A conserved RNA structure within the HCV IRES eIF3-binding site

Abstract

The hepatitis C virus (HCV) internal ribosome entry site (IRES) is recognized specifically by the small ribosomal subunit and eukaryotic initiation factor 3 (eIF3) before viral translation initiation. Using extensive mutagenesis and structure probing analysis, we show that the eIF3-binding domain of the HCV IRES contains an internal loop structure (loop IIIb) and an adjacent mismatched helix that are important for IRES-dependent initiation of translation. NMR studies reveal a unique three-dimensional structure for this internal loop that is conserved between viral isolates of varying primary sequence in this region. These data indicate that internal loop IIIb may be an attractive target for structure-based design of new antiviral agents.

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Figure 1: Mutational and structural analysis of the HCV IIIb internal loop.
Figure 2: HCV IRES IIIb sequences and NMR spectral data.
Figure 3: Structure of HCV IRES IIIb internal loop.

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References

  1. 1

    Jackson, R.J. & Kaminski, A. RNA 1, 985–1000 (1995).

  2. 2

    Reynolds, J.E. et al. EMBO J. 14, 6010–6020 (1995).

  3. 3

    Sizova, D.V., Kolupaeva, V.G., Pestova, T.V., Shatsky, I.N. & Hellen, C.U.T. J. Virol. 72, 4775–4782 (1998).

  4. 4

    Kieft, J., Zhou, K., Rubin, R. & Doudna, J.A. RNA 7, 194–206 (2001).

  5. 5

    Pestova, T.V., Shatsky, I.N., Fletcher, S.P., Jackson, R.J. & Hellen, C.U.T. Genes Dev. 12, 67–83 (1998)

  6. 6

    Kieft, J.S. et al. J. Mol. Biol. 292, 513–529 (1999).

  7. 7

    Spahn, C.M. et al. Science 291, 1959–1962 (2001).

  8. 8

    Klinck, R. et al. RNA 6, 1423–1431 (2000).

  9. 9

    Lukavsky, P.J., Otto, G.A., Lancaster, A.M., Sarnow, P. & Puglisi, J.D. Nature Struct. Biol. 7, 1105–1110 (2000).

  10. 10

    Odreman-Macchioli, F.E., Tisminetzky, S.G., Zotti, M., Baralle, F.E. & Buratti, E. Nucleic Acids Res. 28, 875–885 (2000).

  11. 11

    Brown, E.A., Zhang, H., Ping, L.H. & Lemon, S.M. Nucleic Acids Res. 20, 5041–5045 (1992).

  12. 12

    Honda, M. et al. Virology 222, 31–42 (1996).

  13. 13

    Allain, F.H.-T. & Varani, G. J. Mol. Biol. 250, 333–353 (1995).

  14. 14

    Wimberly, B., Varani, G. & Tinoco, I. Jr Biochemistry 32, 1078–1087 (1993).

  15. 15

    Szewczak, A.A., Moore, P.B., Chan, Y.-L. & Wool, I.G. Proc. Natl. Acad. Sci. USA 90, 9581–9585 (1993).

  16. 16

    Chang, K.Y., Varani, G., Bhattacharya, S., Choi, H. & McClain, W.H. Proc. Natl. Acad. Sci. USA 96, 11764–11769 (1999).

  17. 17

    Mariappan, S.V., Catasti, P., Silks, L.A.I., Bradbury, E.M. & Gupta, G. J. Mol. Biol. 285, 2035–2052 (1999).

  18. 18

    Hennig, M. & Williamson, J.R. Nucleic Acids Res. 28, 1585–1593 (2000).

  19. 19

    Churcher, M.J. et al. J. Mol. Biol. 230, 90–110 (1993).

  20. 20

    Heaphy, S., Finch, J.T., Gait, M.J., Karn, J. & Singh, M. Proc. Natl. Acad. Sci. USA 88, 7366–7370 (1991).

  21. 21

    Rijnbrand, R.C.A., Abbink, E.M., Joost Haasnoot, P.C., Spaan, W.J.M. & Bredenbeek, P.J. Virology 226, 47–56 (1996)

  22. 22

    Reynolds, J.E. et al. RNA 2, 867–878 (1996)

  23. 23

    Peterson, R.D. & Feigon, J. J. Mol. Biol. 264, 863–877 (1996).

  24. 24

    Zeffman, A., Hassard, S., Varani, G. & Lever, A. J. Mol. Biol. 297, 877–893 (2000).

  25. 25

    Gubser, C.C. & Varani, G. Biochemistry 35, 2253–2267 (1996).

  26. 26

    Price, S.R., Oubridge, C., Varani, G. & Nagai, K. In RNA-protein interaction: practical approach (ed. Smith, C.) 37–74 (Oxford University Press, New York; 1998).

  27. 27

    Delaglio, F. et al. J. Biomol. NMR 6, 277–293 (1995).

  28. 28

    Goddard, T.D. & Kneller, D.G. Sparky 3. (University of California, San Francisco; 2001).

  29. 29

    Varani, G., Aboul-ela, F. & Allain, F.H.-T. Prog. NMR Spectrosc. 29, 51–127 (1996).

  30. 30

    Brünger, A.T. X-PLOR v3.854 (Yale University, New Haven; 1996).

  31. 31

    Honda, M., Rijnbrand, R., Abell, G., Kim, D. & Lemon, S.M. J. Virol. 73, 4941–4951 (1999).

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Acknowledgements

This work was supported by an EU FP5 grant, an EU Marie Curie Fellowship to J.G. and the Medical Research Council. We also wish to thank J. Karn for initiating the project at RiboTargets Ltd.

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Correspondence to Stephen Walker.

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Competing interests

The submitted manuscript reports the results and conclusions of basic research performed by employees of RiboTargets and academic collaborators. Although some of the authors hold share options in the company, the manuscript represents an unbiased interpretation of the research results and data. Because publication of the manuscript has an indirect impact on the value of share options, I declare (on behalf of the authors) a competing interest.

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Collier, A., Gallego, J., Klinck, R. et al. A conserved RNA structure within the HCV IRES eIF3-binding site. Nat Struct Mol Biol 9, 375–380 (2002). https://doi.org/10.1038/nsb785

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