1887

Abstract

We have investigated the possible role of a conserved -acting element, the cryptic AUG, present in the 5′ UTR of coxsackievirus B3 (CVB3 ) RNA. CVB3 5′ UTR contains multiple AUG codons upstream of the initiator AUG, which are not used for the initiation of translation. The 48S ribosomal assembly takes place upstream of the cryptic AUG. We show here that mutation in the cryptic AUG results in reduced efficiency of translation mediated by the CVB3 IRES; mutation also reduces the interaction of mutant IRES with a well characterized IRES -acting factor, the human La protein. Furthermore, partial silencing of the La gene showed a decrease in IRES activity in the case of both the wild-type and mutant. We have demonstrated here that the interaction of the 48S ribosomal complex with mutant RNA was weaker compared with wild-type RNA by ribosome assembly analysis. We have also investigated by chemical and enzymic modifications the possible alteration in secondary structure in the mutant RNA. Results suggest that the secondary structure of mutant RNA was only marginally altered. Additionally, we have demonstrated by generating compensatory and non-specific mutations the specific function of the cryptic AUG in internal initiation. Results suggest that the effect of the cryptic AUG is specific and translation could not be rescued. However, a possibility of tertiary interaction of the cryptic AUG with other -acting elements cannot be ruled out. Taken together, it appears that the integrity of the cryptic AUG is important for efficient translation initiation by the CVB3 IRES RNA.

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2011-10-01
2020-01-27
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References

  1. Agol V. I. . ( 1991; ). The 5′-untranslated region of picornaviral genomes. . Adv Virus Res 40:, 103–180. [CrossRef] [PubMed]
    [Google Scholar]
  2. Bailey J. M. , Tapprich W. E. . ( 2007; ). Structure of the 5′ nontranslated region of the coxsackievirus b3 genome: chemical modification and comparative sequence analysis. . J Virol 81:, 650–668. [CrossRef] [PubMed]
    [Google Scholar]
  3. Belsham G. J. , Sonenberg N. . ( 1996; ). RNA–protein interactions in regulation of picornavirus RNA translation. . Microbiol Rev 60:, 499–511.[PubMed]
    [Google Scholar]
  4. Bhattacharyya S. , Das S. . ( 2005; ). Mapping of secondary structure of the spacer region within the 5′-untranslated region of the coxsackievirus B3 RNA: possible role of an apical GAGA loop in binding La protein and influencing internal initiation of translation. . Virus Res 108:, 89–100. [CrossRef] [PubMed]
    [Google Scholar]
  5. Bhattacharyya S. , Das S. . ( 2006; ). An apical GAGA loop within 5′ UTR of the coxsackievirus B3 RNA maintains structural organization of the IRES element required for efficient ribosome entry. . RNA Biol 3:, 60–68. [CrossRef] [PubMed]
    [Google Scholar]
  6. Bhattacharyya S. , Verma B. , Pandey G. , Das S. . ( 2008; ). The structure and function of a cis-acting element located upstream of the IRES that influences coxsackievirus B3 RNA translation. . Virology 377:, 345–354. [CrossRef] [PubMed]
    [Google Scholar]
  7. Borman A. M. , Jackson R. J. . ( 1992; ). Initiation of translation of human rhinovirus RNA: mapping the internal ribosome entry site. . Virology 188:, 685–696. [CrossRef] [PubMed]
    [Google Scholar]
  8. Chen T. C. , Weng K. F. , Chang S. C. , Lin J. Y. , Huang P. N. , Shih S. R. . ( 2008; ). Development of antiviral agents for enteroviruses. . J Antimicrob Chemother 62:, 1169–1173. [CrossRef] [PubMed]
    [Google Scholar]
  9. Costa-Mattioli M. , Svitkin Y. , Sonenberg N. . ( 2004; ). La autoantigen is necessary for optimal function of the poliovirus and hepatitis C virus internal ribosome entry site in vivo and in vitro . . Mol Cell Biol 24:, 6861–6870. [CrossRef] [PubMed]
    [Google Scholar]
  10. Dhar D. , Roy S. , Das S. . ( 2007; ). Translational control of the interferon regulatory factor 2 mRNA by IRES element. . Nucleic Acids Res 35:, 5409–5421. [CrossRef] [PubMed]
    [Google Scholar]
  11. Dmitriev S. E. , Pisarev A. V. , Rubtsova M. P. , Dunaevsky Y. E. , Shatsky I. N. . ( 2003; ). Conversion of 48S translation preinitiation complexes into 80S initiation complexes as revealed by toeprinting. . FEBS Lett 533:, 99–104. [CrossRef] [PubMed]
    [Google Scholar]
  12. Fitzgerald K. D. , Semler B. L. . ( 2009; ). Bridging IRES elements in mRNAs to the eukaryotic translation apparatus. . Biochim Biophys Acta 1789:, 518–528.[PubMed] [CrossRef]
    [Google Scholar]
  13. Iizuka N. , Yonekawa H. , Nomoto A. . ( 1991; ). Nucleotide sequences important for translation initiation of enterovirus RNA. . J Virol 65:, 4867–4873. [CrossRef] [PubMed]
    [Google Scholar]
  14. Jang S. K. , Kräusslich H. G. , Nicklin M. J. , Duke G. M. , Palmenberg A. C. , Wimmer E. . ( 1988; ). A segment of the 5′ nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. . J Virol 62:, 2636–2643.[PubMed]
    [Google Scholar]
  15. Liu Z. , Carthy C. M. , Cheung P. , Bohunek L. , Wilson J. E. , McManus B. M. , Yang D. . ( 1999; ). Structural and functional analysis of the 5′ untranslated region of coxsackievirus B3 RNA: in vivo translational and infectivity studies of full-length mutants. . Virology 265:, 206–217. [CrossRef] [PubMed]
    [Google Scholar]
  16. Meerovitch K. , Nicholson R. , Sonenberg N. . ( 1991; ). In vitro mutational analysis of cis-acting RNA translational elements within the poliovirus type 2 5′ untranslated region. . J Virol 65:, 5895–5901.[PubMed]
    [Google Scholar]
  17. M’hadheb-Gharbi M. B. , Paulous S. , Aouni M. , Kean K. M. , Gharbi J. . ( 2007; ). The substitution U475→C with Sabin3-like mutation within the IRES attenuate coxsackievirus B3 cardiovirulence. . Mol Biotechnol 36:, 52–60. [CrossRef] [PubMed]
    [Google Scholar]
  18. Nicholson R. , Pelletier J. , Le S. Y. , Sonenberg N. . ( 1991; ). Structural and functional analysis of the ribosome landing pad of poliovirus type 2: in vivo translation studies. . J Virol 65:, 5886–5894.[PubMed]
    [Google Scholar]
  19. Pelletier J. , Flynn M. E. , Kaplan G. , Racaniello V. R. , Sonenberg N. . ( 1988; ). Mutational analysis of upstream AUG codons of poliovirus RNA. . J Virol 62:, 4486–4492.[PubMed]
    [Google Scholar]
  20. Pilipenko E. V. , Gmyl A. P. , Maslova S. V. , Svitkin Y. V. , Sinyakov A. N. , Agol V. I. . ( 1992; ). Prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus RNA. . Cell 68:, 119–131. [CrossRef] [PubMed]
    [Google Scholar]
  21. Pöyry T. , Kinnunen L. , Hovi T. . ( 1992; ). Genetic variation in vivo and proposed functional domains of the 5′ noncoding region of poliovirus RNA. . J Virol 66:, 5313–5319.[PubMed]
    [Google Scholar]
  22. Pudi R. , Srinivasan P. , Das S. . ( 2004; ). La protein binding at the GCAC site near the initiator AUG facilitates the ribosomal assembly on the hepatitis C virus RNA to influence internal ribosome entry site-mediated translation. . J Biol Chem 279:, 29879–29888. [CrossRef] [PubMed]
    [Google Scholar]
  23. Ray P. S. , Das S. . ( 2002; ). La autoantigen is required for the internal ribosome entry site-mediated translation of coxsackievirus B3 RNA. . Nucleic Acids Res 30:, 4500–4508. [CrossRef] [PubMed]
    [Google Scholar]
  24. Skinner M. A. , Racaniello V. R. , Dunn G. , Cooper J. , Minor P. D. , Almond J. W. . ( 1989; ). New model for the secondary structure of the 5′ non-coding RNA of poliovirus is supported by biochemical and genetic data that also show that RNA secondary structure is important in neurovirulence. . J Mol Biol 207:, 379–392. [CrossRef] [PubMed]
    [Google Scholar]
  25. Slobodskaya O. R. , Gmyl A. P. , Maslova S. V. , Tolskaya E. A. , Viktorova E. G. , Agol V. I. . ( 1996; ). Poliovirus neurovirulence correlates with the presence of a cryptic AUG upstream of the initiator codon. . Virology 221:, 141–150. [CrossRef] [PubMed]
    [Google Scholar]
  26. Verma B. , Bhattacharyya S. , Das S. . ( 2010; ). Polypyrimidine tract-binding protein interacts with coxsackievirus B3 RNA and influences its translation. . J Gen Virol 91:, 1245–1255. [CrossRef] [PubMed]
    [Google Scholar]
  27. Yang D. , Wilson J. E. , Anderson D. R. , Bohunek L. , Cordeiro C. , Kandolf R. , McManus B. M. . ( 1997; ). In vitro mutational and inhibitory analysis of the cis-acting translational elements within the 5′ untranslated region of coxsackievirus B3: potential targets for antiviral action of antisense oligomers. . Virology 228:, 63–73. [CrossRef] [PubMed]
    [Google Scholar]
  28. Yang D. , Cheung P. , Sun Y. , Yuan J. , Zhang H. , Carthy C. M. , Anderson D. R. , Bohunek L. , Wilson J. E. , McManus B. M. . ( 2003; ). A shine-dalgarno-like sequence mediates in vitro ribosomal internal entry and subsequent scanning for translation initiation of coxsackievirus B3 RNA. . Virology 305:, 31–43. [CrossRef] [PubMed]
    [Google Scholar]
  29. Zell R. , Ihle Y. , Seitz S. , Gündel U. , Wutzler P. , Görlach M. . ( 2008; ). Poly(rC)-binding protein 2 interacts with the oligo(rC) tract of coxsackievirus B3. . Biochem Biophys Res Commun 366:, 917–921. [CrossRef] [PubMed]
    [Google Scholar]
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vol. , part 10, pp. 2310 - 2319

α-32P-labelled CVB3 5' UTR RNA

Representation of sucrose-gradient sedimentation profiles of [α-32P]UTP-labelled CVB3

α-32P-end labelled primer [Single PDF file](733 KB)



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