Skip to main content
NCBI home page
RNA logoLink to RNA
. 2001 Feb;7(2):194–206. doi: 10.1017/s1355838201001790

Mechanism of ribosome recruitment by hepatitis C IRES RNA.

J S Kieft 1, K Zhou 1, R Jubin 1, J A Doudna 1
PMCID: PMC1370078  PMID: 11233977

Abstract

Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 5' untranslated region, called the internal ribosome entry site (IRES). In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle. Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA-40S-eIF3 ternary complex. Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date.

Full Text

The Full Text of this article is available as a PDF (3.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ali N., Siddiqui A. Interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation. J Virol. 1995 Oct;69(10):6367–6375. doi: 10.1128/jvi.69.10.6367-6375.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ali N., Siddiqui A. The La antigen binds 5' noncoding region of the hepatitis C virus RNA in the context of the initiator AUG codon and stimulates internal ribosome entry site-mediated translation. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2249–2254. doi: 10.1073/pnas.94.6.2249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benne R., Hershey J. W. Purification and characterization of initiation factor IF-E3 from rabbit reticulocytes. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3005–3009. doi: 10.1073/pnas.73.9.3005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Borman A. M., Bailly J. L., Girard M., Kean K. M. Picornavirus internal ribosome entry segments: comparison of translation efficiency and the requirements for optimal internal initiation of translation in vitro. Nucleic Acids Res. 1995 Sep 25;23(18):3656–3663. doi: 10.1093/nar/23.18.3656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown E. A., Zhang H., Ping L. H., Lemon S. M. Secondary structure of the 5' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs. Nucleic Acids Res. 1992 Oct 11;20(19):5041–5045. doi: 10.1093/nar/20.19.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buratti E., Tisminetzky S., Zotti M., Baralle F. E. Functional analysis of the interaction between HCV 5'UTR and putative subunits of eukaryotic translation initiation factor eIF3. Nucleic Acids Res. 1998 Jul 1;26(13):3179–3187. doi: 10.1093/nar/26.13.3179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cameron V., Uhlenbeck O. C. 3'-Phosphatase activity in T4 polynucleotide kinase. Biochemistry. 1977 Nov 15;16(23):5120–5126. doi: 10.1021/bi00642a027. [DOI] [PubMed] [Google Scholar]
  8. Celander D. W., Cech T. R. Visualizing the higher order folding of a catalytic RNA molecule. Science. 1991 Jan 25;251(4992):401–407. doi: 10.1126/science.1989074. [DOI] [PubMed] [Google Scholar]
  9. Chaudhuri J., Chakrabarti A., Maitra U. Biochemical characterization of mammalian translation initiation factor 3 (eIF3). Molecular cloning reveals that p110 subunit is the mammalian homologue of Saccharomyces cerevisiae protein Prt1. J Biol Chem. 1997 Dec 5;272(49):30975–30983. doi: 10.1074/jbc.272.49.30975. [DOI] [PubMed] [Google Scholar]
  10. Christian E. L., Yarus M. Analysis of the role of phosphate oxygens in the group I intron from Tetrahymena. J Mol Biol. 1992 Dec 5;228(3):743–758. doi: 10.1016/0022-2836(92)90861-d. [DOI] [PubMed] [Google Scholar]
  11. Conway L., Wickens M. Modification interference analysis of reactions using RNA substrates. Methods Enzymol. 1989;180:369–379. doi: 10.1016/0076-6879(89)80112-0. [DOI] [PubMed] [Google Scholar]
  12. England T. E., Uhlenbeck O. C. 3'-terminal labelling of RNA with T4 RNA ligase. Nature. 1978 Oct 12;275(5680):560–561. doi: 10.1038/275560a0. [DOI] [PubMed] [Google Scholar]
  13. Fukushi S., Okada M., Kageyama T., Hoshino F. B., Katayama K. Specific interaction of a 25-kilodalton cellular protein, a 40S ribosomal subunit protein, with the internal ribosome entry site of hepatitis C virus genome. Virus Genes. 1999;19(2):153–161. doi: 10.1023/a:1008131325056. [DOI] [PubMed] [Google Scholar]
  14. Garcia-Barrio M. T., Naranda T., Vazquez de Aldana C. R., Cuesta R., Hinnebusch A. G., Hershey J. W., Tamame M. GCD10, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic translation initiation factor-3. Genes Dev. 1995 Jul 15;9(14):1781–1796. doi: 10.1101/gad.9.14.1781. [DOI] [PubMed] [Google Scholar]
  15. Goss D. J., Rounds D. J. A kinetic light-scattering study of the binding of wheat germ protein synthesis initiation factor 3 to 40S ribosomal subunits and 80S ribosomes. Biochemistry. 1988 May 17;27(10):3610–3613. doi: 10.1021/bi00410a012. [DOI] [PubMed] [Google Scholar]
  16. Hahm B., Kim Y. K., Kim J. H., Kim T. Y., Jang S. K. Heterogeneous nuclear ribonucleoprotein L interacts with the 3' border of the internal ribosomal entry site of hepatitis C virus. J Virol. 1998 Nov;72(11):8782–8788. doi: 10.1128/jvi.72.11.8782-8788.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Honda M., Beard M. R., Ping L. H., Lemon S. M. A phylogenetically conserved stem-loop structure at the 5' border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation. J Virol. 1999 Feb;73(2):1165–1174. doi: 10.1128/jvi.73.2.1165-1174.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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. 1996 Oct;2(10):955–968. [PMC free article] [PubMed] [Google Scholar]
  19. Honda M., Ping L. H., Rijnbrand R. C., Amphlett E., Clarke B., Rowlands D., Lemon S. M. Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA. Virology. 1996 Aug 1;222(1):31–42. doi: 10.1006/viro.1996.0395. [DOI] [PubMed] [Google Scholar]
  20. Jubin R., Murray M. G. Activity screening of bacteria containing Renilla luciferase plasmids. Biotechniques. 1998 Feb;24(2):185–188. doi: 10.2144/98242bm02. [DOI] [PubMed] [Google Scholar]
  21. Kamoshita N., Tsukiyama-Kohara K., Kohara M., Nomoto A. Genetic analysis of internal ribosomal entry site on hepatitis C virus RNA: implication for involvement of the highly ordered structure and cell type-specific transacting factors. Virology. 1997 Jun 23;233(1):9–18. doi: 10.1006/viro.1997.8600. [DOI] [PubMed] [Google Scholar]
  22. Kieft J. S., Zhou K., Jubin R., Murray M. G., Lau J. Y., Doudna J. A. The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold. J Mol Biol. 1999 Sep 24;292(3):513–529. doi: 10.1006/jmbi.1999.3095. [DOI] [PubMed] [Google Scholar]
  23. 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. 2000 Jul;74(14):6242–6250. doi: 10.1128/jvi.74.14.6242-6250.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Latham J. A., Cech T. R. Defining the inside and outside of a catalytic RNA molecule. Science. 1989 Jul 21;245(4915):276–282. doi: 10.1126/science.2501870. [DOI] [PubMed] [Google Scholar]
  25. Le S. Y., Sonenberg N., Maizel J. V., Jr Unusual folding regions and ribosome landing pad within hepatitis C virus and pestivirus RNAs. Gene. 1995 Mar 10;154(2):137–143. doi: 10.1016/0378-1119(94)00859-q. [DOI] [PubMed] [Google Scholar]
  26. Odreman-Macchioli F. E., Tisminetzky S. G., Zotti M., Baralle F. E., Buratti E. Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES. Nucleic Acids Res. 2000 Feb 15;28(4):875–885. doi: 10.1093/nar/28.4.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ortoleva-Donnelly L., Szewczak A. A., Gutell R. R., Strobel S. A. The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme. RNA. 1998 May;4(5):498–519. doi: 10.1017/s1355838298980086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pain V. M. Initiation of protein synthesis in eukaryotic cells. Eur J Biochem. 1996 Mar 15;236(3):747–771. doi: 10.1111/j.1432-1033.1996.00747.x. [DOI] [PubMed] [Google Scholar]
