Cellular and Molecular Life Sciences

, Volume 69, Issue 1, pp 103–113 | Cite as

The functional RNA domain 5BSL3.2 within the NS5B coding sequence influences hepatitis C virus IRES-mediated translation

  • Cristina Romero-López
  • Alfredo Berzal-HerranzEmail author
Research article


Hepatitis C virus (HCV) translation is mediated by an internal ribosome entry site (IRES) located at the 5′ end of the genomic RNA. The 3′ untranslatable region (3′UTR) stimulates translation by the recruitment of protein factors that simultaneously bind to the 5′ end of the viral genome. This leads to the formation of a macromolecular complex with a closed loop conformation, similar to that described for the cap-translated mRNAs. We previously demonstrated the existence of a long-range RNA–RNA interaction involving subdomain IIId of the IRES region and the stem–loop 5BSL3.2 of the CRE element at the 3′ end of the viral genome. The present study provides evidence that the enhancement of HCV IRES-dependent translation mediated by the 3′UTR is negatively controlled by the CRE region in the human hepatoma cell lines Huh-7 and Hep-G2 in a time-dependent manner. Domain 5BSL3.2 is the major partner in this process. Mutations in this motif lead to an increase in IRES activity by up to eightfold. These data support the existence of a functional high order structure in the HCV genome that involves two evolutionarily conserved RNA elements, domain IIId in the IRES and stem–loop 5BSL3.2 in the CRE region. This interaction could have a role in the circularisation of the viral genome.


RNA–RNA interaction Hepatitis C virus (HCV) Internal ribosome entry site (IRES) cis-acting replicating element (CRE) Domain 5BSL3.2 Domain IIId IRES-dependent translation 



Hepatitis C virus


Internal ribosome entry site


Untranslatable region


cis-acting replicating element



The Huh-7 cell line was a kind gift of Dr. R. Aldabe. We thank Dr. Alicia Barroso-delJesus for helpful discussions. We also thank Vicente Augustin for excellent technical assistance. This work was supported by Grants BFU2009-08137 from the Spanish Ministerio de Innovación y Ciencia, CTS-5077 from the Junta de Andalucía and FEDER funds from the EU to A.B–H. C.R-L was funded by Grants 2004-20E632 from the Spanish National Research Council (CSIC) and BFU2009-08137.


