Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1992 Mar;66(3):1476–1483. doi: 10.1128/jvi.66.3.1476-1483.1992

Internal ribosome entry site within hepatitis C virus RNA.

K Tsukiyama-Kohara 1, N Iizuka 1, M Kohara 1, A Nomoto 1
PMCID: PMC240872  PMID: 1310759

Abstract

The mechanism of initiation of translation on hepatitis C virus (HCV) RNA was investigated in vitro. HCV RNA was transcribed from the cDNA that corresponded to nucleotide positions 9 to 1772 of the genome by using phage T7 RNA polymerase. Both capped and uncapped RNAs thus transcribed were active as mRNAs in a cell-free protein synthesis system with lysates prepared from HeLa S3 cells or rabbit reticulocytes, and the translation products were detected by anti-gp35 antibodies. The data indicate that protein synthesis starts at the fourth AUG, which was the initiator AUG at position 333 of the HCV RNA used in this study. Efficiency of translation of the capped methylated RNA appeared to be similar to that of the capped unmethylated RNA. However, a capped methylated RNA showed a much higher activity as mRNA than did the capped unmethylated RNA in rabbit reticulocyte lysates when the RNA lacked a nucleotide sequence upstream of position 267. The results strongly suggest that HCV RNA carries an internal ribosome entry site (IRES). Artificial mono- and dicistronic mRNAs were prepared and used to identify the region that carried the IRES. The results indicate that the sequence between nucleotide positions 101 and 332 in the 5' untranslated region of HCV RNA plays an important role in efficient translation. Our data suggest that the IRES resides in this region of the RNA. Furthermore, an IRES in the group II HCV RNA was found to be more efficient than that in the group I HCV RNA.

Full text

PDF
1476

Images in this article

1478Image
on p.1478
1479Image
on p.1479
1479Image
on p.1479
1481Image
on p.1481
1482Image
on p.1482

Selected References

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

  1. Bienkowska-Szewczyk K., Ehrenfeld E. An internal 5'-noncoding region required for translation of poliovirus RNA in vitro. J Virol. 1988 Aug;62(8):3068–3072. doi: 10.1128/jvi.62.8.3068-3072.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Both G. W., Banerjee A. K., Shatkin A. J. Methylation-dependent translation of viral messenger RNAs in vitro. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1189–1193. doi: 10.1073/pnas.72.3.1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Choo Q. L., Richman K. H., Han J. H., Berger K., Lee C., Dong C., Gallegos C., Coit D., Medina-Selby R., Barr P. J. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2451–2455. doi: 10.1073/pnas.88.6.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ensinger M. J., Moss B. Modification of the 5' terminus of mRNA by an RNA (guanine-7-)-methyltransferase from HeLa cells. J Biol Chem. 1976 Sep 10;251(17):5283–5291. [PubMed] [Google Scholar]
  5. Han J. H., Shyamala V., Richman K. H., Brauer M. J., Irvine B., Urdea M. S., Tekamp-Olson P., Kuo G., Choo Q. L., Houghton M. Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5' untranslated region and poly(A) tails at the 3' end. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1711–1715. doi: 10.1073/pnas.88.5.1711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hijikata M., Kato N., Ootsuyama Y., Nakagawa M., Shimotohno K. Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5547–5551. doi: 10.1073/pnas.88.13.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Iizuka N., Kohara M., Hagino-Yamagishi K., Abe S., Komatsu T., Tago K., Arita M., Nomoto A. Construction of less neurovirulent polioviruses by introducing deletions into the 5' noncoding sequence of the genome. J Virol. 1989 Dec;63(12):5354–5363. doi: 10.1128/jvi.63.12.5354-5363.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Iizuka N., Yonekawa H., Nomoto A. Nucleotide sequences important for translation initiation of enterovirus RNA. J Virol. 1991 Sep;65(9):4867–4873. doi: 10.1128/jvi.65.9.4867-4873.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jang S. K., Davies M. V., Kaufman R. J., Wimmer E. Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo. J Virol. 1989 Apr;63(4):1651–1660. doi: 10.1128/jvi.63.4.1651-1660.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Jang S. K., Wimmer E. Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev. 1990 Sep;4(9):1560–1572. doi: 10.1101/gad.4.9.1560. [DOI] [PubMed] [Google Scholar]
  12. Kaminski A., Howell M. T., Jackson R. J. Initiation of encephalomyocarditis virus RNA translation: the authentic initiation site is not selected by a scanning mechanism. EMBO J. 1990 Nov;9(11):3753–3759. doi: 10.1002/j.1460-2075.1990.tb07588.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Sugimura T., Shimotohno K. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9524–9528. doi: 10.1073/pnas.87.24.9524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kozak M. Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems. Mol Cell Biol. 1989 Nov;9(11):5073–5080. doi: 10.1128/mcb.9.11.5073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
  16. Kozak M. Inability of circular mRNA to attach to eukaryotic ribosomes. Nature. 1979 Jul 5;280(5717):82–85. doi: 10.1038/280082a0. [DOI] [PubMed] [Google Scholar]
  17. Kuge S., Nomoto A. Construction of viable deletion and insertion mutants of the Sabin strain of type 1 poliovirus: function of the 5' noncoding sequence in viral replication. J Virol. 1987 May;61(5):1478–1487. doi: 10.1128/jvi.61.5.1478-1487.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lee K. A., Sonenberg N. Inactivation of cap-binding proteins accompanies the shut-off of host protein synthesis by poliovirus. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3447–3451. doi: 10.1073/pnas.79.11.3447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Okamoto H., Okada S., Sugiyama Y., Yotsumoto S., Tanaka T., Yoshizawa H., Tsuda F., Miyakawa Y., Mayumi M. The 5'-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med. 1990 Jun;60(3):167–177. [PubMed] [Google Scholar]
  22. Pelletier J., Flynn M. E., Kaplan G., Racaniello V., Sonenberg N. Mutational analysis of upstream AUG codons of poliovirus RNA. J Virol. 1988 Dec;62(12):4486–4492. doi: 10.1128/jvi.62.12.4486-4492.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Rose J. K., Trachsel H., Leong K., Baltimore D. Inhibition of translation by poliovirus: inactivation of a specific initiation factor. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2732–2736. doi: 10.1073/pnas.75.6.2732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Takeuchi K., Kubo Y., Boonmar S., Watanabe Y., Katayama T., Choo Q. L., Kuo G., Houghton M., Saito I., Miyamura T. The putative nucleocapsid and envelope protein genes of hepatitis C virus determined by comparison of the nucleotide sequences of two isolates derived from an experimentally infected chimpanzee and healthy human carriers. J Gen Virol. 1990 Dec;71(Pt 12):3027–3033. doi: 10.1099/0022-1317-71-12-3027. [DOI] [PubMed] [Google Scholar]
  27. Tsukiyama-Kohara K., Kohara M., Yamaguchi K., Maki N., Toyoshima A., Miki K., Tanaka S., Hattori N., Nomoto A. A second group of hepatitis C viruses. Virus Genes. 1991 Jul;5(3):243–254. doi: 10.1007/BF00568974. [DOI] [PubMed] [Google Scholar]
  28. Tsukiyama K., Yoshikawa Y., Kamata H., Imaoka K., Asano K., Funahashi S., Maruyama T., Shida H., Sugimoto M., Yamanouchi K. Development of heat-stable recombinant rinderpest vaccine. Arch Virol. 1989;107(3-4):225–235. doi: 10.1007/BF01317919. [DOI] [PubMed] [Google Scholar]

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

RESOURCES