad. Sci. USA 109, 5223–5228 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Robert, F. & Brakier-Gingras, L. A functional interaction between ribosomal proteins S7 and S11 within the bacterial ribosome. J. Biol. Chem. 278, 44913–44920 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Galkin, O. et al. Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5. RNA 13, 2116–2128 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Geigenmüller, U. & Nierhaus, K.H. Significance of the third tRNA binding site, the E site, on E. coli ribosomes for the accuracy of translation: an occupied E site prevents the binding of non-cognate aminoacyl-tRNA to the A site. EMBO J. 9, 4527–4533 (1990).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Petropoulos, A.D. & Green, R. Further in vitro exploration fails to support the allosteric three-site model. J. Biol. Chem. 287, 11642–11648 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Uemura, S. et al. Real-time tRNA transit on single translating ribosomes at codon resolution. Nature 464, 1012–1017 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Chen, C. et al. Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis. Proc. Natl. Acad. Sci. USA 108, 16980–16985 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Malygin, A.A., Yanshina, D.D. & Karpova, G.G. Interactions of human ribosomal proteins S16 and S5 with an 18S rRNA fragment containing their binding sites. Biochimie 91, 1180–1186 (2009).

    Article  CAS  PubMed  Google Scholar 

  48. Ian'shina, D.D., Malygin, A.A. & Karpova, G.G. Binding of human ribosomal protein S5 with the 18S rRNA fragment 1203–1236/1521–1698 [in Russian]. Mol. Biol. (Mosk.) 40, 460–467 (2006).

    CAS  Google Scholar 

  49. Yu, Y. et al. Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing. Nucleic Acids Res. 37, 5167–5182 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Antúnez de Mayolo, P. & Woolford, J.L. Jr. Interactions of yeast ribosomal protein rpS14 with RNA. J. Mol. Biol. 333, 697–709 (2003).

    Article  PubMed  CAS  Google Scholar 

  51. Lomakin, I.B., Kolupaeva, V.G., Marintchev, A., Wagner, G. & Pestova, T.V. Position of eukaryotic initiation factor eIF1 on the 40S ribosomal subunit determined by directed hydroxyl radical probing. Genes Dev. 17, 2786–2797 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Acker, M.G. et al. Kinetic analysis of late steps of eukaryotic translation initiation. J. Mol. Biol. 385, 491–506 (2009).

    Article  CAS  PubMed  Google Scholar 

  53. Fringer, J.M., Acker, M.G., Fekete, C.A., Lorsch, J.R. & Dever, T.E. Coupled release of eukaryotic translation initiation factors 5B and 1A from 80S ribosomes following subunit joining. Mol. Cell. Biol. 27, 2384–2397 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. van Heel, M., Harauz, G., Orlova, E.V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996).

    Article  CAS  PubMed  Google Scholar 

  55. Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006).

    Article  CAS  PubMed  Google Scholar 

  56. Ben-Shem, A. et al. The structure of the eukaryotic ribosome at 3.0-Å resolution. Science 334, 1524–1529 (2011).

    Article  CAS  PubMed  Google Scholar 

  57. Stoneley, M., Paulin, F.E., Le Quesne, J.P., Chappell, S.A. & Willis, A.E. C-Myc 5′ untranslated region contains an internal ribosome entry segment. Oncogene 16, 423–428 (1998).

    Article  CAS  PubMed  Google Scholar 

  58. Keel, A.Y., Easton, L.E., Lukavsky, P.J. & Kieft, J.S. Large-scale native preparation of in vitro transcribed RNA. Methods Enzymol. 469, 3–25 (2009).

    Article  CAS  PubMed  Google Scholar 

  59. Ohi, M., Li, Y., Cheng, Y. & Walz, T. Negative Staining and Image Classification - Powerful Tools in Modern Electron Microscopy. Biol. Proced. Online 6, 23–34 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Mindell, J.A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  PubMed  Google Scholar 

  61. Grigorieff, N. FREALIGN: high-resolution refinement of single particle structures. J. Struct. Biol. 157, 117–125 (2007).

    Article  CAS  PubMed  Google Scholar 

  62. Sousa, D. & Grigorieff, N. Ab initio resolution measurement for single particle structures. J. Struct. Biol. 157, 201–210 (2007).

    Article  CAS  PubMed  Google Scholar 

  63. Kleywegt, G.J. & Jones, T.A. xdlMAPMAN and xdlDATAMAN - programs for reformatting, analysis and manipulation of biomacromolecular electron-density maps and reflection data sets. Acta Crystallogr. D Biol. Crystallogr. 52, 826–828 (1996).

    Article  CAS  PubMed  Google Scholar 

  64. Pettersen, E.F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

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

Authors
  1. Megan E Filbin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Breanna S Vollmar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dan Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tamir Gonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeffrey S Kieft
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

Correspondence to Tamir Gonen or Jeffrey S Kieft.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2465

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing