Skip to main content
PMC home page
RNA logoLink to RNA
. 2000 Oct;6(10):1380–1392. doi: 10.1017/s1355838200000753

Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo.

S López de Quinto 1, E Martínez-Salas 1
PMCID: PMC1370009  PMID: 11073214

Abstract

The strategies developed by internal ribosome entry site (IRES) elements to recruit the translational machinery are poorly understood. In this study we show that protein-RNA interaction of the eIF4G translation initiation factor with sequences of the foot-and-mouth disease virus (FMDV) IRES is a key determinant of internal translation initiation in living cells. Moreover, we have identified the nucleotides required for eIF4G-RNA functional interaction, using native proteins from FMDV-susceptible cell extracts. Substitutions in the conserved internal AA loop of the base of domain 4 led to strong impairment of both eIF4G-RNA interaction in vitro and IRES-dependent translation initiation in vivo. Conversely, substitutions in the vicinity of the internal AA loop that did not impair IRES activity retained their ability to interact with eIF4G. Direct UV-crosslinking as well as competition assays indicated that domains 1-2, 3, and 5 of the IRES did not contribute to this interaction. In agreement with this, binding to domain 4 alone was as efficient as to the full-length IRES. The C-terminal fragment of eIF4G, proteolytically processed by the FMDV Lb protease, was sufficient to interact with the IRES or to its domain 4 alone. Additionally, we show here that binding of the eIF4B initiation factor to the IRES required domain 5 sequences. Moreover, eIF4G-IRES interaction was detected in the absence of eIF4B-IRES binding, suggesting that both initiation factors interact with the 3' region of the IRES but use different residues. The strong correlation found between eIF4G-RNA interaction and IRES activity in transfected cells suggests that eIF4G acts as a linker to recruit the translational machinery in IRES-dependent initiation.

Full Text

The Full Text of this article is available as a PDF (728.4 KB).

Selected References

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

  1. Andino R., Böddeker N., Silvera D., Gamarnik A. V. Intracellular determinants of picornavirus replication. Trends Microbiol. 1999 Feb;7(2):76–82. doi: 10.1016/s0966-842x(98)01446-2. [DOI] [PubMed] [Google Scholar]
  2. Aragón T., de la Luna S., Novoa I., Carrasco L., Ortín J., Nieto A. Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus. Mol Cell Biol. 2000 Sep;20(17):6259–6268. doi: 10.1128/mcb.20.17.6259-6268.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Belsham G. J., Brangwyn J. K. A region of the 5' noncoding region of foot-and-mouth disease virus RNA directs efficient internal initiation of protein synthesis within cells: involvement with the role of L protease in translational control. J Virol. 1990 Nov;64(11):5389–5395. doi: 10.1128/jvi.64.11.5389-5395.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Block K. L., Vornlocher H. P., Hershey J. W. Characterization of cDNAs encoding the p44 and p35 subunits of human translation initiation factor eIF3. J Biol Chem. 1998 Nov 27;273(48):31901–31908. doi: 10.1074/jbc.273.48.31901. [DOI] [PubMed] [Google Scholar]
  5. Borman A. M., Kirchweger R., Ziegler E., Rhoads R. E., Skern T., Kean K. M. elF4G and its proteolytic cleavage products: effect on initiation of protein synthesis from capped, uncapped, and IRES-containing mRNAs. RNA. 1997 Feb;3(2):186–196. [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. Dever T. E. Translation initiation: adept at adapting. Trends Biochem Sci. 1999 Oct;24(10):398–403. doi: 10.1016/s0968-0004(99)01457-7. [DOI] [PubMed] [Google Scholar]
  8. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gamarnik A. V., Andino R. Two functional complexes formed by KH domain containing proteins with the 5' noncoding region of poliovirus RNA. RNA. 1997 Aug;3(8):882–892. [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. Goyer C., Altmann M., Lee H. S., Blanc A., Deshmukh M., Woolford J. L., Jr, Trachsel H., Sonenberg N. TIF4631 and TIF4632: two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function. Mol Cell Biol. 1993 Aug;13(8):4860–4874. doi: 10.1128/mcb.13.8.4860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gradi A., Imataka H., Svitkin Y. V., Rom E., Raught B., Morino S., Sonenberg N. A novel functional human eukaryotic translation initiation factor 4G. Mol Cell Biol. 1998 Jan;18(1):334–342. doi: 10.1128/mcb.18.1.334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gradi A., Svitkin Y. V., Imataka H., Sonenberg N. Proteolysis of human eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein synthesis after poliovirus infection. Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11089–11094. doi: 10.1073/pnas.95.19.11089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hentze M. W. eIF4G: a multipurpose ribosome adapter? Science. 1997 Jan 24;275(5299):500–501. doi: 10.1126/science.275.5299.500. [DOI] [PubMed] [Google Scholar]
  15. Imataka H., Gradi A., Sonenberg N. A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation. EMBO J. 1998 Dec 15;17(24):7480–7489. doi: 10.1093/emboj/17.24.7480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Imataka H., Sonenberg N. Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A. Mol Cell Biol. 1997 Dec;17(12):6940–6947. doi: 10.1128/mcb.17.12.6940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jackson R. J., Kaminski A. Internal initiation of translation in eukaryotes: the picornavirus paradigm and beyond. RNA. 1995 Dec;1(10):985–1000. [PMC free article] [PubMed] [Google Scholar]
  18. Johannes G., Carter M. S., Eisen M. B., Brown P. O., Sarnow P. Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray. Proc Natl Acad Sci U S A. 1999 Nov 9;96(23):13118–13123. doi: 10.1073/pnas.96.23.13118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Johannes G., Sarnow P. Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites. RNA. 1998 Dec;4(12):1500–1513. doi: 10.1017/s1355838298981080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kim C. Y., Takahashi K., Nguyen T. B., Roberts J. K., Webster C. Identification of a nucleic acid binding domain in eukaryotic initiation factor eIFiso4G from wheat. J Biol Chem. 1999 Apr 9;274(15):10603–10608. doi: 10.1074/jbc.274.15.10603. [DOI] [PubMed] [Google Scholar]
  21. Kirchweger R., Ziegler E., Lamphear B. J., Waters D., Liebig H. D., Sommergruber W., Sobrino F., Hohenadl C., Blaas D., Rhoads R. E. Foot-and-mouth disease virus leader proteinase: purification of the Lb form and determination of its cleavage site on eIF-4 gamma. J Virol. 1994 Sep;68(9):5677–5684. doi: 10.1128/jvi.68.9.5677-5684.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kolupaeva V. G., Hellen C. U., Shatsky I. N. Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs. RNA. 1996 Dec;2(12):1199–1212. [PMC free article] [PubMed] [Google Scholar]
  23. Kolupaeva V. G., Pestova T. V., Hellen C. U., Shatsky I. N. Translation eukaryotic initiation factor 4G recognizes a specific structural element within the internal ribosome entry site of encephalomyocarditis virus RNA. J Biol Chem. 1998 Jul 17;273(29):18599–18604. doi: 10.1074/jbc.273.29.18599. [DOI] [PubMed] [Google Scholar]
  24. Kühn R., Luz N., Beck E. Functional analysis of the internal translation initiation site of foot-and-mouth disease virus. J Virol. 1990 Oct;64(10):4625–4631. doi: 10.1128/jvi.64.10.4625-4631.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lamphear B. J., Kirchweger R., Skern T., Rhoads R. E. Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation. J Biol Chem. 1995 Sep 15;270(37):21975–21983. doi: 10.1074/jbc.270.37.21975. [DOI] [PubMed] [Google Scholar]
  26. Lamphear B. J., Yan R., Yang F., Waters D., Liebig H. D., Klump H., Kuechler E., Skern T., Rhoads R. E. Mapping the cleavage site in protein synthesis initiation factor eIF-4 gamma of the 2A proteases from human Coxsackievirus and rhinovirus. J Biol Chem. 1993 Sep 15;268(26):19200–19203. [PubMed] [Google Scholar]
  27. Le S. Y., Maizel J. V., Jr A common RNA structural motif involved in the internal initiation of translation of cellular mRNAs. Nucleic Acids Res. 1997 Jan 15;25(2):362–369. doi: 10.1093/nar/25.2.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Luz N., Beck E. Interaction of a cellular 57-kilodalton protein with the internal translation initiation site of foot-and-mouth disease virus. J Virol. 1991 Dec;65(12):6486–6494. doi: 10.1128/jvi.65.12.6486-6494.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. López de Quinto S., Martínez-Salas E. Conserved structural motifs located in distal loops of aphthovirus internal ribosome entry site domain 3 are required for internal initiation of translation. J Virol. 1997 May;71(5):4171–4175. doi: 10.1128/jvi.71.5.4171-4175.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. López de Quinto S., Martínez-Salas E. Involvement of the aphthovirus RNA region located between the two functional AUGs in start codon selection. Virology. 1999 Mar 15;255(2):324–336. doi: 10.1006/viro.1999.9598. [DOI] [PubMed] [Google Scholar]
  31. López de Quinto S., Martínez-Salas E. Parameters influencing translational efficiency in aphthovirus IRES-based bicistronic expression vectors. Gene. 1998 Sep 14;217(1-2):51–56. doi: 10.1016/s0378-1119(98)00379-5. [DOI] [PubMed] [Google Scholar]
  32. Martínez-Salas E. Internal ribosome entry site biology and its use in expression vectors. Curr Opin Biotechnol. 1999 Oct;10(5):458–464. doi: 10.1016/s0958-1669(99)00010-5. [DOI] [PubMed] [Google Scholar]
  33. Martínez-Salas E., Regalado M. P., Domingo E. Identification of an essential region for internal initiation of translation in the aphthovirus internal ribosome entry site and implications for viral evolution. J Virol. 1996 Feb;70(2):992–998. doi: 10.1128/jvi.70.2.992-998.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Martínez-Salas E., Sáiz J. C., Dávila M., Belsham G. J., Domingo E. A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo. J Virol. 1993 Jul;67(7):3748–3755. doi: 10.1128/jvi.67.7.3748-3755.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Medina M., Domingo E., Brangwyn J. K., Belsham G. J. The two species of the foot-and-mouth disease virus leader protein, expressed individually, exhibit the same activities. Virology. 1993 May;194(1):355–359. doi: 10.1006/viro.1993.1267. [DOI] [PubMed] [Google Scholar]
  36. Methot N., Pickett G., Keene J. D., Sonenberg N. In vitro RNA selection identifies RNA ligands that specifically bind to eukaryotic translation initiation factor 4B: the role of the RNA remotif. RNA. 1996 Jan;2(1):38–50. [PMC free article] [PubMed] [Google Scholar]
  37. Meyer K., Petersen A., Niepmann M., Beck E. Interaction of eukaryotic initiation factor eIF-4B with a picornavirus internal translation initiation site. J Virol. 1995 May;69(5):2819–2824. doi: 10.1128/jvi.69.5.2819-2824.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Morley S. J., Curtis P. S., Pain V. M. eIF4G: translation's mystery factor begins to yield its secrets. RNA. 1997 Oct;3(10):1085–1104. [PMC free article] [PubMed] [Google Scholar]
  39. Méthot N., Pause A., Hershey J. W., Sonenberg N. The translation initiation factor eIF-4B contains an RNA-binding region that is distinct and independent from its ribonucleoprotein consensus sequence. Mol Cell Biol. 1994 Apr;14(4):2307–2316. doi: 10.1128/mcb.14.4.2307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Méthot N., Song M. S., Sonenberg N. A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3. Mol Cell Biol. 1996 Oct;16(10):5328–5334. doi: 10.1128/mcb.16.10.5328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Naranda T., Strong W. B., Menaya J., Fabbri B. J., Hershey J. W. Two structural domains of initiation factor eIF-4B are involved in binding to RNA. J Biol Chem. 1994 May 20;269(20):14465–14472. [PubMed] [Google Scholar]
  42. Ohlmann T., Rau M., Pain V. M., Morley S. J. The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E. EMBO J. 1996 Mar 15;15(6):1371–1382. [PMC free article] [PubMed] [Google Scholar]
  43. Pestova T. V., Hellen C. U. Ribosome recruitment and scanning: what's new? Trends Biochem Sci. 1999 Mar;24(3):85–87. doi: 10.1016/s0968-0004(99)01356-0. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Pestova T. V., Shatsky I. N., Hellen C. U. Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes. Mol Cell Biol. 1996 Dec;16(12):6870–6878. doi: 10.1128/mcb.16.12.6870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Piron M., Vende P., Cohen J., Poncet D. Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F. EMBO J. 1998 Oct 1;17(19):5811–5821. doi: 10.1093/emboj/17.19.5811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Preiss T., Hentze M. W. From factors to mechanisms: translation and translational control in eukaryotes. Curr Opin Genet Dev. 1999 Oct;9(5):515–521. doi: 10.1016/s0959-437x(99)00005-2. [DOI] [PubMed] [Google Scholar]
  49. Ramos R., Martínez-Salas E. Long-range RNA interactions between structural domains of the aphthovirus internal ribosome entry site (IRES). RNA. 1999 Oct;5(10):1374–1383. doi: 10.1017/s1355838299991240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rose J. K., Buonocore L., Whitt M. A. A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques. 1991 Apr;10(4):520–525. [PubMed] [Google Scholar]
  51. 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]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES