Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Oct;69(10):6400–6407. doi: 10.1128/jvi.69.10.6400-6407.1995

An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors.

C Berlioz 1, C Torrent 1, J L Darlix 1
PMCID: PMC189539  PMID: 7666541

Abstract

The genetic organization of the 5' genomic RNA domain of the highly oncogenic Harvey murine sarcoma virus appears to be unusual in that a multifunctional untranslated leader precedes the v-ras oncogene. This 5' leader is 1,076 nucleotides in length and is formed of independent regions involved in key steps of the viral life cycle: (i) the Moloney murine leukemia virus 5' repeat, untranslated 5' region, and primer binding site sequences necessary for the first steps of proviral DNA synthesis, (ii) the virus-like 30S (VL30)-derived sequence containing a functional dimerization-packaging signal (E/DLS) directing viral RNA dimerization and packaging into MLV virions, and (iii) an Alu-like sequence preceding the 5' untranslated sequence of v-rasH which contains the initiation codon of the p21ras oncoprotein. These functional features, the unusual length of this leader (1,076 nucleotides), and the presence of stable secondary structures between the cap and the v-ras initiation codon might well cause a premature stop of the scanning ribosomes and thus inhibit v-ras translation. In order to understand how Harvey murine sarcoma virus achieves a high level of expression of the ras oncogene, we asked whether the rat VL30 sequence, 5' to v-ras, could contribute to an efficient synthesis of the ras oncoprotein. The implications of the VL30 sequence in the translation initiation of Ha-ras were investigated in the rabbit reticulocyte lysate system and in murine cells. Results show that the rat VL30 sequence allows a cap-independent translation of a downstream reporter gene both in vitro and in murine cells. Additional experiments performed with dicistronic neo.VL30.lacZ mRNAs indicate that the 5' VL30 sequence (positions 380 to 794) contains an internal ribosomal entry signal. This finding led us to construct a new dicistronic retroviral vector with which the rat VL30 sequence was able to direct the efficient expression of a 3' cistron and packaging of recombinant dicistronic RNA into murine leukemia virus virions.

Full Text

The Full Text of this article is available as a PDF (333.9 KB).

Selected References

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

  1. Adam M. A., Miller A. D. Identification of a signal in a murine retrovirus that is sufficient for packaging of nonretroviral RNA into virions. J Virol. 1988 Oct;62(10):3802–3806. doi: 10.1128/jvi.62.10.3802-3806.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adam M. A., Ramesh N., Miller A. D., Osborne W. R. Internal initiation of translation in retroviral vectors carrying picornavirus 5' nontranslated regions. J Virol. 1991 Sep;65(9):4985–4990. doi: 10.1128/jvi.65.9.4985-4990.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson G. R., Robbins K. C. Rat sequences of the Kirsten and Harvey murine sarcoma virus genomes: nature, origin, and expression in rat tumor RNA. J Virol. 1976 Feb;17(2):335–351. doi: 10.1128/jvi.17.2.335-351.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Basso J., Dallaire P., Charest P. J., Devantier Y., Laliberté J. F. Evidence for an internal ribosome entry site within the 5' non-translated region of turnip mosaic potyvirus RNA. J Gen Virol. 1994 Nov;75(Pt 11):3157–3165. doi: 10.1099/0022-1317-75-11-3157. [DOI] [PubMed] [Google Scholar]
  5. Baudin F., Marquet R., Isel C., Darlix J. L., Ehresmann B., Ehresmann C. Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains. J Mol Biol. 1993 Jan 20;229(2):382–397. doi: 10.1006/jmbi.1993.1041. [DOI] [PubMed] [Google Scholar]
  6. Bender M. A., Palmer T. D., Gelinas R. E., Miller A. D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol. 1987 May;61(5):1639–1646. doi: 10.1128/jvi.61.5.1639-1646.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Berlioz C., Darlix J. L. An internal ribosomal entry mechanism promotes translation of murine leukemia virus gag polyprotein precursors. J Virol. 1995 Apr;69(4):2214–2222. doi: 10.1128/jvi.69.4.2214-2222.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bieth E., Gabus C., Darlix J. L. A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro. Nucleic Acids Res. 1990 Jan 11;18(1):119–127. doi: 10.1093/nar/18.1.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Borman A., Jackson R. J. Initiation of translation of human rhinovirus RNA: mapping the internal ribosome entry site. Virology. 1992 Jun;188(2):685–696. doi: 10.1016/0042-6822(92)90523-r. [DOI] [PubMed] [Google Scholar]
  10. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Darlix J. L., Zuker M., Spahr P. F. Structure-function relationship of Rous sarcoma virus leader RNA. Nucleic Acids Res. 1982 Sep 11;10(17):5183–5196. doi: 10.1093/nar/10.17.5183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dolph P. J., Huang J. T., Schneider R. J. Translation by the adenovirus tripartite leader: elements which determine independence from cap-binding protein complex. J Virol. 1990 Jun;64(6):2669–2677. doi: 10.1128/jvi.64.6.2669-2677.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Duke G. M., Hoffman M. A., Palmenberg A. C. Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation. J Virol. 1992 Mar;66(3):1602–1609. doi: 10.1128/jvi.66.3.1602-1609.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Firulli B. A., Anderson G. R., Stoler D. L., Estes S. D. Anoxia-inducible rat VL30 elements and their relationship to ras-containing sarcoma viruses. J Virol. 1993 Nov;67(11):6857–6862. doi: 10.1128/jvi.67.11.6857-6862.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fouillot N., Tlouzeau S., Rossignol J. M., Jean-Jean O. Translation of the hepatitis B virus P gene by ribosomal scanning as an alternative to internal initiation. J Virol. 1993 Aug;67(8):4886–4895. doi: 10.1128/jvi.67.8.4886-4895.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ghattas I. R., Sanes J. R., Majors J. E. The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Mol Cell Biol. 1991 Dec;11(12):5848–5859. doi: 10.1128/mcb.11.12.5848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glass M. J., Jia X. Y., Summers D. F. Identification of the hepatitis A virus internal ribosome entry site: in vivo and in vitro analysis of bicistronic RNAs containing the HAV 5' noncoding region. Virology. 1993 Apr;193(2):842–852. doi: 10.1006/viro.1993.1193. [DOI] [PubMed] [Google Scholar]
  18. HARVEY J. J. AN UNIDENTIFIED VIRUS WHICH CAUSES THE RAPID PRODUCTION OF TUMOURS IN MICE. Nature. 1964 Dec 12;204:1104–1105. doi: 10.1038/2041104b0. [DOI] [PubMed] [Google Scholar]
  19. Hellen C. U., Fäcke M., Kräusslich H. G., Lee C. K., Wimmer E. Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220. J Virol. 1991 Aug;65(8):4226–4231. doi: 10.1128/jvi.65.8.4226-4231.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ilves H., Kahre O., Speek M. Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting. Mol Cell Biol. 1992 Sep;12(9):4242–4248. doi: 10.1128/mcb.12.9.4242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jackson R. J., Howell M. T., Kaminski A. The novel mechanism of initiation of picornavirus RNA translation. Trends Biochem Sci. 1990 Dec;15(12):477–483. doi: 10.1016/0968-0004(90)90302-r. [DOI] [PubMed] [Google Scholar]
  22. Jackson R. J. Potassium salts influence the fidelity of mRNA translation initiation in rabbit reticulocyte lysates: unique features of encephalomyocarditis virus RNA translation. Biochim Biophys Acta. 1991 Mar 26;1088(3):345–358. doi: 10.1016/0167-4781(91)90124-5. [DOI] [PubMed] [Google Scholar]
  23. Jang S. K., Kräusslich H. G., Nicklin M. J., Duke G. M., Palmenberg A. C., Wimmer E. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol. 1988 Aug;62(8):2636–2643. doi: 10.1128/jvi.62.8.2636-2643.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kalnins A., Otto K., Rüther U., Müller-Hill B. Sequence of the lacZ gene of Escherichia coli. EMBO J. 1983;2(4):593–597. doi: 10.1002/j.1460-2075.1983.tb01468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kozak M. Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol Cell Biol. 1989 Nov;9(11):5134–5142. doi: 10.1128/mcb.9.11.5134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol. 1987 Oct;7(10):3438–3445. doi: 10.1128/mcb.7.10.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kozak M. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci U S A. 1986 May;83(9):2850–2854. doi: 10.1073/pnas.83.9.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lang M., Treinies I., Duesberg P. H., Kurth R., Cichutek K. Development of transforming function during transduction of proto-ras into Harvey sarcoma virus. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):654–658. doi: 10.1073/pnas.91.2.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ledbetter J., Nowinski R. C. Identification of the Gross cell surface antigen associated with murine leukemia virus-infected cells. J Virol. 1977 Aug;23(2):315–322. doi: 10.1128/jvi.23.2.315-322.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Macejak D. G., Sarnow P. Internal initiation of translation mediated by the 5' leader of a cellular mRNA. Nature. 1991 Sep 5;353(6339):90–94. doi: 10.1038/353090a0. [DOI] [PubMed] [Google Scholar]
  33. Makris A., Patriotis C., Bear S. E., Tsichlis P. N. Structure of a Moloney murine leukemia virus-virus-like 30 recombinant: implications for transduction of the c-Ha-ras proto-oncogene. J Virol. 1993 Mar;67(3):1286–1291. doi: 10.1128/jvi.67.3.1286-1291.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Manly K. F., Anderson G. R., Stoler D. L. Harvey sarcoma virus genome contains no extensive sequences unrelated to those of other retroviruses except ras. J Virol. 1988 Sep;62(9):3540–3543. doi: 10.1128/jvi.62.9.3540-3543.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Markowitz D., Goff S., Bank A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol. 1988 Apr;62(4):1120–1124. doi: 10.1128/jvi.62.4.1120-1124.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McMillan J. P., Singer M. F. Translation of the human LINE-1 element, L1Hs. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11533–11537. doi: 10.1073/pnas.90.24.11533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Merrick W. C. Mechanism and regulation of eukaryotic protein synthesis. Microbiol Rev. 1992 Jun;56(2):291–315. doi: 10.1128/mr.56.2.291-315.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Morgan R. A., Couture L., Elroy-Stein O., Ragheb J., Moss B., Anderson W. F. Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy. Nucleic Acids Res. 1992 Mar 25;20(6):1293–1299. doi: 10.1093/nar/20.6.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mougel M., Tounekti N., Darlix J. L., Paoletti J., Ehresmann B., Ehresmann C. Conformational analysis of the 5' leader and the gag initiation site of Mo-MuLV RNA and allosteric transitions induced by dimerization. Nucleic Acids Res. 1993 Oct 11;21(20):4677–4684. doi: 10.1093/nar/21.20.4677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Murphy J. E., Goff S. P. Construction and analysis of deletion mutations in the U5 region of Moloney murine leukemia virus: effects on RNA packaging and reverse transcription. J Virol. 1989 Jan;63(1):319–327. doi: 10.1128/jvi.63.1.319-327.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nassal M., Junker-Niepmann M., Schaller H. Translational inactivation of RNA function: discrimination against a subset of genomic transcripts during HBV nucleocapsid assembly. Cell. 1990 Dec 21;63(6):1357–1363. doi: 10.1016/0092-8674(90)90431-d. [DOI] [PubMed] [Google Scholar]
  42. Oh S. K., Scott M. P., Sarnow P. Homeotic gene Antennapedia mRNA contains 5'-noncoding sequences that confer translational initiation by internal ribosome binding. Genes Dev. 1992 Sep;6(9):1643–1653. doi: 10.1101/gad.6.9.1643. [DOI] [PubMed] [Google Scholar]
  43. Pelletier J., Kaplan G., Racaniello V. R., Sonenberg N. Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region. Mol Cell Biol. 1988 Mar;8(3):1103–1112. doi: 10.1128/mcb.8.3.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pelletier J., Kaplan G., Racaniello V. R., Sonenberg N. Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5' noncoding region. J Virol. 1988 Jul;62(7):2219–2227. doi: 10.1128/jvi.62.7.2219-2227.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pelletier J., Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 1988 Jul 28;334(6180):320–325. doi: 10.1038/334320a0. [DOI] [PubMed] [Google Scholar]
  46. Rotman G., Itin A., Keshet E. Promoter and enhancer activities of long terminal repeats associated with cellular retrovirus-like (VL30) elements. Nucleic Acids Res. 1986 Jan 24;14(2):645–658. doi: 10.1093/nar/14.2.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Savatier P., Morgenstern J., Beddington R. S. Permissiveness to murine leukemia, virus expression during preimplantation and early postimplantation mouse development. Development. 1990 Jul;109(3):655–665. doi: 10.1242/dev.109.3.655. [DOI] [PubMed] [Google Scholar]
  48. Swain A., Coffin J. M. Mechanism of transduction by retroviruses. Science. 1992 Feb 14;255(5046):841–845. doi: 10.1126/science.1371365. [DOI] [PubMed] [Google Scholar]
  49. Thiel V., Siddell S. G. Internal ribosome entry in the coding region of murine hepatitis virus mRNA 5. J Gen Virol. 1994 Nov;75(Pt 11):3041–3046. doi: 10.1099/0022-1317-75-11-3041. [DOI] [PubMed] [Google Scholar]
  50. Thomas A. A., ter Haar E., Wellink J., Voorma H. O. Cowpea mosaic virus middle component RNA contains a sequence that allows internal binding of ribosomes and that requires eukaryotic initiation factor 4F for optimal translation. J Virol. 1991 Jun;65(6):2953–2959. doi: 10.1128/jvi.65.6.2953-2959.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Torrent C., Bordet T., Darlix J. L. Analytical study of rat retrotransposon VL30 RNA dimerization in vitro and packaging in murine leukemia virus. J Mol Biol. 1994 Jul 29;240(5):434–444. doi: 10.1006/jmbi.1994.1459. [DOI] [PubMed] [Google Scholar]
  52. Torrent C., Gabus C., Darlix J. L. A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer. J Virol. 1994 Feb;68(2):661–667. doi: 10.1128/jvi.68.2.661-667.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tounekti N., Mougel M., Roy C., Marquet R., Darlix J. L., Paoletti J., Ehresmann B., Ehresmann C. Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA. J Mol Biol. 1992 Jan 5;223(1):205–220. doi: 10.1016/0022-2836(92)90726-z. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Vagner S., Gensac M. C., Maret A., Bayard F., Amalric F., Prats H., Prats A. C. Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes. Mol Cell Biol. 1995 Jan;15(1):35–44. doi: 10.1128/mcb.15.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Velu T. J., Vass W. C., Lowy D. R., Tambourin P. E. Harvey murine sarcoma virus: influences of coding and noncoding sequences on cell transformation in vitro and oncogenicity in vivo. J Virol. 1989 Mar;63(3):1384–1392. doi: 10.1128/jvi.63.3.1384-1392.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weber L. A., Hickey E. D., Baglioni C. Influence of potassium salt concentration and temperature on inhibition of mRNA translation by 7-methylguanosine5'-monophosphate. J Biol Chem. 1978 Jan 10;253(1):178–183. [PubMed] [Google Scholar]
  58. Weber L. A., Hickey E. D., Maroney P. A., Baglioni C. Inhibition of protein synthesis by Cl-. J Biol Chem. 1977 Jun 10;252(11):4007–4010. [PubMed] [Google Scholar]
  59. Wei C. M., Lowy D. R., Scolnick E. M. Mapping of transforming region of the Harvey murine sarcoma virus genome by using insertion-deletion mutants constructed in vitro. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4674–4678. doi: 10.1073/pnas.77.8.4674. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES