Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Inhibition of the Replication of Hepatitis C Virus Replicon with Nuclease-Resistant RNA Aptamers

  • Shin, Kyung-Sook (Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University) ;
  • Lim, Jong-Hoon (Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University) ;
  • Kim, Jung-Hye (Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University) ;
  • Myung, Hee-Joon (Department of Bioscience and Biotechnology, Hankook University of Foreign Studies) ;
  • Lee, Seong-Wook (Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University)
  • Published : 2006.10.31
Download PDF
⟨ Previous Next ⟩

Abstract

Hepatitis C virus (HCV)-encoded nonstructural protein 5B (NS5B) possesses RNA-dependent RNA polymerase activity, which is considered essential for viral proliferation. Thus, HCV NS5B is a good therapeutic target protein for the development of anti-HCV agents. In this study, we isolated two different kinds of nuclease-resistant RNA aptamers with 2'-fluoro pyrimidines against the HCV NS5B from a combinatorial RNA library with 40 nucleotide random sequences, using SELEX technology. The isolated RNA aptamers were observed to specifically and avidly bind the HCV NS5B with an apparent $K_d$ of 5 nM and 18 nM, respectively, in contrast with the original RNA library that hardly bound the target protein. Moreover, these aptamers could partially inhibit RNA synthesis of the HCV subgenomic replicon when transfected into Huh-7 hepatoma cell lines. These results suggest that the RNA aptamers selected in vitro could be useful not only as therapeutic agents of HCV infection but also as a powerful tool for the study of the HCV RNA-dependent RNA polymerase mechanism.

Keywords

References

  1. Bae, S.-J., J.-H. Oum, S. Sharma, J. Park, and S.-W. Lee. 2002. In vitro selection of specific RNA inhibitors of NFATc. Biochem. Biophys. Res. Commun. 298: 486-492 https://doi.org/10.1016/S0006-291X(02)02490-7
  2. Behrens, S.-E., L. Tomei, and R. De Francesco. 1996. Identification and properties of the RNA-dependent RNA polymerase of the hepatitis C virus. EMBO J. 15: 12-22
  3. Biroccio, A., J. Hamm, I. Incitti, R. De Francesco, and L. Tomei. 2002. Selection of RNA aptamers that are specific and high-affinity ligands of the hepatitis C virus RNA-dependent RNA polymerase. J. Virol. 76: 3688-3696 https://doi.org/10.1128/JVI.76.8.3688-3696.2002
  4. Burgstaller, P., A. Girod, and M. Blind. 2002. Aptamers as tools for target prioritization and lead identification. Drug Discov. Today 7: 1221-1228 https://doi.org/10.1016/S1359-6446(02)02522-9
  5. Cload, S. T., T. G. McCauley, A. D. Keefe, J. M. Healy, and C. Wilson. 2006. Properties of therapeutic aptamers, pp. 363-416. In S. Klussmann (ed.). The Aptamer Handbook. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
  6. De Francesco, R. 1999. Molecular virology of the hepatitis C virus. J. Hepatol. 312: 47-53
  7. Ellington, A. D. and J. W. Szostak. 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346: 818-822 https://doi.org/10.1038/346818a0
  8. Gold, L., P. Allen, J. Binkley, D. Brown, D. Schneider, S. R. Eddy, C. Tuerk, L. Green, S. Macdougal, and D. Tasset. 1993. RNA: The shape of things to come, pp. 497-510. In: R. F. Gestelend and J. F. Atkins (eds.). The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y
  9. Hwang, B., K. Han, and S. W. Lee. 2003. Prevention of passively transferred experimental autoimmune myasthenia gravis by an in vitro selected RNA aptamer. FEBS Lett. 548: 85-89 https://doi.org/10.1016/S0014-5793(03)00745-2
  10. Hwang, B., J. S. Cho, H. J. Yeo, J.-H. Kim, K. M. Chung, K. Han, S. K. Jang, and S.-W. Lee. 2004. Isolation of specific and high-affinity RNA aptamers against NS3 helicase domain of hepatitis C virus. RNA 10: 1277-1290 https://doi.org/10.1261/rna.7100904
  11. Hwang, B. and S.-W. Lee. 2005. Analysis of in vivo interaction of HCV NS3 protein and specific RNA aptamer with yeast three-hybrid system. J. Microbiol. Biotechnol. 15: 660-664
  12. Krieger, N., V. Lohmann, and R. Bartenschlager. 2001. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J. Virol. 75: 4614-4624 https://doi.org/10.1128/JVI.75.10.4614-4624.2001
  13. Kumar, P. K., K. Machida, P. T. Urvil, N. Kakiuchi, D. Vishnuvardhan, K. Shimotohno, K. Taira, and S. Nishikawa. 1997. Isolation of RNA aptamers specific to the NS3 protein of hepatitis C virus from a pool of completely random RNA. Virology 237: 270-282 https://doi.org/10.1006/viro.1997.8773
  14. Lauer, G. M. and B. D. Walker. 2001. Hepatitis C virus infection. N. Engl. J. Med. 345: 41-52 https://doi.org/10.1056/NEJM200107053450107
  15. Lee, S. W. and B. Sullenger. 1996. Isolation of a nuclease resistant decoy RNA that selectively blocks autoantibody binding to insulin receptors on human lymphocytes. J. Exp. Med. 194: 315-324
  16. Lee, S. W. and B. Sullenger. 1997. Isolation of a nuclease-resistant decoy RNA that can protect human acetylcholine receptors from myasthenic antibodies. Nature Biotechnol. 15: 41-45 https://doi.org/10.1038/nbt0197-41
  17. Lohmann, V., F. Korner, J. Koch, U. Herian, L. Theilmann, and R. Bartenschlager. 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285: 110-113 https://doi.org/10.1126/science.285.5424.110
  18. Moradpour, D., V. Brass, R. Gosert, B.Wolk, and H. Blum. 2002. Hepatitis C: Molecular virology and antiviral targets. Trends Mol. Med. 8: 476-482 https://doi.org/10.1016/S1471-4914(02)02395-X
  19. Ng, E. W. M., D. T. Shima, P. Calias, E. T. Cunningham Jr., D. R. Guyer, and A. P. Adamis. 2006. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nature Rev. Drug Discov. 5: 123-132 https://doi.org/10.1038/nrd1955
  20. Nishikawa, F., N. Kakiuchi, K. Funaji, K. Fukuda, S. Sekiya, and S. Nishikawa. 2003. Inhibition of HCV NS3 protease by RNA aptamers in cells. Nucleic Acids Res. 31: 1935-1943 https://doi.org/10.1093/nar/gkg291
  21. Pagratis, N. C., C. Bell, Y. F. Chang, S. Jennings, T. Fitzwater, D. Jellinek, and C. Dang. 1997. Potent 2'-amino, and 2'-fluoro-2'deoxyribonucleotide RNA inhibitors of keratinocyte growth factor. Nature Biotechnol. 15: 68-73 https://doi.org/10.1038/nbt0197-68
  22. Park, C.-H., Y.-H. Kee, J.-H. Lee, J.-H. Oh, J.-C. Park, and H.-J. Myung. 1999. Purification and characterization of recombinant hepatitis C virus replicase. J. Microbiol. Biotechnol. 9: 881-884
  23. Purcell, R. 1997. The hepatitis C virus: Overview. Hepatology 26(Suppl 1): S11-S14 https://doi.org/10.1002/hep.510260702
  24. Rusconi, C. P., J. D. Roberts, G. A. Pitoc, S. M. Nimjee, R. R. White, G. Jr. Quick, E. Scardino, W. P. Fay, and B. A. Sullenger. 2004. Antidote-mediated control of an anticoagulant aptamer in vivo. Nature Biotechnol. 22: 1423-1428 https://doi.org/10.1038/nbt1023
  25. Seo, H. S. and S. W. Lee. 2000. In vitro selection of the 2'-fluoro-2'-deoxyribonucleotide decoy RNA inhibitor of myasthenic autoantibodies. J. Microbiol. Biotechnol. 10: 707-713
  26. Sullenger, B. A. and E. Gilboa. 2002. Emerging clinical applications of RNA. Nature 418: 252-258 https://doi.org/10.1038/418252a
  27. Thiel, K. 2004. Oligo oligarch - the surprisingly small world of aptamers. Nat. Biotechnol. 22: 649-651 https://doi.org/10.1038/nbt0604-649
  28. Tuerk, C. and L. Gold. 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249: 505-510 https://doi.org/10.1126/science.2200121
  29. Vo, N. V., J. W. Oh, and M. M. Lai. 2003. Identification of RNA ligands that bind hepatitis C virus polymerase selectively and inhibit its RNA synthesis from the natural viral RNA templates. Virology 307: 301-316 https://doi.org/10.1016/S0042-6822(02)00095-8
  30. Zuker, M. 2003. Mfold Web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31: 3406-3415 https://doi.org/10.1093/nar/gkg595