1887

Abstract

Translational readthrough-promoting drugs enhance the incorporation of amino acids at stop codons and can thus bypass premature termination during protein synthesis. The polymerase (Pol) proteins of Moloney murine leukemia virus (MoMLV) are synthesized as a large Gag–Pol fusion protein, formed by the readthrough of a stop codon at the end of the ORF. The downstream ORF lacks its own start codon, and Pol protein synthesis is wholly dependent on translation of the upstream gene and the readthrough event for expression. Here, we explored the effects of readthrough-promoting drugs – aminoglycoside antibiotics and the small molecule ataluren – on the efficiency of readthrough of the stop codon in the context of the MoMLV genome. We showed that these compounds increased readthrough of the stop codon at the MoMLV junction above the already high basal level and that the resulting elevated readthrough had deleterious effects on virus replication. We also showed that readthrough efficiency could be driven to even higher levels , and that the combination of the small molecules and the RNA structure at the MoMLV stop codon could achieve extremely high readthrough efficiencies.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000284
2015-11-01
2019-10-14
Loading full text...

Full text loading...

/deliver/fulltext/jgv/96/11/3411.html?itemId=/content/journal/jgv/10.1099/jgv.0.000284&mimeType=html&fmt=ahah

References

  1. Al-Tawfiq J.A. , Antony A. , Abed M.S. . ( 2009;). Antimicrobial resistance rates of Enterobacter spp.: a seven-year surveillance study. Med Princ Pract 18: 100–104 [CrossRef] [PubMed].
    [Google Scholar]
  2. Alam S.L. , Wills N.M. , Ingram J.A. , Atkins J.F. , Gesteland R.F. . ( 1999;). Structural studies of the RNA pseudoknot required for readthrough of the gag-termination codon of murine leukemia virus. J Mol Biol 288: 837–852 [CrossRef] [PubMed].
    [Google Scholar]
  3. Auld D.S. , Thorne N. , Maguire W.F. , Inglese J. . ( 2009;). Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression. Proc Natl Acad Sci U S A 106: 3585–3590 [CrossRef] [PubMed].
    [Google Scholar]
  4. Auld D.S. , Lovell S. , Thorne N. , Lea W.A. , Maloney D.J. , Shen M. , Rai G. , Battaile K.P. , Thomas C.J. , other authors . ( 2010;). Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124. Proc Natl Acad Sci U S A 107: 4878–4883 [CrossRef] [PubMed].
    [Google Scholar]
  5. Banerjee A. , De M. , Ali N. . ( 2011;). Combination therapy with paromomycin-associated stearylamine-bearing liposomes cures experimental visceral leishmaniasis through Th1-biased immunomodulation. Antimicrob Agents Chemother 55: 1661–1670 [CrossRef] [PubMed].
    [Google Scholar]
  6. Bernacchi S. , Freisz S. , Maechling C. , Spiess B. , Marquet R. , Dumas P. , Ennifar E. . ( 2007;). Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion. Nucleic Acids Res 35: 7128–7139 [CrossRef] [PubMed].
    [Google Scholar]
  7. Burke J.F. , Mogg A.E. . ( 1985;). Suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside antibiotics G-418 and paromomycin. Nucleic Acids Res 13: 6265–6272 [CrossRef] [PubMed].
    [Google Scholar]
  8. Cabada M.M. , White A.C. Jr . ( 2010;). Treatment of cryptosporidiosis: do we know what we think we know?. Curr Opin Infect Dis 23: 494–499 [CrossRef] [PubMed].
    [Google Scholar]
  9. Caminero J.A. , Sotgiu G. , Zumla A. , Migliori G.B. . ( 2010;). Best drug treatment for multidrug-resistant and extensively drug-resistant tuberculosis. Lancet Infect Dis 10: 621–629 [CrossRef] [PubMed].
    [Google Scholar]
  10. Carter A.P. , Clemons W.M. , Brodersen D.E. , Morgan-Warren R.J. , Wimberly B.T. , Ramakrishnan V. . ( 2000;). Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407: 340–348 [CrossRef] [PubMed].
    [Google Scholar]
  11. Chamorro M. , Parkin N. , Varmus H.E. . ( 1992;). An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA. Proc Natl Acad Sci U S A 89: 713–717 [CrossRef] [PubMed].
    [Google Scholar]
  12. Chen X. , Chamorro M. , Lee S.I. , Shen L.X. , Hines J.V. , Tinoco I. Jr , Varmus H.E. . ( 1995;). Structural and functional studies of retroviral RNA pseudoknots involved in ribosomal frameshifting: nucleotides at the junction of the two stems are important for efficient ribosomal frameshifting. EMBO J 14: 842–852 [PubMed].
    [Google Scholar]
  13. Csibra E. , Brierley I. , Irigoyen N. . ( 2014;). Modulation of stop codon read-through efficiency and its effect on the replication of murine leukemia virus. J Virol 88: 10364–10376 [CrossRef] [PubMed].
    [Google Scholar]
  14. Du Z. , Holland J.A. , Hansen M.R. , Giedroc D.P. , Hoffman D.W. . ( 1997;). Base-pairings within the RNA pseudoknot associated with the simian retrovirus-1 gag–pro frameshift site. J Mol Biol 270: 464–470 [CrossRef] [PubMed].
    [Google Scholar]
  15. Du M. , Liu X. , Welch E.M. , Hirawat S. , Peltz S.W. , Bedwell D.M. . ( 2008;). PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model. Proc Natl Acad Sci U S A 105: 2064–2069 [CrossRef] [PubMed].
    [Google Scholar]
  16. Du L. , Damoiseaux R. , Nahas S. , Gao K. , Hu H. , Pollard J.M. , Goldstine J. , Jung M.E. , Henning S.M. , other authors . ( 2009;). Nonaminoglycoside compounds induce readthrough of nonsense mutations. J Exp Med 206: 2285–2297 [CrossRef] [PubMed].
    [Google Scholar]
  17. Ennifar E. , Paillart J.C. , Marquet R. , Ehresmann B. , Ehresmann C. , Dumas P. , Walter P. . ( 2003;). HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics. J Biol Chem 278: 2723–2730 [CrossRef] [PubMed].
    [Google Scholar]
  18. Ennifar E. , Paillart J.C. , Bodlenner A. , Walter P. , Weibel J.M. , Aubertin A.M. , Pale P. , Dumas P. , Marquet R. . ( 2006;). Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell. Nucleic Acids Res 34: 2328–2339 [CrossRef] [PubMed].
    [Google Scholar]
  19. Fan-Minogue H. , Bedwell D.M. . ( 2008;). Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity. RNA 14: 148–157 [CrossRef] [PubMed].
    [Google Scholar]
  20. Felsenstein K.M. , Goff S.P. . ( 1988;). Expression of the gag–pol fusion protein of Moloney murine leukemia virus without gag protein does not induce virion formation or proteolytic processing. J Virol 62: 2179–2182 [PubMed].
    [Google Scholar]
  21. Felsenstein K.M. , Goff S.P. . ( 1992;). Mutational analysis of the gag–pol junction of Moloney murine leukemia virus: requirements for expression of the gag–pol fusion protein. J Virol 66: 6601–6608 [PubMed].
    [Google Scholar]
  22. Feng Y.X. , Hatfield D.L. , Rein A. , Levin J.G. . ( 1989a;). Translational readthrough of the murine leukemia virus gag gene amber codon does not require virus-induced alteration of tRNA. J Virol 63: 2405–2410 [PubMed].
    [Google Scholar]
  23. Feng Y.X. , Levin J.G. , Hatfield D.L. , Schaefer T.S. , Gorelick R.J. , Rein A. . ( 1989b;). Suppression of UAA and UGA termination codons in mutant murine leukemia viruses. J Virol 63: 2870–2873 [PubMed].
    [Google Scholar]
  24. Feng Y.X. , Copeland T.D. , Oroszlan S. , Rein A. , Levin J.G. . ( 1990;). Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag–pol junction of Moloney murine leukemia virus. Proc Natl Acad Sci USA 87: 8860–8863 [CrossRef] [PubMed].
    [Google Scholar]
  25. Feng Y.X. , Yuan H. , Rein A. , Levin J.G. . ( 1992;). Bipartite signal for read-through suppression in murine leukemia virus mRNA: an eight-nucleotide purine-rich sequence immediately downstream of the gag termination codon followed by an RNA pseudoknot. J Virol 66: 5127–5132 [PubMed].
    [Google Scholar]
  26. Filippini P. , Iona E. , Piccaro G. , Peyron P. , Neyrolles O. , Fattorini L. . ( 2010;). Activity of drug combinations against dormant Mycobacterium tuberculosis . Antimicrob Agents Chemother 54: 2712–2715 [CrossRef] [PubMed].
    [Google Scholar]
  27. Fourmy D. , Recht M.I. , Blanchard S.C. , Puglisi J.D. . ( 1996;). Structure of the A site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside antibiotic. Science 274: 1367–1371 [CrossRef] [PubMed].
    [Google Scholar]
  28. Green L. , Houck-Loomis B. , Yueh A. , Goff S.P. . ( 2012;). Large ribosomal protein 4 increases efficiency of viral recoding sequences. J Virol 86: 8949–8958 [CrossRef] [PubMed].
    [Google Scholar]
  29. Grentzmann G. , Ingram J.A. , Kelly P.J. , Gesteland R.F. , Atkins J.F. . ( 1998;). A dual-luciferase reporter system for studying recoding signals. RNA 4: 479–486 [PubMed].[CrossRef]
    [Google Scholar]
  30. Hermann T. . ( 2005;). Drugs targeting the ribosome. Curr Opin Struct Biol 15: 355–366 [CrossRef] [PubMed].
    [Google Scholar]
  31. Honigman A. , Falk H. , Mador N. , Rosental T. , Panet A. . ( 1995;). Translation efficiency of the human T-cell leukemia virus (HTLV-2) gag gene modulates the frequency of ribosomal frameshifting. Virology 208: 312–318 [CrossRef] [PubMed].
    [Google Scholar]
  32. Houck-Loomis B. , Durney M.A. , Salguero C. , Shankar N. , Nagle J.M. , Goff S.P. , D'Souza V.M. . ( 2011;). An equilibrium-dependent retroviral mRNA switch regulates translational recoding. Nature 480: 561–564 [PubMed].
    [Google Scholar]
  33. Jacks T. , Varmus H.E. . ( 1985;). Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting. Science 230: 1237–1242 [CrossRef] [PubMed].
    [Google Scholar]
  34. Jones D.S. , Nemoto F. , Kuchino Y. , Masuda M. , Yoshikura H. , Nishimura S. . ( 1989;). The effect of specific mutations at and around the gag-pol gene junction of Moloney murine leukaemia virus. Nucleic Acids Res 17: 5933–5945 [CrossRef] [PubMed].
    [Google Scholar]
  35. Karasawa T. , Steyger P.S. . ( 2011;). Intracellular mechanisms of aminoglycoside-induced cytotoxicity. Integr Biol (Camb) 3: 879–886 [CrossRef] [PubMed].
    [Google Scholar]
  36. Kerem E. , Hirawat S. , Armoni S. , Yaakov Y. , Shoseyov D. , Cohen M. , Nissim-Rafinia M. , Blau H. , Rivlin J. , other authors . ( 2008;). Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial. Lancet 372: 719–727 [CrossRef] [PubMed].
    [Google Scholar]
  37. Le S.Y. , Shapiro B.A. , Chen J.H. , Nussinov R. , Maizel J.V. . ( 1991;). RNA pseudoknots downstream of the frameshift sites of retroviruses. Genet Anal Tech Appl 8: 191–205 [CrossRef] [PubMed].
    [Google Scholar]
  38. Levin J.G. , Hatfield D.L. , Oroszlan S. , Rein A. . ( 1993;). Mechanisms of Translational Suppression Used in the Biosynthesis of Reverse Transcriptaser. , pp. 5-31. In Reverse Transcriptase. Edited by Skalka A.-M. , Goff S. P. . Cold Spring Harbor: Cold Spring Harbor Press;.
    [Google Scholar]
  39. Manuvakhova M. , Keeling K. , Bedwell D.M. . ( 2000;). Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. RNA 6: 1044–1055 [CrossRef] [PubMed].
    [Google Scholar]
  40. Moore R. , Dixon M. , Smith R. , Peters G. , Dickson C. . ( 1987;). Complete nucleotide sequence of a milk-transmitted mouse mammary tumor virus: two frameshift suppression events are required for translation of gag and pol. J Virol 61: 480–490 [PubMed].
    [Google Scholar]
  41. Murphy E.C. Jr , Arlinghaus R.B. . ( 1978;). Cell-free synthesis of Rauscher murine leukemia virus “gag” and “gag-pol” precursor polyproteins from virion 35S RNA in a mRNA-dependent translation system derived from mouse tissue culture cells. Virology 86: 329–343 [CrossRef] [PubMed].
    [Google Scholar]
  42. Murphy E.C. Jr , Kopchick J.J. , Watson K.F. , Arlinghaus R.B. . ( 1978;). Cell-free synthesis of a precursor polyprotein containing both gag and pol gene products by Rauscher murine leukemia virus 35S RNA. Cell 13: 359–369 [CrossRef] [PubMed].
    [Google Scholar]
  43. Murphy G.J. , Mostoslavsky G. , Kotton D.N. , Mulligan R.C. . ( 2006;). Exogenous control of mammalian gene expression via modulation of translational termination. Nat Med 12: 1093–1099 [CrossRef] [PubMed].
    [Google Scholar]
  44. Musa A.M. , Younis B. , Fadlalla A. , Royce C. , Balasegaram M. , Wasunna M. , Hailu A. , Edwards T. , Omollo R. , other authors . ( 2010;). Paromomycin for the treatment of visceral leishmaniasis in Sudan: a randomized, open-label, dose-finding study. PLoS Negl Trop Dis 4: e855 [CrossRef] [PubMed].
    [Google Scholar]
  45. Nam S.H. , Copeland T.D. , Hatanaka M. , Oroszlan S. . ( 1993;). Characterization of ribosomal frameshifting for expression of pol gene products of human T-cell leukemia virus type I. J Virol 67: 196–203 [PubMed].
    [Google Scholar]
  46. Nudelman I. , Rebibo-Sabbah A. , Cherniavsky M. , Belakhov V. , Hainrichson M. , Chen F. , Schacht J. , Pilch D.S. , Ben-Yosef T. , Baasov T. . ( 2009;). Development of novel aminoglycoside (NB54) with reduced toxicity and enhanced suppression of disease-causing premature stop mutations. J Med Chem 52: 2836–2845 [CrossRef] [PubMed].
    [Google Scholar]
  47. Odawara T. , Yoshikura H. , Ohshima M. , Tanaka T. , Jones D.S. , Nemoto F. , Kuchino Y. , Iwamoto A. . ( 1991;). Analysis of Moloney murine leukemia virus revertants mutated at the gag–pol junction. J Virol 65: 6376–6379 [PubMed].
    [Google Scholar]
  48. Ogle J.M. , Brodersen D.E. , Clemons W.M. Jr , Tarry M.J. , Carter A.P. , Ramakrishnan V. . ( 2001;). Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science 292: 897–902 [CrossRef] [PubMed].
    [Google Scholar]
  49. Oppermann H. , Bishop J.M. , Varmus H.E. , Levintow L. . ( 1977;). A joint produce of the genes gag and pol of avian sarcoma virus: a possible precursor of reverse transcriptase. Cell 12: 993–1005 [CrossRef] [PubMed].
    [Google Scholar]
  50. Panganiban A.T. . ( 1988;). Retroviral gag gene amber codon suppression is caused by an intrinsic cis-acting component of the viral mRNA. J Virol 62: 3574–3580 [PubMed].
    [Google Scholar]
  51. Parkin N.T. , Chamorro M. , Varmus H.E. . ( 1992;). Human immunodeficiency virus type 1 gag–pol frameshifting is dependent on downstream mRNA secondary structure: demonstration by expression in vivo. J Virol 66: 5147–5151 [PubMed].
    [Google Scholar]
  52. Plant E.P. , Dinman J.D. . ( 2008;). The role of programmed-1 ribosomal frameshifting in coronavirus propagation. Front Biosci 13: 4873–4881 [CrossRef] [PubMed].
    [Google Scholar]
  53. Rein A. , Levin J.G. . ( 1992;). Readthrough suppression in the mammalian type C retroviruses and what it has taught us. New Biol 4: 283–289 [PubMed].
    [Google Scholar]
  54. Rowe S.M. , Clancy J.P. . ( 2009;). Pharmaceuticals targeting nonsense mutations in genetic diseases: progress in development. BioDrugs 23: 165–174 [CrossRef] [PubMed].
    [Google Scholar]
  55. Sermet-Gaudelus I. , Boeck K.D. , Casimir G.J. , Vermeulen F. , Leal T. , Mogenet A. , Roussel D. , Fritsch J. , Hanssens L. , other authors . ( 2010;). Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis. Am J Respir Crit Care Med 182: 1262–1272 [CrossRef] [PubMed].
    [Google Scholar]
  56. Shehu-Xhilaga M. , Crowe S.M. , Mak J. . ( 2001;). Maintenance of the Gag/Gag–Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity. J Virol 75: 1834–1841 [CrossRef] [PubMed].
    [Google Scholar]
  57. Southern P.J. , Berg P. . ( 1982;). Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet 1: 327–341 [PubMed].
    [Google Scholar]
  58. Welch E.M. , Barton E.R. , Zhuo J. , Tomizawa Y. , Friesen W.J. , Trifillis P. , Paushkin S. , Patel M. , Trotta C.R. , other authors . ( 2007;). PTC124 targets genetic disorders caused by nonsense mutations. Nature 447: 87–91 [CrossRef] [PubMed].
    [Google Scholar]
  59. Wills N.M. , Gesteland R.F. , Atkins J.F. . ( 1991;). Evidence that a downstream pseudoknot is required for translational read-through of the Moloney murine leukemia virus gag stop codon. Proc Natl Acad Sci U S A 88: 6991–6995 [CrossRef] [PubMed].
    [Google Scholar]
  60. Wilschanski M. , Miller L.L. , Shoseyov D. , Blau H. , Rivlin J. , Aviram M. , Cohen M. , Armoni S. , Yaakov Y. , other authors . ( 2011;). Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis. Eur Respir J 38: 59–69 [CrossRef] [PubMed].
    [Google Scholar]
  61. Wilson W. , Braddock M. , Adams S.E. , Rathjen P.D. , Kingsman S.M. , Kingsman A.J. . ( 1988;). HIV expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems. Cell 55: 1159–1169 [CrossRef] [PubMed].
    [Google Scholar]
  62. Xie Y. , Dix A.V. , Tor Y. . ( 2010;). Antibiotic selectivity for prokaryotic vs. eukaryotic decoding sites. Chem Commun (Camb) 46: 5542–5544 [CrossRef] [PubMed].
    [Google Scholar]
  63. Yoshinaka Y. , Katoh I. , Copeland T.D. , Oroszlan S. . ( 1985a;). Murine leukemia virus protease is encoded by the gagpol gene and is synthesized through suppression of an amber termination codon. Proc Natl Acad Sci U S A 82: 1618–1622 [CrossRef] [PubMed].
    [Google Scholar]
  64. Yoshinaka Y. , Katoh I. , Copeland T.D. , Oroszlan S. . ( 1985b;). Translational readthrough of an amber termination codon during synthesis of feline leukemia virus protease. J Virol 55: 870–873 [PubMed].
    [Google Scholar]
  65. Zaher H.S. , Green R. . ( 2009;). Fidelity at the molecular level: lessons from protein synthesis. Cell 136: 746–762 [CrossRef] [PubMed].
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000284
Loading
/content/journal/jgv/10.1099/jgv.0.000284
Loading

Data & Media loading...

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error