Skip to main content
PMC home page
RNA logoLink to RNA
. 2002 Jul;8(7):913–923. doi: 10.1017/s1355838202022057

Ribosomal proteins mediate the hepatitis C virus IRES-HeLa 40S interaction.

Geoff A Otto 1, Peter J Lukavsky 1, Alissa M Lancaster 1, Peter Sarnow 1, Joseph D Puglisi 1
PMCID: PMC1370308  PMID: 12166646

Abstract

Translation of the hepatitis C virus genomic RNA is mediated by an internal ribosome entry site (IRES). The 330-nt IRES RNA forms a binary complex with the small 40S ribosomal subunit as a first step in translation initiation. Here chemical probing and 4-thiouridine-mediated crosslinking are used to characterize the interaction of the HCV IRES with the HeLa 40S subunit. No IRES-18S rRNA contacts were detected, but several specific crosslinks to 40S ribosomal proteins were observed. The identity of the crosslinked proteins agrees well with available structural information and provides new insights into HCV IRES function. The protein-rich surface of the 40S subunit thus mediates the IRES-ribosome interaction.

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

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

  1. Agafonov D. E., Kolb V. A., Nazimov I. V., Spirin A. S. A protein residing at the subunit interface of the bacterial ribosome. Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12345–12349. doi: 10.1073/pnas.96.22.12345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blobel G., Sabatini D. Dissociation of mammalian polyribosomes into subunits by puromycin. Proc Natl Acad Sci U S A. 1971 Feb;68(2):390–394. doi: 10.1073/pnas.68.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brow D. A., Noller H. F. Protection of ribosomal RNA from kethoxal in polyribosomes. Implication of specific sites in ribosome function. J Mol Biol. 1983 Jan 5;163(1):27–46. doi: 10.1016/0022-2836(83)90028-1. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Carter A. P., Clemons W. M., Brodersen D. E., Morgan-Warren R. J., Wimberly B. T., Ramakrishnan V. Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature. 2000 Sep 21;407(6802):340–348. doi: 10.1038/35030019. [DOI] [PubMed] [Google Scholar]
  6. Carter A. P., Clemons W. M., Jr, Brodersen D. E., Morgan-Warren R. J., Hartsch T., Wimberly B. T., Ramakrishnan V. Crystal structure of an initiation factor bound to the 30S ribosomal subunit. Science. 2001 Jan 19;291(5503):498–501. doi: 10.1126/science.1057766. [DOI] [PubMed] [Google Scholar]
  7. Frank J., Agrawal R. K. A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature. 2000 Jul 20;406(6793):318–322. doi: 10.1038/35018597. [DOI] [PubMed] [Google Scholar]
  8. Fukushi S., Okada M., Stahl J., Kageyama T., Hoshino F. B., Katayama K. Ribosomal protein S5 interacts with the internal ribosomal entry site of hepatitis C virus. J Biol Chem. 2001 Apr 30;276(24):20824–20826. doi: 10.1074/jbc.C100206200. [DOI] [PubMed] [Google Scholar]
  9. Gabashvili I. S., Agrawal R. K., Grassucci R., Squires C. L., Dahlberg A. E., Frank J. Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA. EMBO J. 1999 Nov 15;18(22):6501–6507. doi: 10.1093/emboj/18.22.6501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gingras A. C., Raught B., Sonenberg N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem. 1999;68:913–963. doi: 10.1146/annurev.biochem.68.1.913. [DOI] [PubMed] [Google Scholar]
  11. Graifer D. M., Juzumiene D. I., Wollenzien P., Karpova G. G. Cross-linking of mRNA analogues containing 4-thiouridine residues on the 3'- or 5'-side of the coding triplet to the mRNA binding center of the human ribosome. Biochemistry. 1994 Apr 5;33(13):3878–3884. doi: 10.1021/bi00179a012. [DOI] [PubMed] [Google Scholar]
  12. Green R., Noller H. F. Ribosomes and translation. Annu Rev Biochem. 1997;66:679–716. doi: 10.1146/annurev.biochem.66.1.679. [DOI] [PubMed] [Google Scholar]
  13. Gressner A. M. Human liver ribosomal proteins: characterization by two-dimensional electrophoresis and molecular weight determinations. Biochem Med. 1980 Jun;23(3):350–357. doi: 10.1016/0006-2944(80)90045-9. [DOI] [PubMed] [Google Scholar]
  14. Han H., Schepartz A., Pellegrini M., Dervan P. B. Mapping RNA regions in eukaryotic ribosomes that are accessible to methidiumpropyl-EDTA.Fe(II) and EDTA.Fe(II). Biochemistry. 1994 Aug 23;33(33):9831–9844. doi: 10.1021/bi00199a004. [DOI] [PubMed] [Google Scholar]
  15. Hardy S. J., Kurland C. G., Voynow P., Mora G. The ribosomal proteins of Escherichia coli. I. Purification of the 30S ribosomal proteins. Biochemistry. 1969 Jul;8(7):2897–2905. doi: 10.1021/bi00835a031. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Jubin R., Vantuno N. E., Kieft J. S., Murray M. G., Doudna J. A., Lau J. Y., Baroudy B. M. Hepatitis C virus internal ribosome entry site (IRES) stem loop IIId contains a phylogenetically conserved GGG triplet essential for translation and IRES folding. J Virol. 2000 Nov;74(22):10430–10437. doi: 10.1128/jvi.74.22.10430-10437.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Karimi R., Ehrenberg M. Dissociation rates of peptidyl-tRNA from the P-site of E.coli ribosomes. EMBO J. 1996 Mar 1;15(5):1149–1154. [PMC free article] [PubMed] [Google Scholar]
  20. Kieft J. S., Zhou K., Jubin R., Doudna J. A. Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA. 2001 Feb;7(2):194–206. doi: 10.1017/s1355838201001790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Klinck R., Westhof E., Walker S., Afshar M., Collier A., Aboul-Ela F. A potential RNA drug target in the hepatitis C virus internal ribosomal entry site. RNA. 2000 Oct;6(10):1423–1431. doi: 10.1017/s1355838200000935. [DOI] [PMC free article] [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. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  25. Link A. J., Eng J., Schieltz D. M., Carmack E., Mize G. J., Morris D. R., Garvik B. M., Yates J. R., 3rd Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol. 1999 Jul;17(7):676–682. doi: 10.1038/10890. [DOI] [PubMed] [Google Scholar]
  26. Long K. S., Crothers D. M. Interaction of human immunodeficiency virus type 1 Tat-derived peptides with TAR RNA. Biochemistry. 1995 Jul 11;34(27):8885–8895. doi: 10.1021/bi00027a041. [DOI] [PubMed] [Google Scholar]
  27. Lorsch J. R., Herschlag D. Kinetic dissection of fundamental processes of eukaryotic translation initiation in vitro. EMBO J. 1999 Dec 1;18(23):6705–6717. doi: 10.1093/emboj/18.23.6705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lukavsky P. J., Otto G. A., Lancaster A. M., Sarnow P., Puglisi J. D. Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function. Nat Struct Biol. 2000 Dec;7(12):1105–1110. doi: 10.1038/81951. [DOI] [PubMed] [Google Scholar]
  29. Lutsch G., Stahl J., Kärgel H. J., Noll F., Bielka H. Immunoelectron microscopic studies on the location of ribosomal proteins on the surface of the 40S ribosomal subunit from rat liver. Eur J Cell Biol. 1990 Feb;51(1):140–150. [PubMed] [Google Scholar]
  30. Madjar J. J., Arpin M., Buisson M., Reboud J. P. Spot position of rat liver ribosomal proteins by four different two-dimensional electrophoreses in polyacrylamide gel. Mol Gen Genet. 1979 Mar 20;171(2):121–134. doi: 10.1007/BF00269998. [DOI] [PubMed] [Google Scholar]
