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. 1995 Oct;69(10):6367–6375. doi: 10.1128/jvi.69.10.6367-6375.1995

Interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation.

N Ali 1, A Siddiqui 1
PMCID: PMC189536  PMID: 7666538

Abstract

Initiation of translation of the human hepatitis C virus (HCV) RNA genome occurs by internal ribosome entry into the 5' noncoding region (5'NCR) in a cap-independent manner. The internal ribosome entry site of the HCV 5'NCR has been previously defined to encompass almost the entire 5'NCR. Here we report the interaction of polypyrimidine tract-binding protein (PTB) at three distinct regions within the 5'NCR by UV cross-linking assays. All three regions contain a consensus polypyrimidine tract motif. The evidence for the interaction of recombinant PTB at multiple sites within the 5'NCR is based on the use of 5'NCR mutants as competitors and by direct UV cross-linking of the mutant RNAs. Furthermore, the PTB isomers from HeLa nuclear extracts interact with the HCV 5'NCR, as shown by immunoprecipitation of a UV cross-linked complex with anti-PTB serum. Immunodepletion of PTB from translation lysates suggested the functional requirement for PTB during translation initiation of the HCV RNA. Addition of purified PTB to immunodepleted lysates did not restore translation mediated by the HCV 5'NCR, indicating the requirement of PTB-associated factors that were removed during immunodepletion.

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Selected References

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  1. Agol V. I. The 5'-untranslated region of picornaviral genomes. Adv Virus Res. 1991;40:103–180. doi: 10.1016/S0065-3527(08)60278-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borman A., Howell M. T., Patton J. G., Jackson R. J. The involvement of a spliceosome component in internal initiation of human rhinovirus RNA translation. J Gen Virol. 1993 Sep;74(Pt 9):1775–1788. doi: 10.1099/0022-1317-74-9-1775. [DOI] [PubMed] [Google Scholar]
  3. Borovjagin A. V., Ezrokhi M. V., Rostapshov V. M., Ugarova TYu, Bystrova T. F., Shatsky I. N. RNA--protein interactions within the internal translation initiation region of encephalomyocarditis virus RNA. Nucleic Acids Res. 1991 Sep 25;19(18):4999–5005. doi: 10.1093/nar/19.18.4999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bothwell A. L., Ballard D. W., Philbrick W. M., Lindwall G., Maher S. E., Bridgett M. M., Jamison S. F., Garcia-Blanco M. A. Murine polypyrimidine tract binding protein. Purification, cloning, and mapping of the RNA binding domain. J Biol Chem. 1991 Dec 25;266(36):24657–24663. [PubMed] [Google Scholar]
  5. 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]
  6. Bukh J., Purcell R. H., Miller R. H. Sequence analysis of the 5' noncoding region of hepatitis C virus. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):4942–4946. doi: 10.1073/pnas.89.11.4942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang K. H., Brown E. A., Lemon S. M. Cell type-specific proteins which interact with the 5' nontranslated region of hepatitis A virus RNA. J Virol. 1993 Nov;67(11):6716–6725. doi: 10.1128/jvi.67.11.6716-6725.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Dignam J. D. Preparation of extracts from higher eukaryotes. Methods Enzymol. 1990;182:194–203. doi: 10.1016/0076-6879(90)82017-v. [DOI] [PubMed] [Google Scholar]
  10. Ehrenfeld E., Gebhard J. G. Interaction of cellular proteins with the poliovirus 5' noncoding region. Arch Virol Suppl. 1994;9:269–277. doi: 10.1007/978-3-7091-9326-6_27. [DOI] [PubMed] [Google Scholar]
  11. García-Blanco M. A., Jamison S. F., Sharp P. A. Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns. Genes Dev. 1989 Dec;3(12A):1874–1886. doi: 10.1101/gad.3.12a.1874. [DOI] [PubMed] [Google Scholar]
  12. Ghetti A., Piñol-Roma S., Michael W. M., Morandi C., Dreyfuss G. hnRNP I, the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs. Nucleic Acids Res. 1992 Jul 25;20(14):3671–3678. doi: 10.1093/nar/20.14.3671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gil A., Sharp P. A., Jamison S. F., Garcia-Blanco M. A. Characterization of cDNAs encoding the polypyrimidine tract-binding protein. Genes Dev. 1991 Jul;5(7):1224–1236. doi: 10.1101/gad.5.7.1224. [DOI] [PubMed] [Google Scholar]
  14. Hambidge S. J., Sarnow P. Translational enhancement of the poliovirus 5' noncoding region mediated by virus-encoded polypeptide 2A. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10272–10276. doi: 10.1073/pnas.89.21.10272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Hellen C. U., Pestova T. V., Litterst M., Wimmer E. The cellular polypeptide p57 (pyrimidine tract-binding protein) binds to multiple sites in the poliovirus 5' nontranslated region. J Virol. 1994 Feb;68(2):941–950. doi: 10.1128/jvi.68.2.941-950.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hellen C. U., Witherell G. W., Schmid M., Shin S. H., Pestova T. V., Gil A., Wimmer E. A cytoplasmic 57-kDa protein that is required for translation of picornavirus RNA by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7642–7646. doi: 10.1073/pnas.90.16.7642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Houghton M., Weiner A., Han J., Kuo G., Choo Q. L. Molecular biology of the hepatitis C viruses: implications for diagnosis, development and control of viral disease. Hepatology. 1991 Aug;14(2):381–388. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. Meerovitch K., Svitkin Y. V., Lee H. S., Lejbkowicz F., Kenan D. J., Chan E. K., Agol V. I., Keene J. D., Sonenberg N. La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate. J Virol. 1993 Jul;67(7):3798–3807. doi: 10.1128/jvi.67.7.3798-3807.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Miller R. H., Purcell R. H. Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2057–2061. doi: 10.1073/pnas.87.6.2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mulligan G. J., Guo W., Wormsley S., Helfman D. M. Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin pre-mRNA. J Biol Chem. 1992 Dec 15;267(35):25480–25487. [PubMed] [Google Scholar]
  28. 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]
  29. Okamoto H., Okada S., Sugiyama Y., Kurai K., Iizuka H., Machida A., Miyakawa Y., Mayumi M. Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human carrier: comparison with reported isolates for conserved and divergent regions. J Gen Virol. 1991 Nov;72(Pt 11):2697–2704. doi: 10.1099/0022-1317-72-11-2697. [DOI] [PubMed] [Google Scholar]
  30. Patton J. G., Mayer S. A., Tempst P., Nadal-Ginard B. Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing. Genes Dev. 1991 Jul;5(7):1237–1251. doi: 10.1101/gad.5.7.1237. [DOI] [PubMed] [Google Scholar]
  31. Pestova T. V., Hellen C. U., Wimmer E. Translation of poliovirus RNA: role of an essential cis-acting oligopyrimidine element within the 5' nontranslated region and involvement of a cellular 57-kilodalton protein. J Virol. 1991 Nov;65(11):6194–6204. doi: 10.1128/jvi.65.11.6194-6204.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pilipenko E. V., Gmyl A. P., Maslova S. V., Svitkin Y. V., Sinyakov A. N., Agol V. I. Prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus RNA. Cell. 1992 Jan 10;68(1):119–131. doi: 10.1016/0092-8674(92)90211-t. [DOI] [PubMed] [Google Scholar]
  33. Schmid M., Wimmer E. IRES-controlled protein synthesis and genome replication of poliovirus. Arch Virol Suppl. 1994;9:279–289. doi: 10.1007/978-3-7091-9326-6_28. [DOI] [PubMed] [Google Scholar]
  34. Sonenberg N. Poliovirus translation. Curr Top Microbiol Immunol. 1990;161:23–47. doi: 10.1007/978-3-642-75602-3_2. [DOI] [PubMed] [Google Scholar]
  35. Stade K., Rinke-Appel J., Brimacombe R. Site-directed cross-linking of mRNA analogues to the Escherichia coli ribosome; identification of 30S ribosomal components that can be cross-linked to the mRNA at various points 5' with respect to the decoding site. Nucleic Acids Res. 1989 Dec 11;17(23):9889–9908. doi: 10.1093/nar/17.23.9889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Svitkin Y. V., Meerovitch K., Lee H. S., Dholakia J. N., Kenan D. J., Agol V. I., Sonenberg N. Internal translation initiation on poliovirus RNA: further characterization of La function in poliovirus translation in vitro. J Virol. 1994 Mar;68(3):1544–1550. doi: 10.1128/jvi.68.3.1544-1550.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Takamizawa A., Mori C., Fuke I., Manabe S., Murakami S., Fujita J., Onishi E., Andoh T., Yoshida I., Okayama H. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol. 1991 Mar;65(3):1105–1113. doi: 10.1128/jvi.65.3.1105-1113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Tsukuma H., Hiyama T., Tanaka S., Nakao M., Yabuuchi T., Kitamura T., Nakanishi K., Fujimoto I., Inoue A., Yamazaki H. Risk factors for hepatocellular carcinoma among patients with chronic liver disease. N Engl J Med. 1993 Jun 24;328(25):1797–1801. doi: 10.1056/NEJM199306243282501. [DOI] [PubMed] [Google Scholar]
  40. Wakita T., Wands J. R. Specific inhibition of hepatitis C virus expression by antisense oligodeoxynucleotides. In vitro model for selection of target sequence. J Biol Chem. 1994 May 13;269(19):14205–14210. [PubMed] [Google Scholar]
  41. Wang C., Le S. Y., Ali N., Siddiqui A. An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region. RNA. 1995 Jul;1(5):526–537. [PMC free article] [PubMed] [Google Scholar]
  42. Wang C., Sarnow P., Siddiqui A. A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA. J Virol. 1994 Nov;68(11):7301–7307. doi: 10.1128/jvi.68.11.7301-7307.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Wang C., Siddiqui A. Structure and function of the hepatitis C virus internal ribosome entry site. Curr Top Microbiol Immunol. 1995;203:99–115. doi: 10.1007/978-3-642-79663-0_5. [DOI] [PubMed] [Google Scholar]
  45. Witherell G. W., Gil A., Wimmer E. Interaction of polypyrimidine tract binding protein with the encephalomyocarditis virus mRNA internal ribosomal entry site. Biochemistry. 1993 Aug 17;32(32):8268–8275. doi: 10.1021/bi00083a030. [DOI] [PubMed] [Google Scholar]
  46. Witherell G. W., Wimmer E. Encephalomyocarditis virus internal ribosomal entry site RNA-protein interactions. J Virol. 1994 May;68(5):3183–3192. doi: 10.1128/jvi.68.5.3183-3192.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]

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