  1. 1.
    Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359–362PubMedCrossRefGoogle Scholar
  2. 2.
    Kuo G, Choo QL, Alter HJ et al (1989) An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 244:362–364PubMedCrossRefGoogle Scholar
  3. 3.
    Hoofnagle JH (1997) Hepatitis C: the clinical spectrum of disease. Hepatology 26:15S–20SPubMedCrossRefGoogle Scholar
  4. 4.
    Kato N, Hijikata M, Ootsuyama Y, Nakagawa M, Ohkoshi S, Sugimura T, Shimotohno K (1990) Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci USA 87:9524–9528PubMedCrossRefGoogle Scholar
  5. 5.
    Takamizawa A, Mori C, Fuke I, Manabe S, Murakami S, Fujita J, Onishi E, Andoh T, Yoshida I, Okayama H (1991) Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol 65:1105–1113PubMedGoogle Scholar
  6. 6.
    Tsukiyama-Kohara K, Iizuka N, Kohara M, Nomoto A (1992) Internal ribosome entry site within hepatitis C virus RNA. J Virol 66:1476–1483PubMedGoogle Scholar
  7. 7.
    Wang C, Sarnow P, Siddiqui A (1993) Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. J Virol 67:3338–3344PubMedGoogle Scholar
  8. 8.
    Kolykhalov AA, Mihalik K, Feinstone SM, Rice CM (2000) Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3’ nontranslated region are essential for virus replication in vivo. J Virol 74:2046–2051PubMedCrossRefGoogle Scholar
  9. 9.
    Friebe P, Bartenschlager R (2002) Genetic analysis of sequences in the 3’ nontranslated region of hepatitis C virus that are important for RNA replication. J Virol 76:5326–5338PubMedCrossRefGoogle Scholar
  10. 10.
    Yi M, Lemon SM (2003) 3’ nontranslated RNA signals required for replication of hepatitis C virus RNA. J Virol 77:3557–3568PubMedCrossRefGoogle Scholar
  11. 11.
    Yi M, Lemon SM (2003) Structure-function analysis of the 3’ stem–loop of hepatitis C virus genomic RNA and its role in viral RNA replication. RNA 9:331–345PubMedCrossRefGoogle Scholar
  12. 12.
    Song Y, Friebe P, Tzima E, Junemann C, Bartenschlager R, Niepmann M (2006) The hepatitis C virus RNA 3’-untranslated region strongly enhances translation directed by the internal ribosome entry site. J Virol 80:11579–11588PubMedCrossRefGoogle Scholar
  13. 13.
    Reynolds JE, Kaminski A, Kettinen HJ, Grace K, Clarke BE, Carroll AR, Rowlands DJ, Jackson RJ (1995) Unique features of internal initiation of hepatitis C virus RNA translation. EMBO J 14:6010–6020PubMedGoogle Scholar
  14. 14.
    Wang TH, Rijnbrand RC, Lemon SM (2000) Core protein-coding sequence, but not core protein, modulates the efficiency of cap-independent translation directed by the internal ribosome entry site of hepatitis C virus. J Virol 74:11347–11358PubMedCrossRefGoogle Scholar
  15. 15.
    Lytle JR, Wu L, Robertson HD (2002) Domains on the hepatitis C virus internal ribosome entry site for 40S subunit binding. RNA 8:1045–1055PubMedCrossRefGoogle Scholar
  16. 16.
    Ji H, Fraser CS, Yu Y, Leary J, Doudna JA (2004) Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA. Proc Natl Acad Sci USA 101:16990–16995PubMedCrossRefGoogle Scholar
  17. 17.
    Otto GA, Puglisi JD (2004) The pathway of HCV IRES-mediated translation initiation. Cell 119:369–380PubMedCrossRefGoogle Scholar
  18. 18.
    Ito T, Tahara SM, Lai MM (1998) The 3’-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site. J Virol 72:8789–8796PubMedGoogle Scholar
  19. 19.
    Bradrick SS, Walters RW, Gromeier M (2006) The hepatitis C virus 3’-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase. Nucleic Acids Res 34:1293–1303PubMedCrossRefGoogle Scholar
  20. 20.
    Morikawa K, Ito T, Nozawa H, Inokuchi M, Uchikoshi M, Saito T, Mitamura K, Imawari M (2006) Translational enhancement of HCV RNA genotype 1b by 3’-untranslated and envelope 2 protein-coding sequences. Virology 345:404–415PubMedCrossRefGoogle Scholar
  21. 21.
    Lourenço S, Costa F, Debarges B, Andrieu T, Cahour A (2008) Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by cis-acting RNA elements and trans-acting viral factors. FEBS J 275:4179–4197PubMedCrossRefGoogle Scholar
  22. 22.
    Bung C, Bochkaeva Z, Terenin I, Zinovkin R, Shatsky IN, Niepmann M (2010) Influence of the hepatitis C virus 3’-untranslated region on IRES-dependent and cap-dependent translation initiation. FEBS Lett 584:837–842PubMedCrossRefGoogle Scholar
  23. 23.
    Petrik J, Parker H, Alexander GJ (1999) Human hepatic glyceraldehyde-3-phosphate dehydrogenase binds to the poly(U) tract of the 3’ non-coding region of hepatitis C virus genomic RNA. J Gen Virol 80:3109–3113PubMedGoogle Scholar
  24. 24.
    Spangberg K, Goobar-Larsson L, Wahren-Herlenius M, Schwartz S (1999) The La protein from human liver cells interacts specifically with the U-rich region in the hepatitis C virus 3’ untranslated region. J Hum Virol 2:296–307PubMedGoogle Scholar
  25. 25.
    Wood J, Frederickson RM, Fields S, Patel AH (2001) Hepatitis C virus 3’X region interacts with human ribosomal proteins. J Virol 75:1348–1358PubMedCrossRefGoogle Scholar
  26. 26.
    McCaffrey AP, Ohashi K, Meuse L, Shen S, Lancaster AM, Lukavsky PJ, Sarnow P, Kay MA (2002) Determinants of hepatitis C translational initiation in vitro, in cultured cells and mice. Mol Ther 5:676–684PubMedCrossRefGoogle Scholar
  27. 27.
    Huang L, Hwang J, Sharma SD, Hargittai MR, Chen Y, Arnold JJ, Raney KD, Cameron CE (2005) Hepatitis C virus non-structural protein 5A (NS5A) is a RNA-binding protein. J Biol Chem 280:36417–36428PubMedCrossRefGoogle Scholar
  28. 28.
    Wang H, Shen XT, Ye R, Lan SY, Xiang L, Yuan ZH (2005) Roles of the polypyrimidine tract and 3’ noncoding region of hepatitis C virus RNA in the internal ribosome entry site-mediated translation. Arch Virol 150:1085–1099PubMedCrossRefGoogle Scholar
  29. 29.
    Scheller N, Mina LB, Galao RP, Chari A, Gimenez-Barcons M, Noueiry A, Fischer U, Meyerhans A, Díez J (2009) Translation and replication of hepatitis C virus genomic RNA depends on ancient cellular proteins that control mRNA fates. Proc Natl Acad Sci USA 106:13517–13522PubMedCrossRefGoogle Scholar
  30. 30.
    Weinlich S, Huttelmaier S, Schierhorn A, Behrens SE, Ostareck-Lederer A, Ostareck DH (2009) IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3’UTR. RNA 15:1528–1542PubMedCrossRefGoogle Scholar
  31. 31.
    Yu KL, Jang SI, You JC (2009) Identification of in vivo interaction between Hepatitis C Virus core protein and 5’ and 3’ UTR RNA. Virus Res 145:285–292PubMedCrossRefGoogle Scholar
  32. 32.
    Spangberg K, Wiklund L, Schwartz S (2001) Binding of the La autoantigen to the hepatitis C virus 3’ untranslated region protects the RNA from rapid degradation in vitro. J Gen Virol 82:113–120PubMedGoogle Scholar
  33. 33.
    Sachs AB, Sarnow P, Hentze MW (1997) Starting at the beginning, middle, and end: translation initiation in eukaryotes. Cell 89:831–838PubMedCrossRefGoogle Scholar
  34. 34.
    Edgil D, Harris E (2006) End-to-end communication in the modulation of translation by mammalian RNA viruses. Virus Res 119:43–51PubMedCrossRefGoogle Scholar
  35. 35.
    Harris E, Holden KL, Edgil D, Polacek C, Clyde K (2006) Molecular biology of flaviviruses. Novartis Found Symp 277:23–39 Discussion 40, 71–23, 251–253PubMedCrossRefGoogle Scholar
  36. 36.
    Serrano P, Pulido MR, Saiz M, Martínez-Salas E (2006) The 3’ end of the foot-and-mouth disease virus genome establishes two distinct long-range RNA–RNA interactions with the 5’ end region. J Gen Virol 87:3013–3022PubMedCrossRefGoogle Scholar
  37. 37.
    Romero-López C, Berzal-Herranz A (2009) A long-range RNA–RNA interaction between the 5’ and 3’ ends of the HCV genome. RNA 15:1740–1752PubMedCrossRefGoogle Scholar
  38. 38.
    Kolupaeva VG, Pestova TV, Hellen CU (2000) An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus. J Virol 74:6242–6250PubMedCrossRefGoogle Scholar
  39. 39.
    Lukavsky PJ, Otto GA, Lancaster AM, Sarnow P, Puglisi JD (2000) Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function. Nat Struct Biol 7:1105–1110PubMedCrossRefGoogle Scholar
  40. 40.
    Babaylova E, Graifer D, Malygin A, Stahl J, Shatsky I, Karpova G (2009) Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome. Nucleic Acids Res 37:1141–1151PubMedCrossRefGoogle Scholar
  41. 41.
    Barria MI, González A, Vera-Otarola J, Leon U, Vollrath V, Marsac D, Monasterio O, Pérez-Acle T, Soza A, López-Lastra M (2009) Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role on cap-independent translation. Nucleic Acids Res 37:957–971PubMedCrossRefGoogle Scholar
  42. 42.
    Jubin R, Vantuno NE, Kieft JS, Murray MG, Doudna JA, Lau JY, Baroudy BM (2000) 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 74:10430–10437PubMedCrossRefGoogle Scholar
  43. 43.
    Klinck R, Westhof E, Walker S, Afshar M, Collier A, Aboul-Ela F (2000) A potential RNA drug target in the hepatitis C virus internal ribosomal entry site. RNA 6:1423–1431PubMedCrossRefGoogle Scholar
  44. 44.
    Lee H, Shin H, Wimmer E, Paul AV (2004) cis-acting RNA signals in the NS5B C-terminal coding sequence of the hepatitis C virus genome. J Virol 78:10865–10877PubMedCrossRefGoogle Scholar
  45. 45.
    You S, Stump DD, Branch AD, Rice CM (2004) A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication. J Virol 78:1352–1366PubMedCrossRefGoogle Scholar
  46. 46.
    Friebe P, Boudet J, Simorre JP, Bartenschlager R (2005) Kissing-loop interaction in the 3’ end of the hepatitis C virus genome essential for RNA replication. J Virol 79:380–392PubMedCrossRefGoogle Scholar
  47. 47.
    Romero-López C, Díaz-González R, Berzal-Herranz A (2007) Inhibition of hepatitis C virus internal ribosome entry site-mediated translation by an RNA targeting the conserved IIIf domain. Cell Mol Life Sci 64:2994–3006PubMedCrossRefGoogle Scholar
  48. 48.
    Romero-López C, Barroso-delJesus A, Puerta-Fernández E, Berzal-Herranz A (2005) Interfering with hepatitis C virus IRES activity using RNA molecules identified by a novel in vitro selection method. Biol Chem 386:183–190PubMedCrossRefGoogle Scholar
  49. 49.
    Romero-López C, Díaz-González R, Barroso-delJesus A, Berzal-Herranz A (2009) Inhibition of HCV replication and IRES-dependent translation by an RNA molecule. J Gen Virol 90:1659–1669PubMedCrossRefGoogle Scholar
  50. 50.
    Diviney S, Tuplin A, Struthers M, Armstrong V, Elliott RM, Simmonds P, Evans DJ (2008) A hepatitis C virus cis-acting replication element forms a long-range RNA–RNA interaction with upstream RNA sequences in NS5B. J Virol 82:9008–9022PubMedCrossRefGoogle Scholar
  51. 51.
    Komarova AV, Brocard M, Kean KM (2006) The case for mRNA 5’ and 3’ end cross talk during translation in a eukaryotic cell. Prog Nucleic Acid Res Mol Biol 81:331–367PubMedCrossRefGoogle Scholar
  52. 52.
    Corver J, Lenches E, Smith K, Robison RA, Sando T, Strauss EG, Strauss JH (2003) Fine mapping of a cis-acting sequence element in yellow fever virus RNA that is required for RNA replication and cyclization. J Virol 77:2265–2270PubMedCrossRefGoogle Scholar
  53. 53.
    Markoff L (2003) 5’- and 3’-noncoding regions in flavivirus RNA. Adv Virus Res 59:177–228PubMedCrossRefGoogle Scholar
  54. 54.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415PubMedCrossRefGoogle Scholar
  55. 55.
    Junemann C, Song Y, Bassili G, Goergen D, Henke J, Niepmann M (2007) Picornavirus internal ribosome entry site elements can stimulate translation of upstream genes. J Biol Chem 282:132–141PubMedCrossRefGoogle Scholar
  56. 56.
    Henke JI, Goergen D, Zheng J, Song Y, Schuttler CG, Fehr C, Junemann C, Niepmann M (2008) microRNA-122 stimulates translation of hepatitis C virus RNA. EMBO J 27:3300–3310PubMedCrossRefGoogle Scholar
  57. 57.
    Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P (2005) Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA. Science 309:1577–1581PubMedCrossRefGoogle Scholar
  58. 58.
    Cai Z, Zhang C, Chang KS, Jiang J, Ahn BC, Wakita T, Liang TJ, Luo G (2005) Robust production of infectious hepatitis C virus (HCV) from stably HCV cDNA-transfected human hepatoma cells. J Virol 79:13963–13973PubMedCrossRefGoogle Scholar
  59. 59.
    Wakita T, Pietschmann T, Kato T et al (2005) Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796PubMedCrossRefGoogle Scholar
  60. 60.
    Boonstra A, van der Laan LJ, Vanwolleghem T, Janssen HL (2009) Experimental models for hepatitis C viral infection. Hepatology 50:1646–1655PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel AG 2011

Authors and Affiliations

  • Cristina Romero-López
    • 1
  • Alfredo Berzal-Herranz
    • 1
    Email author
  1. 1.Instituto de Parasitología y Biomedicina “López-Neyra” IPBLN-CSICCSICArmillaSpain

Personalised recommendations