  31. Mets L. J., Bogorad L. Two-dimensional polyacrylamide gel electrophoresis: an improved method for ribosomal proteins. Anal Biochem. 1974 Jan;57(1):200–210. doi: 10.1016/0003-2697(74)90065-7. [DOI] [PubMed] [Google Scholar]
  32. Moazed D., Noller H. F. Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell. 1986 Dec 26;47(6):985–994. doi: 10.1016/0092-8674(86)90813-5. [DOI] [PubMed] [Google Scholar]
  33. Moazed D., Stern S., Noller H. F. Rapid chemical probing of conformation in 16 S ribosomal RNA and 30 S ribosomal subunits using primer extension. J Mol Biol. 1986 Feb 5;187(3):399–416. doi: 10.1016/0022-2836(86)90441-9. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Puglisi J. D., Blanchard S. C., Green R. Approaching translation at atomic resolution. Nat Struct Biol. 2000 Oct;7(10):855–861. doi: 10.1038/79603. [DOI] [PubMed] [Google Scholar]
  37. Reynolds J. E., Kaminski A., Kettinen H. J., Grace K., Clarke B. E., Carroll A. R., Rowlands D. J., Jackson R. J. Unique features of internal initiation of hepatitis C virus RNA translation. EMBO J. 1995 Dec 1;14(23):6010–6020. doi: 10.1002/j.1460-2075.1995.tb00289.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Schiffmann D., Horak I. Ribosomal proteins of HeLa cells. Eur J Biochem. 1978 Jan 2;82(1):91–95. doi: 10.1111/j.1432-1033.1978.tb11999.x. [DOI] [PubMed] [Google Scholar]
  40. Schluenzen F., Tocilj A., Zarivach R., Harms J., Gluehmann M., Janell D., Bashan A., Bartels H., Agmon I., Franceschi F. Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution. Cell. 2000 Sep 1;102(5):615–623. doi: 10.1016/s0092-8674(00)00084-2. [DOI] [PubMed] [Google Scholar]
  41. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Spahn C. M., Kieft J. S., Grassucci R. A., Penczek P. A., Zhou K., Doudna J. A., Frank J. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit. Science. 2001 Mar 9;291(5510):1959–1962. doi: 10.1126/science.1058409. [DOI] [PubMed] [Google Scholar]
  44. Tsukiyama-Kohara K., Iizuka N., Kohara M., Nomoto A. Internal ribosome entry site within hepatitis C virus RNA. J Virol. 1992 Mar;66(3):1476–1483. doi: 10.1128/jvi.66.3.1476-1483.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang C., Sarnow P., Siddiqui A. Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. J Virol. 1993 Jun;67(6):3338–3344. doi: 10.1128/jvi.67.6.3338-3344.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wimberly B. T., Brodersen D. E., Clemons W. M., Jr, Morgan-Warren R. J., Carter A. P., Vonrhein C., Hartsch T., Ramakrishnan V. Structure of the 30S ribosomal subunit. Nature. 2000 Sep 21;407(6802):327–339. doi: 10.1038/35030006. [DOI] [PubMed] [Google Scholar]
  47. Wollenzien P., Expert-Bezançon A., Favre A. Sites of contact of mRNA with 16S rRNA and 23S rRNA in the Escherichia coli ribosome. Biochemistry. 1991 Feb 19;30(7):1788–1795. doi: 10.1021/bi00221a009. [DOI] [PubMed] [Google Scholar]
  48. Yusupov M. M., Yusupova G. Z., Baucom A., Lieberman K., Earnest T. N., Cate J. H., Noller H. F. Crystal structure of the ribosome at 5.5 A resolution. Science. 2001 Mar 29;292(5518):883–896. doi: 10.1126/science.1060089. [DOI] [PubMed] [Google Scholar]
  49. Yusupova G. Z., Yusupov M. M., Cate J. H., Noller H. F. The path of messenger RNA through the ribosome. Cell. 2001 Jul 27;106(2):233–241. doi: 10.1016/s0092-8674(01)00435-4. [DOI] [PubMed] [Google Scholar]
  50. Zhao W. D., Wimmer E. Genetic analysis of a poliovirus/hepatitis C virus chimera: new structure for domain II of the internal ribosomal entry site of hepatitis C virus. J Virol. 2001 Apr;75(8):3719–3730. doi: 10.1128/JVI.75.8.3719-3730.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES