1887

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Volume 86, Issue 11

Antiviral activity of morpholino oligomers designed to block various aspects of amplification in cell culture Free

Abstract

The antiviral efficacy of ten antisense phosphorodiamidate morpholino oligomers (PMOs) directed against (EAV), a nidovirus belonging to the family , was evaluated in mammalian (Vero-E6) cells. Peptide-conjugated PMOs (P-PMOs) supplied in cell culture medium at micromolar concentrations were efficiently taken up by Vero-E6 cells and were minimally cytotoxic. The P-PMOs were designed to base pair to RNA sequences involved in different aspects of EAV amplification: genome replication, subgenomic mRNA synthesis, and translation of genome and subgenomic mRNAs. A novel recombinant EAV, expressing green fluorescent protein as part of its replicase polyproteins, was used to facilitate drug screening. A moderate reduction of EAV amplification was observed with relatively high concentrations of P-PMOs designed to anneal to the 3′-terminal regions of the viral genome or antigenome. To determine if the synthesis of subgenomic mRNAs could be specifically reduced, transcription-regulating sequences essential for their production, but not for the production of genomic RNA, were targeted, but these P-PMOs were found to be ineffective at transcription interference. In contrast, all four P-PMOs designed to base pair with targets in the genomic 5′ untranslated region markedly reduced virus amplification in a sequence-specific and dose-responsive manner. At concentrations in the low micromolar range, some of the P-PMOs tested completely inhibited virus amplification. translation assays showed that these P-PMOs were potent inhibitors of translation. The data suggest that these compounds could be useful as reagents for exploring the molecular mechanics of nidovirus translation and have anti-EAV potential at relatively low concentrations.

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2005-11-01
2024-04-19
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References

  1. Albina E. 1997; Epidemiology of porcine reproductive and respiratory syndrome (PRRS): an overview. Vet Microbiol 55:309–316 [CrossRef]
     [Google Scholar]
  2. Boguslavsky D., Ierusalimsky V., Malyshev A., Balaban P., Belyavsky A. 2003; Selective blockade of gene expression in a single identified snail neuron. Neuroscience 119:15–18 [CrossRef]
     [Google Scholar]
  3. Cook J. 2002; In Poultry Diseases . , 5th edn. pp  298–306 Edited by Jordan F., Pattison M., Alezander D., Faragher T. London: W. B. Saunders;
  4. Deas T. S., Binduga-Gajewska I., Tilgner M. 7 other authors 2005; Inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and RNA replication. J Virol 79:4599–4609 [CrossRef]
     [Google Scholar]
  5. den Boon J. A., Kleijnen M. F., Spaan W. J. M., Snijder E. J. 1996; Equine arteritis virus subgenomic mRNA synthesis: analysis of leader-body junctions and replicative-form RNAs. J Virol 70:4291–4298
     [Google Scholar]
  6. González J. M., Gomez-Puertas P., Cavanagh D., Gorbalenya A. E., Enjuanes L. 2003; A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae . Arch Virol 148:2207–2235 [CrossRef]
     [Google Scholar]
  7. Heasman J. 2002; Morpholino oligos: making sense of antisense?. Dev Biol 243:209–214 [CrossRef]
     [Google Scholar]
  8. Jubin R., Vantuno N. E., Kieft J. S., Murray M. G., Doudna J. A., Lau J. Y., Baroudy B. M. 2000; Hepatitis C virus internal ribosome entry site (IRES) stem loop IIId contains a phylogenetically conserved GGG triplet essential for translation and IRES folding. J Virol 74:10430–10437 [CrossRef]
     [Google Scholar]
  9. Kinney R. M., Huang C. Y., Rose B. C., Kroeker A. D., Dreher T. W., Iversen P. L., Stein D. A. 2005; Inhibition of dengue virus serotypes 1 to 4 in vero cell cultures with morpholino oligomers. J Virol 79:5116–5128 [CrossRef]
     [Google Scholar]
  10. Liu Y., Sinha S., Owens G. 2003; A transforming growth factor- β control element required for SM α -actin expression in vivo also partially mediates GKLF-dependent transcriptional repression. J Biol Chem 278:48004–48011 [CrossRef]
     [Google Scholar]
  11. Makela M. J., Puhakka T., Ruuskanen O., Leinonen M., Saikku P., Kimpimaki M., Blomqvist S., Hyypia T., Arstila P. 1998; Viruses and bacteria in the etiology of the common cold. J Clin Microbiol 36:539–542
     [Google Scholar]
  12. McCaffrey A. P., Meuse L., Karimi M., Contag C. H., Kay M. A. 2003; A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice. Hepatology 38:503–508
     [Google Scholar]
  13. Molenkamp R., Greve S., Spaan W. J. M., Snijder E. J. 2000a; Efficient homologous RNA recombination and requirement for an open reading frame during replication of equine arteritis virus defective interfering RNAs. J Virol 74:9062–9070 [CrossRef]
     [Google Scholar]
  14. Molenkamp R., Rozier B. C., Greve S., Spaan W. J. M., Snijder E. J. 2000b; Isolation and characterization of an arterivirus defective interfering RNA genome. J Virol 74:3156–3165 [CrossRef]
     [Google Scholar]
  15. Molenkamp R., van Tol H., Rozier B. C., van der Meer Y., Spaan W. J. M., Snijder E. J. 2000c; The arterivirus replicase is the only viral protein required for genome replication and subgenomic mRNA transcription. J Gen Virol 81:2491–2496
     [Google Scholar]
  16. Moulton H. M., Hase M. C., Smith K. M., Iversen P. L. 2003; HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers. Antisense Nucleic Acid Drug Dev 13:31–43 [CrossRef]
     [Google Scholar]
  17. Moulton H. M., Nelson M. H., Hatlevig S. A., Reddy M. T., Iversen P. L. 2004; Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides. Bioconjug Chem 15:290–299 [CrossRef]
     [Google Scholar]
  18. Muto E., Tabata Y., Taneda T., Aoki Y., Muto A., Arai K., Watanabe S. 2004; Identification and characterization of Veph, a novel gene encoding a PH domain-containing protein expressed in the developing central nervous system of vertebrates. Biochimie 86:523–531 [CrossRef]
     [Google Scholar]
  19. Nasevicius A., Ekker S. C. 2000; Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet 26:216–220 [CrossRef]
     [Google Scholar]
  20. Neuman B. W., Stein D. A., Kroeker A. D., Paulino A. D., Moulton H. M., Iversen P. L., Buchmeier M. J. 2004; Antisense morpholino-oligomers directed against the 5′ end of the genome inhibit coronavirus proliferation and growth. J Virol 78:5891–5899 [CrossRef]
     [Google Scholar]
  21. Panavas T., Pogany J., Nagy P. D. 2002; Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase. Virology 296:263–274 [CrossRef]
     [Google Scholar]
  22. Pasternak A. O., van den Born E., Spaan W. J. M., Snijder E. J. 2001; Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis. EMBO J 20:7220–7228 [CrossRef]
     [Google Scholar]
  23. Pasternak A. O., van den Born E., Spaan W. J. M., Snijder E. J. 2003; The stability of the duplex between sense and antisense transcription-regulating sequences is a crucial factor in arterivirus subgenomic mRNA synthesis. J Virol 77:1175–1183 [CrossRef]
     [Google Scholar]
  24. Pedersen K. W., van der Meer Y., Roos N., Snijder E. J. 1999; Open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex. J Virol 73:2016–2026
     [Google Scholar]
  25. Peiris J. S., Lai S. T., Poon L. L. 13 other authors; 2003; Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361:1319–1325 [CrossRef]
     [Google Scholar]
  26. Saif L. J., Redman D. R., Brock K. V., Kohler E. M., Heckert R. A. 1988; Winter dysentery in adult dairy cattle: detection of coronavirus in the faeces. Vet Rec 123:300–301 [CrossRef]
     [Google Scholar]
  27. Sawicki S. G., Sawicki D. L. 1995; Coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands. Adv Exp Med Biol 380:499–506
     [Google Scholar]
  28. Snijder E. J., Meulenberg J. J. M. 2001; In Fields Virology . , 4th edn. pp  1205–1220 Edited by Knipe D. M., Howley P. M. Philadelphia, PA: Lippincott Williams & Wilkins;
  29. Summerton J. 1999; Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1489141–158 [CrossRef]
     [Google Scholar]
  30. Summerton J., Weller D. 1997; Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 7:187–195 [CrossRef]
     [Google Scholar]
  31. Tijms M. A., van Dinten L. C., Gorbalenya A. E., Snijder E. J. 2001; A zinc finger-containing papain-like protease couples subgenomic mRNA synthesis to genome translation in a positive-stranded RNA virus. Proc Natl Acad Sci U S A 98:1889–1894 [CrossRef]
     [Google Scholar]
  32. Tortorici M. A., Broering T. J., Nibert M. L., Patton J. T. 2003; Template recognition and formation of initiation complexes by the replicase of a segmented double-stranded RNA virus. J Biol Chem 278:32673–32682 [CrossRef]
     [Google Scholar]
  33. van den Born E., Gultyaev A. P., Snijder E. J. 2004; Secondary structure and function of the 5′-proximal region of the equine arteritis virus RNA genome. RNA 10:424–437 [CrossRef]
     [Google Scholar]
  34. van den Born E., Posthuma C. C., Gultyaev A. P., Snijder E. J. 2005; Discontinuous subgenomic RNA synthesis in arteriviruses is guided by an RNA hairpin structure located in the genomic leader region. J Virol 79:6312–6324 [CrossRef]
     [Google Scholar]
  35. van Marle G., Dobbe J. C., Gultyaev A. P., Luytjes W., Spaan W. J. M., Snijder E. J. 1999; Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences. Proc Natl Acad Sci U S A 96:12056–12061 [CrossRef]
     [Google Scholar]
  36. Zuniga S., Sola I., Alonso S., Enjuanes L. 2004; Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis. J Virol 78:980–994 [CrossRef]
     [Google Scholar]
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Antiviral activity of morpholino oligomers designed to block various aspects of Equine arteritis virus amplification in cell culture
J. Gen. Virol. 86, 3081 (2005); https://doi.org/10.1099/vir.0.81158-0
/content/journal/jgv/10.1099/vir.0.81158-0
/content/journal/jgv/10.1099/vir.0.81158-0
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