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Abstract

We have previously documented the inhibitory activity of RNA aptamers to the RNA-dependent RNA polymerase of foot-and-mouth disease virus (3D). Here we report their modification and use with a subgenomic replicon incorporating GFP (pGFP-PAC replicon), allowing replication to be monitored and quantified in real-time. GFP expression in transfected BHK-21 cells reached a maximum at approximately 8 h post-transfection, at which time change in morphology of the cells was consistent with a virus-induced cytopathic effect. However, transfection of replicon-bearing cells with a 3D aptamer RNA resulted in inhibition of GFP expression and maintenance of normal cell morphology, whereas a control aptamer RNA had little effect. The inhibition was correlated with a reduction in 3D (detected by immunoblotting) and shown to be dose dependent. The 3D aptamers appeared to be more effective than 2′--methylcytidine (2′CMC). Aptamers to components of the replication complex are therefore useful molecular tools for studying viral replication and also have potential as diagnostic molecules in the future.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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2014-12-01
2024-03-28
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References

  1. Alexandersen S., Zhang Z., Donaldson A. I. 2002; Aspects of the persistence of foot-and-mouth disease virus in animals – the carrier problem. Microbes Infect 4:1099–1110 [View Article][PubMed]
    [Google Scholar]
  2. Belsham G. J., Brangwyn J. K. 1990; A region of the 5′ noncoding region of foot-and-mouth disease virus RNA directs efficient internal initiation of protein synthesis within cells: involvement with the role of L protease in translational control. J Virol 64:5389–5395[PubMed]
    [Google Scholar]
  3. Belyaeva T. A., Nicol C., Cesur Ö., Travé G., Blair G. E., Stonehouse N. J. 2014; An RNA aptamer targets the PDZ-binding motif of the HPV16 E6 oncoprotein. Cancers (Basel) 6:1553–1569 [View Article][PubMed]
    [Google Scholar]
  4. Bentham M., Holmes K., Forrest S., Rowlands D. J., Stonehouse N. J. 2012; Formation of higher-order foot-and-mouth disease virus 3Dpol complexes is dependent on elongation activity. J Virol 86:2371–2374 [View Article][PubMed]
    [Google Scholar]
  5. Brody E. N., Gold L. 2000; Aptamers as therapeutic and diagnostic agents. J Biotechnol 74:5–13[PubMed]
    [Google Scholar]
  6. Doble R., McDermott M. F., Cesur O., Stonehouse N. J., Wittmann M. 2014; IL-17A RNA aptamer: possible therapeutic potential in some cells, more than we bargained for in others?. J Invest Dermatol 134:852–855 [View Article][PubMed]
    [Google Scholar]
  7. Domingo E., Holland J. J. 1997; RNA virus mutations and fitness for survival. Annu Rev Microbiol 51:151–178 [View Article][PubMed]
    [Google Scholar]
  8. Domingo E., Baranowski E., Escarmís C., Sobrino F. 2002; Foot-and-mouth disease virus. Comp Immunol Microbiol Infect Dis 25:297–308 [View Article][PubMed]
    [Google Scholar]
  9. Ellingham M., Bunka D. H. J., Rowlands D. J., Stonehouse N. J. 2006; Selection and characterization of RNA aptamers to the RNA-dependent RNA polymerase from foot-and-mouth disease virus. RNA 12:1970–1979 [View Article][PubMed]
    [Google Scholar]
  10. Ferrer-Orta C., Arias A., Perez-Luque R., Escarmís C., Domingo E., Verdaguer N. 2004; Structure of foot-and-mouth disease virus RNA-dependent RNA polymerase and its complex with a template-primer RNA. J Biol Chem 279:47212–47221 [View Article][PubMed]
    [Google Scholar]
