Journal of Microbiology and Biotechnology

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

Selection and Target-Site Mapping of Peptides Inhibiting HCV NS5B Polymerase Using Phage Display

  • Kim, Min-Soo (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Park, Chan-Hee (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Lee, Jong-Ho (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Myung, Hee-Joon (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
  • Published : 2008.02.29
Download PDF
⟨ Previous Next ⟩

Abstract

A series of pep tides binding to the HCV NS5B polymerase was selected from phage display peptide libraries. A conserved motif of Ser-Arg-X-Arg/Leu was identified among the selected peptides, and Pep2 (Trp-Ser-Arg-Pro-Arg-Ser-Leu) was chosen for further characterization. The binding of Pep2 to HCV NS5B in vivo was shown by a yeast two-hybrid assay and by subcellular colocalization analysis using immunofluorescence confocal microscopy. The in vitro interaction was also confirmed by GST pulldown assay. The replication of the HCV 1b subgenomic replicon was efficiently inhibited by the presence of the peptide. By using a subtractive biopanning against Pep2, the binding site of the peptide was mapped at the pocket of Pro388 to Pro391 in the thumb subdomain of the polymerase. A yeast two-hybrid analysis using Pro388Ala and Pro391Ala mutants of NS5B confirmed the binding.

Keywords

References

  1. Adachi, T., H. Ago, N. Habuka, K. Okuda, M. Komatsu, S. Ikeda, and K. Yatsunami. 2002. The essential role of C-terminal residues in regulating the activity of hepatitis C virus RNAdependent RNA polymerase. Biochim. Biophys. Acta 1601: 38-48 https://doi.org/10.1016/S1570-9639(02)00433-8
  2. Adda, C. G., R. F. Anders, L. Tilley, and M. Foley. 2002. Random sequence libraries displayed on phage: Identification of biologically important molecules. Comb. Chem. High Throughput Screen. 5: 1-14 https://doi.org/10.2174/1386207023330561
  3. Amin, A., J. Zaccardi, S. Mullen, S. Olland, M. Orlowski, B. Feld, P. Labonte, and P. Mak. 2003. Identification of constrained peptides that bind to and preferentially inhibit the activity of the hepatitis C viral RNA-dependent RNA polymerase. Virology 15: 158-169
  4. Beaulieu, P. L. and Y. S. Tsantrizos. 2004. Inhibitors of the HCV NS5B polymerase: New hope for the treatment of hepatitis C infections. Curr. Opin. Investig. Drugs 5: 838-850
  5. Bressanelli, S., L. Tomei, A. Roussel, I. Incitti, R. L. Vitale, M. Mathieu, R. De Francesco, and F. A. Rey. 1999. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc. Natl. Acad. Sci. USA 96: 13034-13039
  6. Bressanelli, S., L. Tomei, F. A. Rey, and R. De Francesco. 2002. Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. J. Virol. 76: 3482-3492 https://doi.org/10.1128/JVI.76.7.3482-3492.2002
  7. Chan, L., O. Pereira, T. J. Reddy, S. K. Das, C. Poisson, M. Courchesne, M. Proulx, A. Siddiqui, C. G. Yannopoulos, et al. 2004. Discovery of thiophene-2-carboxylic acids as potent inhibitors of HCV NS5B polymerase and HCV subgenomic RNA replication. Part 2: Tertiary amides. Bioorg. Med. Chem. Lett. 14: 797-800 https://doi.org/10.1016/j.bmcl.2003.10.068
  8. Cohen, J. 1999. The scientific challenge of hepatitis C. Science 258: 26-30
  9. De Francesco, R. and G. Migliaccio. 2005. Challenges and successes in developing new therapies for hepatitis C. Nature 436: 953-960 https://doi.org/10.1038/nature04080
  10. Gowans, E. J., K. L. Jones, M. Bharadwaj, and C. C. Jackson. 2004. Prospects for dendritic cell vaccination in persistent infection with hepatitis C virus. J. Clin. Virol. 30: 283-290 https://doi.org/10.1016/j.jcv.2004.03.006
  11. Hantusch, B., S. Krieger, E. Untersmayr, I. Scho, R. Knittelfelder, S. Flicker, S. Spitzauer, R. Valenta, G. Boltz-Nitulescu, et al. 2004. Mapping of conformational IgE epitopes on Phl p 5a by using mimotopes from a phage display library. J. Allergy Clin. Immunol. 114: 1294-1300 https://doi.org/10.1016/j.jaci.2004.06.048
  12. 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
  13. Ichii, K., Y. Tanaka, C.-C. Yap, H. Aizaki, Y. Matsumura, and T. Miyamura. 1999. Expression of hepatitis C virus NS5B protein: Characterization of its RNA polymerase activity and RNA binding. Hepatology 29: 1227-1235 https://doi.org/10.1002/hep.510290448
  14. Kim, J., H. Park, K. Choi, J. Lee, J. Park, and M. Yoon. 2006. Screening of peptides bound to anthrax protective antigen by phage display. J. Microbiol. Biotechnol. 16: 1784-1790
  15. Kofler, M., K. Motzny, M. Beyermann, and C. Freund. 2005. Novel interaction partners of the CD2BP2-GYF domain. J. Biol. Chem. 280: 33397-33402 https://doi.org/10.1074/jbc.M503989200
  16. 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
  17. Lecut, C., V. Arocas, H. Ulrichts, A. Elbaz, J. Villeval, J. Lacapere, H. Deckmyn, and M. Jandrot-Perrus. 2004. Identification of residues within human glycoprotein VI involved in the binding to collagen. J. Biol. Chem. 279: 52293-52299 https://doi.org/10.1074/jbc.M406342200
  18. Lee, J.-H., I. Y. Nam, and H. Myung. 2006. Nonstructural protein 5B of hepatitis C virus. Mol. Cells 21: 330-336
  19. Leveque, V. J.-P. and Q. M. Wang. 2002. RNA-dependent RNA polymerase encoded by hepatitis C virus: Biomedical applications. Cell. Mol. Life Sci. 59: 909-919 https://doi.org/10.1007/s00018-002-8478-7
  20. Lohmann, V., A. Roos, F. Korner, J.-O. Koch, and R. Bartenschlager. 2000. Biochemical and structural analysis of the NS5B RNA-dependent RNA polymerase of the hepatitis C virus. J. Viral Hepat. 7: 167-174 https://doi.org/10.1046/j.1365-2893.2000.00218.x
  21. McHutchison, J. G. and T. Poynard. 1999. Combination therapy with interferon plus ribavirin for the initial treatment of chronic hepatitis C. Semin. Liver Dis. 19: 57-65
  22. Moradpour, D. and H. E. Blum. 2004. A primer on the molecular virology of hepatitis C. Liver Int. 24: 519-525 https://doi.org/10.1111/j.1478-3231.2004.0965.x
  23. Nguyen, T. T., A. T. Gates, L. L. Gutshall, V. K. Johnston, B. Gu, K. J. Duffy, and R. T. Sarisky. 2003. Resistance profile of a hepatitis C virus RNA-dependent RNA polymerase benzothiadiazine inhibitor. Antimicrob. Agents Chemother. 47: 3525-3530 https://doi.org/10.1128/AAC.47.11.3525-3530.2003
  24. Pogam, S. L., H. Kang, S. F. Harris, V. Leveque, A. M. Giannetti, S. Ali, W.-R. Jiang, S. Rajyaguru, G. Tavares, et al. 2006. Selection and characterization of replicon variants dually resistant to thumb- and palm-binding nonnucleoside polymerase inhibitors of the hepatitis C virus. J. Virol. 80: 6146-6154 https://doi.org/10.1128/JVI.02628-05
  25. Purcell, R. 1994. Hepatitis viruses: Changing patterns of human disease. Proc. Natl. Acad. Sci. USA 91: 2401-2406
  26. Rasmusen, U. B., V. Schreiber, H. Schultz, F. Mischler, and K. Schughart. 2002. Tumor cell-targeting by phage-displayed peptides. Cancer Gene Ther. 9: 606-612 https://doi.org/10.1038/sj.cgt.7700476
  27. Rowley, M. J., M. Scealy, J. C. Whisstock, J. A. Jois, L. C. Wijeyewickrema, and I. R. Mackay. 2000. Prediction of the immunodominant epitope of the pyruvate dehydrogenase complex E2 in primary biliary cirrhosis using phage display. J. Immunol. 164: 3413-3419 https://doi.org/10.4049/jimmunol.164.6.3413
  28. Seo, M., J. Lee, M. Kim, H. Chae, and H. Myung. 2006. Selection and characterization of peptides specifically binding to $$TiO_{2}$$ nanoparticles. J. Microbiol. Biotechnol. 16: 303-307
  29. Shin, K., J. Lim, J. Kim, H. Myung, and S.-W. Lee. 2006. Inhibition of replication of hepatitis C virus replicon with nuclease-resistant RNA aptamers. J. Microbiol. Biotechnol. 16: 1634-1639
  30. Sidhu, S., W. Fairbrother, and K. Deshayes. 2003. Exploring protein-protein interactions with phage display. ChemBioChem 4: 14-25 https://doi.org/10.1002/cbic.200390008
  31. Smith, G. P. 1985. Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315-1317 https://doi.org/10.1126/science.4001944
  32. Suzuki, R., T. Suzuki, K. Ishii, Y. Matsuura, and T. Miyamura. 1999. Processing and functions of hepatitis C virus proteins. Intervirology 42: 145-152 https://doi.org/10.1159/000024973
  33. Tomei, L., S. Altamura, L. Bartholomew, A. Biroccio, A. Ceccacci, L. Pacini, F. Narjes, N. Gennari, M. Bisbocci, et al. 2003. Mechanism of action and antiviral activity of benzimidazolebased allosteric inhibitors of the hepatitis C virus RNA-dependent RNA polymerase. J. Virol. 77: 13225-13231 https://doi.org/10.1128/JVI.77.24.13225-13231.2003
  34. Tomei, L., S. Altamura, L. Bartholomew, M. Bisbocci, C. Bailey, M. Bosserman, A. Cellucci, E. Forte, I. Incitti, et al. 2004. Characterization of the inhibition of hepatitis C virus RNA replication by nonnucleosides. J. Virol. 78: 938-946 https://doi.org/10.1128/JVI.78.2.938-946.2004
  35. Wang, C., P. Sarnow, and A. Siddiqui. 1993. Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. J. Virol. 67: 3338-3344
  36. Wang, M., K. K. Ng, M. M. Cherney, L. Chan, C. G. Yannopoulos, J. Bedard, N. Morin, N. Nguyen-Ba, M. H. Alaoui-ismaili, et al. 2003. Non-nucleoside analogue inhibitors bind to an allosteric site on HCV NS5B polymerase. Crystal structures and mechanism of inhibition. J. Biol. Chem. 278: 9489-9495 https://doi.org/10.1074/jbc.M209397200
  37. Wasley, A. and M. J. Alter. 2000. Epidemiology of hepatitis C: Geographic differences and temporal trends. Semin. Liver Dis. 20: 1-16 https://doi.org/10.1055/s-2000-9506
  38. Yang, Z., W. Shm, M. Kim, K. Lee, K. Kim, K. Kim, C. Kim, S. Ha, and D. Chung. 2007. Production and characterization of monoclonal and recombinant antibodies against antimicrobial sulfamethazine. J. Microbiol. Biotechnol. 17: 571-578