  29. Pestova T. V., Hellen C. U., Shatsky I. N. Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol Cell Biol. 1996 Dec;16(12):6859–6869. doi: 10.1128/mcb.16.12.6859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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. 1998 Jan 1;12(1):67–83. doi: 10.1101/gad.12.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pickering J. M., Thomas H. C., Karayiannis P. Predicted secondary structure of the hepatitis G virus and GB virus-A 5'untranslated regions consistent with an internal ribosome entry site. J Viral Hepat. 1997 May;4(3):175–184. doi: 10.1046/j.1365-2893.1997.00143.x. [DOI] [PubMed] [Google Scholar]
  32. Psaridi L., Georgopoulou U., Varaklioti A., Mavromara P. Mutational analysis of a conserved tetraloop in the 5' untranslated region of hepatitis C virus identifies a novel RNA element essential for the internal ribosome entry site function. FEBS Lett. 1999 Jun 18;453(1-2):49–53. doi: 10.1016/s0014-5793(99)00662-6. [DOI] [PubMed] [Google Scholar]
  33. Reynolds J. E., Kaminski A., Carroll A. R., Clarke B. E., Rowlands D. J., Jackson R. J. Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon. RNA. 1996 Sep;2(9):867–878. [PMC free article] [PubMed] [Google Scholar]
  34. Rijnbrand R. C., Lemon S. M. Internal ribosome entry site-mediated translation in hepatitis C virus replication. Curr Top Microbiol Immunol. 2000;242:85–116. doi: 10.1007/978-3-642-59605-6_5. [DOI] [PubMed] [Google Scholar]
  35. Rijnbrand R., Bredenbeek P., van der Straaten T., Whetter L., Inchauspé G., Lemon S., Spaan W. Almost the entire 5' non-translated region of hepatitis C virus is required for cap-independent translation. FEBS Lett. 1995 May 29;365(2-3):115–119. doi: 10.1016/0014-5793(95)00458-l. [DOI] [PubMed] [Google Scholar]
  36. Sachs A. B., Sarnow P., Hentze M. W. Starting at the beginning, middle, and end: translation initiation in eukaryotes. Cell. 1997 Jun 13;89(6):831–838. doi: 10.1016/s0092-8674(00)80268-8. [DOI] [PubMed] [Google Scholar]
  37. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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. 1998 Jun;72(6):4775–4782. doi: 10.1128/jvi.72.6.4775-4782.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smith D. B., Mellor J., Jarvis L. M., Davidson F., Kolberg J., Urdea M., Yap P. L., Simmonds P. Variation of the hepatitis C virus 5' non-coding region: implications for secondary structure, virus detection and typing. The International HCV Collaborative Study Group. J Gen Virol. 1995 Jul;76(Pt 7):1749–1761. doi: 10.1099/0022-1317-76-7-1749. [DOI] [PubMed] [Google Scholar]
  40. Tang S., Collier A. J., Elliott R. M. Alterations to both the primary and predicted secondary structure of stem-loop IIIc of the hepatitis C virus 1b 5' untranslated region (5'UTR) lead to mutants severely defective in translation which cannot be complemented in trans by the wild-type 5'UTR sequence. J Virol. 1999 Mar;73(3):2359–2364. doi: 10.1128/jvi.73.3.2359-2364.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Varaklioti A., Georgopoulou U., Kakkanas A., Psaridi L., Serwe M., Caselmann W. H., Mavromara P. Mutational analysis of two unstructured domains of the 5' untranslated region of HCV RNA. Biochem Biophys Res Commun. 1998 Dec 30;253(3):678–685. doi: 10.1006/bbrc.1998.9842. [DOI] [PubMed] [Google Scholar]
  42. Wang C., Le S. Y., Ali N., Siddiqui A. An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region. RNA. 1995 Jul;1(5):526–537. [PMC free article] [PubMed] [Google Scholar]
  43. Wang C., Sarnow P., Siddiqui A. A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA. J Virol. 1994 Nov;68(11):7301–7307. doi: 10.1128/jvi.68.11.7301-7307.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yen J. H., Chang S. C., Hu C. R., Chu S. C., Lin S. S., Hsieh Y. S., Chang M. F. Cellular proteins specifically bind to the 5'-noncoding region of hepatitis C virus RNA. Virology. 1995 Apr 20;208(2):723–732. doi: 10.1006/viro.1995.1204. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES