1887

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.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.067751-0
2014-12-01
2019-11-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/95/12/2649.html?itemId=/content/journal/jgv/10.1099/vir.0.067751-0&mimeType=html&fmt=ahah

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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
  7. Domingo E. , Holland J. J. . ( 1997; ). RNA virus mutations and fitness for survival. . Annu Rev Microbiol 51:, 151–178. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
  14. Jamal S. M. , Belsham G. J. . ( 2013; ). Foot-and-mouth disease: past, present and future. . Vet Res 44:, 116. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [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. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.067751-0
Loading
/content/journal/jgv/10.1099/vir.0.067751-0
Loading

Data & Media loading...

Supplements

Supplementary Data 

Supplementary Data 

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