  11. Ferrer-Orta C., Arias A., Escarmís C., Verdaguer N. 2006; A comparison of viral RNA-dependent RNA polymerases. Curr Opin Struct Biol 16:27–34 [View Article][PubMed]
    [Google Scholar]
  12. Ferrer-Orta C., Agudo R. R., Domingo E., Verdaguer N. 2009; Structural insights into replication initiation and elongation processes by the FMDV RNA-dependent RNA polymerase. Curr Opin Struct Biol 19:752–758 [View Article][PubMed]
    [Google Scholar]
  13. Green L., Waugh S., Binkley J. P., Hostomska Z., Hostomsky Z., Tuerk C. 1995; Comprehensive chemical modification interference and nucleotide substitution analysis of an RNA pseudoknot inhibitor to HIV-1 reverse transcriptase. J Mol Biol 247:60–68 [View Article][PubMed]
    [Google Scholar]
  14. Jamal S. M., Belsham G. J. 2013; Foot-and-mouth disease: past, present and future. Vet Res 44:116 [View Article][PubMed]
    [Google Scholar]
  15. Licursi M., Christian S. L., Pongnopparat T., Hirasawa K. 2011; In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression. Gene Ther 18:631–636 [View Article][PubMed]
    [Google Scholar]
  16. Lohmann V., Körner F., Koch J., Herian U., Theilmann L., Bartenschlager R. 1999; Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110–113 [View Article][PubMed]
    [Google Scholar]
  17. McInerney G. M., King A. M., Ross-Smith N., Belsham G. J. 2000; Replication-competent foot-and-mouth disease virus RNAs lacking capsid coding sequences. J Gen Virol 81:1699–1702[PubMed]
    [Google Scholar]
  18. Nayak A., Goodfellow I. G., Belsham G. J. 2005; Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3Dpol) in vitro. J Virol 79:7698–7706 [View Article][PubMed]
    [Google Scholar]
  19. Ng E. W. M., Shima D. T., Calias P., Cunningham E. T. Jr, Guyer D. R., Adamis A. P. 2006; Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov 5:123–132 [View Article][PubMed]
    [Google Scholar]
  20. Osborne S. E., Ellington A. D. 1997; Nucleic acid selection and the challenge of combinatorial chemistry. Chem Rev 97:349–370 [View Article][PubMed]
    [Google Scholar]
  21. Ruckman J., Green L. S., Beeson J., Waugh S., Gillette W. L., Henninger D. D., Claesson-Welsh L., Janjić N. 1998; 2′-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J Biol Chem 273:20556–20567 [View Article][PubMed]
    [Google Scholar]
  22. Stuyver L. J., McBrayer T. R., Tharnish P. M., Clark J., Hollecker L., Lostia S., Nachman T., Grier J., Bennett M. A. other authors 2006; Inhibition of hepatitis C replicon RNA synthesis by β-d-2′-deoxy-2′-fluoro-2′-C-methylcytidine: a specific inhibitor of hepatitis C virus replication. Antivir Chem Chemother 17:79–87[PubMed] [CrossRef]
    [Google Scholar]
  23. Thompson D., Muriel P., Russell D., Osborne P., Bromley A., Rowland M., Creigh-Tyte S., Brown C. 2002; Economic costs of the foot and mouth disease outbreak in the United Kingdom in 2001. Rev Sci Tech 21:675–687[PubMed]
    [Google Scholar]
  24. Tulloch F., Pathania U., Luke G. A., Nicholson J., Stonehouse N. J., Rowlands D. J., Jackson T., Tuthill T., Haas J., Lamond. A., Ryan. M. 2014; Quantification of FMDV replicon-derived RNA replication by live-cell fluorescence imaging. J Virol Methods (in press)
    [Google Scholar]
  25. Yang P. C., Chu R. M., Chung W. B., Sung H. T. 1999; Epidemiological characteristics and financial costs of the 1997 foot-and-mouth disease epidemic in Taiwan. Vet Rec 145:731–734[PubMed]
    [Google Scholar]
  26. Yazbeck D. R., Min K. L., Damha M. J. 2002; Molecular requirements for degradation of a modified sense RNA strand by Escherichia coli ribonuclease H1. Nucleic Acids Res 30:3015–3025 [View Article][PubMed]
    [Google Scholar]
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