1887

Journal of General Virology header logo

Volume 90, Issue 10

Inhibition of coxsackievirus B3 and related enteroviruses by antiviral short interfering RNA pools produced using 6 RNA-dependent RNA polymerase Free

Abstract

Coxsackievirus B3 (CBV3) is a member of the human enterovirus B species and a common human pathogen. Even though much is known about the enteroviral life cycle, no specific drugs are available to treat enterovirus infections. RNA interference (RNAi) has evolved to be an important tool for antiviral experimental therapies and gene function studies. We describe here a novel approach for RNAi against CBVs by using a short interfering (siRNA) pool covering 3.5 kb of CBV3 genomic sequence. The RNA-dependent RNA polymerase (RdRP) of bacteriophage 6 was used to synthesize long double-stranded RNA (dsRNA) from a cloned region (nt 3837–7399) of the CBV3 genome. The dsRNA was cleaved using Dicer, purified and introduced to cells by transfection. The siRNA pool synthesized using the 6 RdRP (6–siRNAs) was considerably more effective than single-site siRNAs. The 6–siRNA pool also inhibited replication of other enterovirus B species, such as coxsackievirus B4 and coxsackievirus A9.

  • Received:
  • Accepted:
  • Published Online:
SGM
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.011338-0
2009-10-01
2023-06-10
Loading full text...

Full text loading...

/deliver/fulltext/jgv/90/10/2468.html?itemId=/content/journal/jgv/10.1099/vir.0.011338-0&mimeType=html&fmt=ahah

References

  1. Aalto A. P., Sarin L. P., van Dijk A. A., Saarma M. M., Poranen M., Arumäe U., Bamford D. H. 2007; Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage φ 6 RNA-dependent RNA polymerase. RNA 13:422–429 [CrossRef]
     [Google Scholar]
  2. Ahn J., Jun E., Lee H., Yoon S., Kim D., Joo C., Kim Y., Lee H. 2005; A small interfering RNA targeting coxsackievirus B3 protects permissive HeLa cells from viral challenge. J Virol 79:8620–8624 [CrossRef]
     [Google Scholar]
  3. Bowles N. E., Richardson P. J., Olsen E. G., Archard L. C. 1986; Detection of Coxsackie-B-virus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet 1:1120–1123
     [Google Scholar]
  4. Buchholz F., Kittler R., Slabicki M., Theis M. 2006; Enzymatically prepared RNAi libraries. Nat Methods 3:696–700 [CrossRef]
     [Google Scholar]
  5. Carthew R. W., Sontheimer E. J. 2009; Origins and mechanisms of miRNAs and siRNAs. Cell 136:642–655 [CrossRef]
     [Google Scholar]
  6. Castanotto D., Rossi J. 2009; The promises and pitfalls of RNA-interference-based therapeutics. Nature 457:426–433 [CrossRef]
     [Google Scholar]
  7. Fechner H., Sipo I., Westermann D., Pinkert S., Wang X., Suckau L., Kurreck J., Zeichhardt H., Müller O. other authors 2008; Cardiac-targeted RNA interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3 cardiomyopathy. J Mol Med 86:987–997 [CrossRef]
     [Google Scholar]
  8. Gitlin L., Karelsky S., Andino R. 2002; Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 418:430–434 [CrossRef]
     [Google Scholar]
  9. Haasnoot J., Westerhout E., Berkhout B. 2007; RNA interference against viruses: strike and counterstrike. Nat Biotechnol 25:1435–1443 [CrossRef]
     [Google Scholar]
  10. Harvala H. 2003; Molecular pathogenesis of Coxsackievirus infections. The specific role of the RGD motif in CAV9 . PhD dissertation University of Turku; Turku, Finland: Annales Universitatis Turkuensis D564
  11. Hyöty H., Taylor K. 2002; The role of viruses in human diabetes. Diabetologia 45:1353–1361 [CrossRef]
     [Google Scholar]
  12. Judge A., MacLachlan I. 2008; Overcoming the innate immune response to small interfering RNA. Hum Gene Ther 19:111–124 [CrossRef]
     [Google Scholar]
  13. Kim D. H., Longo M., Han Y., Lundberg P., Cantin E., Rossi J. J. 2004; Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat Biotechnol 22:321–325 [CrossRef]
     [Google Scholar]
  14. Kim Y. J., Ahn J., Jeung S. Y., Kim D. S., Na H. N., Cho Y. J., Yun S. H., Jee Y., Jeon E. S. other authors 2008; Recombinant lentivirus-delivered short hairpin RNAs targeted to conserved coxsackievirus sequences protect against viral myocarditis and improve survival rate in an animal model. Virus Genes 36:141–146 [CrossRef]
     [Google Scholar]
  15. Klingel K., McManus B., Kandolf R. 1995; Enterovirus-infected immune cells of spleen and lymph nodes in the murine model of chronic myocarditis: a role in pathogenesis?. Eur Heart J 16:Suppl. O42–45
     [Google Scholar]
  16. Krönke J., Kittler R., Buchholz F., Windisch M., Pietschmann T., Bartenschlager R., Frese M. 2004; Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs. J Virol 78:3436–3446 [CrossRef]
     [Google Scholar]
  17. Lee H., Ahn J., Jee Y., Seo I., Jeon E., Jeon E., Joo C., Kim Y., Lee H. 2007; Universal and mutation-resistant anti-enteroviral activity: potency of small interfering RNA complementary to the conserved cis -acting replication element within the enterovirus coding region. J Gen Virol 88:2003–2012 [CrossRef]
     [Google Scholar]
  18. Leslie K., Blay R., Haisch C., Lodge A., Weller A., Huber S. 1989; Clinical and experimental aspects of viral myocarditis. Clin Microbiol Rev 2:191–203
     [Google Scholar]
  19. Lönnrot M., Sjöroos M., Salminen K., Maaronen M., Hyypiä T., Hyöty H. 1999; Diagnosis of enterovirus and rhinovirus infections by RT-PCR and time-resolved fluorometry with lanthanide chelate labeled probes. J Med Virol 59:378–384 [CrossRef]
     [Google Scholar]
  20. López-Fraga M., Wright N., Jiménez A. 2008; RNA interference-based therapeutics: new strategies to fight infectious disease. Infect Disord Drug Targets 8:262–273 [CrossRef]
     [Google Scholar]
  21. Lu W. W., Hsu Y. Y., Yang J. Y., Kung S. H. 2004; Selective inhibition of enterovirus 71 replication by short hairpin RNAs. Biochem Biophys Res Commun 325:494–499 [CrossRef]
     [Google Scholar]
  22. Makeyev E. V., Bamford D. H. 2000a; Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage φ 6. EMBO J 19:124–133 [CrossRef]
     [Google Scholar]
  23. Makeyev E. V., Bamford D. H. 2000b; The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism. EMBO J 19:6275–6284 [CrossRef]
     [Google Scholar]
  24. Makeyev E. V., Bamford D. H. 2001; Primer-independent RNA sequencing with bacteriophage φ 6 RNA polymerase and chain terminators. RNA 7:774–781 [CrossRef]
     [Google Scholar]
  25. McManus B. M., Chow L. H., Radio S. J., Tracy S. M., Beck M. A., Chapman N. M., Klingel K., Kandolf R. 1991; Progress and challenges in the pathological diagnosis of myocarditis. Eur Heart J 12:Suppl. D18–21
     [Google Scholar]
  26. Merl S., Wessely R. 2007; Anti-coxsackieviral efficacy of RNA interference is highly dependent on genomic target selection and emergence of escape mutants. Oligonucleotides 17:44–53 [CrossRef]
     [Google Scholar]
  27. Merl S., Michaelis C., Jaschke B., Vorpahl M., Seidl S., Wessely R. 2005; Targeting 2A protease by RNA interference attenuates coxsackieviral cytopathogenicity and promotes survival in highly susceptible mice. Circulation 111:1583–1592 [CrossRef]
     [Google Scholar]
  28. Mindich L., Nemhauser I., Gottlieb P., Romantschuk M., Carton J., Frucht S., Strassman J., Bamford D., Kalkkinen N. 1988; Nucleotide sequence of the large double-stranded RNA segment of bacteriophage φ 6: genes specifying the viral replicase and transcriptase. J Virol 62:1180–1185
     [Google Scholar]
  29. Rossi J. J. 2009; Innate immunity confounds the clinical efficacy of small interfering RNAs (siRNAs). Gene Ther 16:579–580 [CrossRef]
     [Google Scholar]
  30. Schubert S., Grunert H., Zeichhardt H., Werk D., Erdmann V., Kurreck J. 2005; Maintaining inhibition: siRNA double expression vectors against coxsackieviral RNAs. J Mol Biol 346:457–465 [CrossRef]
     [Google Scholar]
  31. Schubert S., Rothe D., Werk D., Grunert H., Zeichhardt H., Erdmann V., Kurreck J. 2007; Strand-specific silencing of a picornavirus by RNA interference: evidence for the superiority of plus-strand specific siRNAs. Antiviral Res 73:197–205 [CrossRef]
     [Google Scholar]
  32. Sim A. C., Luhur A., Tan T. M., Chow V. T., Poh C. L. 2005; RNA interference against enterovirus 71 infection. Virology 341:72–79 [CrossRef]
     [Google Scholar]
  33. Sledz C. A., Holko M., de Veer M. J., Silverman R. H., Williams B. R. 2003; Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5:834–839 [CrossRef]
     [Google Scholar]
  34. Tan E. L., Tan T. M., Chow V. T., Poh C. L. 2007a; Enhanced potency and efficacy of 29-mer shRNAs in inhibition of Enterovirus 71. Antiviral Res 74:9–15 [CrossRef]
     [Google Scholar]
  35. Tan E. L., Tan T. M., Tak Kwong Chow V., Poh C. L. 2007b; Inhibition of enterovirus 71 in virus-infected mice by RNA interference. Mol Ther 15:1931–1938 [CrossRef]
     [Google Scholar]
  36. Tan E. L., Marcus K. F., Poh C. L. 2008; Development of RNA interference (RNAi) as potential antiviral strategy against enterovirus 70. J Med Virol 80:1025–1032 [CrossRef]
     [Google Scholar]
  37. Vidaver A. K., Koski R. K., Van Etten J. L. 1973; Bacteriophage φ 6: a lipid-containing virus of Pseudomonas phaseolicola . J Virol 11:799–805
     [Google Scholar]
  38. Vuorinen T., Vainionpää R., Heino J., Hyypiä T. 1999; Enterovirus receptors and virus replication in human leukocytes. J Gen Virol 80:921–927
     [Google Scholar]
  39. Werk D., Schubert S., Lindig V., Grunert H., Zeichhardt H., Erdmann V., Kurreck J. 2005; Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor. Biol Chem 386:857–863
     [Google Scholar]
  40. Woodruff J. F. 1980; Viral myocarditis. A review. Am J Pathol 101:425–484
     [Google Scholar]
  41. Wu Z., Gao Y., Sun L., Tien P., Jin Q. 2008; Quick identification of effective small interfering RNAs that inhibit the replication of coxsackievirus A16. Antiviral Res 80:295–301 [CrossRef]
     [Google Scholar]
  42. Yuan J., Cheung P., Zhang H., Chau D., Yang D. 2005; Inhibition of coxsackievirus B3 replication by small interfering RNAs requires perfect sequence match in the central region of the viral positive strand. J Virol 79:2151–2159 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.011338-0
Loading
Inhibition of coxsackievirus B3 and related enteroviruses by antiviral short interfering RNA pools produced using φ6 RNA-dependent RNA polymerase
J. Gen. Virol. 90, 2468 (2009); https://doi.org/10.1099/vir.0.011338-0
/content/journal/jgv/10.1099/vir.0.011338-0
/content/journal/jgv/10.1099/vir.0.011338-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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