Nonstructural Protein 5B of Hepatitis C Virus

  • Lee, Jong-Ho (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Nam, In Young (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Myung, Heejoon (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
  • Received : 2006.04.04
  • Accepted : 2006.04.07
  • Published : 2006.06.30

Abstract

Since its identification in 1989, hepatitis C virus has been the subject of extensive research. The biology of the virus and the development of antiviral drugs are closely related. The RNA polymerase activity of nonstructural protein 5B was first demonstrated in 1996. NS5B is believed to localize to the perinuclear region, forming a replicase complex with other viral proteins. It has a typical polymerase structure with thumb, palm, and finger domains encircling the active site. A de novo replication initiation mechanism has been suggested. To date, many small molecule inhibitors are known including nucleoside analogues, non-nucleoside analogues, and pyrophosphate mimics. NS5B interacts with other viral proteins such as core, NS3, 4A, 4B, and 5A. The helicase activity of NS3 seems necessary for RNA strand unwinding during replication, with other nonstructural proteins performing modulatory roles. Cellular proteins interacting with NS5B include VAMP-associated proteins, heIF4AII, hPLIC1, nucleolin, PRK2, ${\alpha}$-actinin, and p68 helicase. The interactions of NS5B with these proteins might play roles in cellular trafficking, signal transduction, and RNA polymerization, as well as the regulation of replication/translation processes.

Keywords

Hepatitis C Virus;Inhibitor;Interacting Proteins;NS5B;RNA Dependent RNA Polymerase;Structure

Acknowledgement

Supported by : HUFS

References

  1. Alexopoulou, L., Holt, A. C., Medzhitov, R., and Flavell, R. A. (2001) Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413, 732-738 https://doi.org/10.1038/35099560
  2. Bost, A. G., Venable, D., Liu, L., and Heinz, B. A. (2003) Cytoskeletal requirements for hepatitis C virus (HCV) RNA synthesis in the HCV replicon cell culture system. J. Virol. 77, 4401-4408
  3. Bressanelli, S., Tomei, L., Rey, F. A., and De Francesco, R. (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
  4. Causevic, M., Hislop, R. G., Kernohan, N. M., Carey, F. A., Kay, R. A., et al. (2001) Overexpression and poly-ubiquitylation of the DEAD-box RNA helicase p68 in colorectal tumors. Oncogene 20, 7734-7743 https://doi.org/10.1038/sj.onc.1204976
  5. Choi, K. H., Groarke, J. M., Young, D. C., Kuhn, R. J., Smith, J. L., et al. (2004) The structure of the RNA-dependent RNA polymerase from bovine viral diarrhea virus establishes the role of GTP in de novo initiation. Proc. Nat. Acad. Sci. USA 101, 4425-4430 https://doi.org/10.1073/pnas.0400660101
  6. Endoh, H., Maruyama, K., Masuhiro, Y., Kobayashi, Y., Goto, M., et al. (1999) Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor $\alpha$. Mol. Cell. Biol. 19, 5363-5372 https://doi.org/10.1128/MCB.19.8.5363
  7. Ferrari, E., Wright-Minogue, J., Fang, J. W., Baroudy, B. M., Lau, J. Y., et al. (1999) Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli. J. Virol. 73, 1649-1654
  8. Goh, P.-Y., Tan, Y.-J., Lim, S. P., Han, Y. H., Lim, S. G., et al. (2004) Cellular RNA helicase p68 relocalization and interaction with the hepatitis C virus (HCV) NS5B protein and the potential role of p68 in HCV RNA replication. J. Virol. 78, 5288-52 https://doi.org/10.1128/JVI.78.10.5288-5298.2004
  9. Hagedorn, C. H., Beers, E. H., and De Staercke, C. (2000) Hepatitis C virus RNA-dependent RNA polymerase. Curr. Top. Microbiol. Immunol. 242, 225-260
  10. Hong, Z., Cameron, C. E., Walker, M. P., Castro, C., Yao, N., et al. (2001) A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase. Virology 285, 6-11 https://doi.org/10.1006/viro.2001.0948
  11. Ishido, S., Fujita, T., and Hotta, H. (1998) Complex formation of NS5B with NS3 and NS4A proteins of hepatitis C virus. Biochem. Biophys. Res. Commun. 244, 35-40 https://doi.org/10.1006/bbrc.1998.8202
  12. Kim, S.-J., Kim, J.-H., Kim, Y.-G., Lim, H.-S., and Oh, J. W. (2004) Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation. 279, 50031-50041 https://doi.org/10.1074/jbc.M408617200
  13. Lee, K. J., Choi, J., Ou, J.-H., and Lai, M. M. C. (2004) The Cterminal transmembrane domain of hepatitis C virus (HCV) RNA polymerase is essential for HCV replication in vivo. J. Virol. 78, 3797-3802 https://doi.org/10.1128/JVI.78.7.3797-3802.2004
  14. Lesburg, C. A., Cable, M. B., Ferrari, E., Hong, Z., Mannarino, A. F., et al. (1999) Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat. Struct. Biol. 6, 937-943 https://doi.org/10.1038/13305
  15. Love, R. A., Parge, H. E., Yu, X., Hickey, M. J., Diehl, W., et al. (2003) Crystallographic identification of a noncompetitive inhibitor binding site on the hepatitis C virus NS5B RNA polymerase enzyme. J. Virol. 77, 7575-7581 https://doi.org/10.1128/JVI.77.13.7575-7581.2003
  16. Love, R. A., Maegley, K. A., Yu, X., Ferre, R. A., Lingardo, L. K., et al. (2004) The crystal structure of the RNA-dependent RNA polymerase from human rhinovirus: a dual function target for common cold antiviral therapy. Structure 12, 1533-1544 https://doi.org/10.1016/j.str.2004.05.024
  17. McHutchison, J. G., Gordon, S. C., Schiff, E. R., Shiffman, M. L., Lee, W. M., et al. (1998) Interferon R-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis. Interventional Therapy Group. N. Engl. J. Med. 339, 1485-1492 https://doi.org/10.1056/NEJM199811193392101
  18. Migliaccio, G., Tomassini, J. E., Carroll, S. S., Tomei, L., Altamura, S., et al. (2003) Characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro. J. Biol. Chem. 278, 49164-49170 https://doi.org/10.1074/jbc.M305041200
  19. Miller, R. H. and Purcell, R. H. (1990) Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc. Natl. Acad. Sci. USA 87, 2057-2061 https://doi.org/10.1073/pnas.87.6.2057
  20. Nisole, S., Krust, B., Callebaut, C., Guichard, G., Muller, S., et al. (1999) The anti-HIV pseudopeptide HB-19 forms a complex with the cell-surface-expressed nucleolin independent of heparah sulfate proteoglycans. J. Biol. Chem. 274, 27875-27884 https://doi.org/10.1074/jbc.274.39.27875
  21. Oh, J. W., Ito, T., and Lai, M. M. (1999) A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA. J. Virol. 73, 7694-7702
  22. Oshiumi, H., Matsumoto, M., Funami, K., Akazawa, T., and Seya, 336 HCV NS5B T. (2003) TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated inferferon-beta induction. Nat. Immunol. 4, 161-167 https://doi.org/10.1038/ni886
  23. Piccininni, S., Varaklioti, A., Nardelli, M., Dave, B., Raney, K. D., et al. (2002) Modulation of the hepatitis C virus RNAdependent RNA polymerase activity by the non-structural (NS) 3 helicase and the NS4B membrane protein. J. Biol. Chem. 277, 45670-45679 https://doi.org/10.1074/jbc.M204124200
  24. Ruggieri, A., Harada, T., Matsuura, Y., and Miyamura, T. (1997) Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein. Virology 229, 68-76 https://doi.org/10.1006/viro.1996.8420
  25. Selby, M. J., Glazer, E., Masiarz, F., and Houghton, M. (1994) Complex processing and protein:protein interactions in the E2:NS2 region of HCV. Virology 204, 114-122 https://doi.org/10.1006/viro.1994.1515
  26. Shim, J., Larson, G., Lai, V., Naim, S., and Wu, J. Z. (2003) Canonical 3′-deoxyribonucleotides as a chain terminator for HCV NS5B RNA-dependent RNA polymerase. Antiviral Res. 58, 243-251 https://doi.org/10.1016/S0166-3542(03)00007-X
  27. Shirota, Y., Luo, H., Qin, W., Kaneko, S., Yamashita, T., et al. (2002) Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity. J. Biol. Chem. 277, 11149- 11155 https://doi.org/10.1074/jbc.M111392200
  28. Summa, V., Petrocchi, A., Pace, P., Matassa, V. G., De Francesco, R., et al. (2004) Discovery of alpha,gamma-diketo acids as potent selective and reversible inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase. J. Med. Chem. 47, 14-17 https://doi.org/10.1021/jm0342109
  29. Tai, C. L., Chi, W. K., Chen, D. S., and Hwang, L. H. (1996) The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). J. Virol. 70, 8477-8484
  30. Thompson, A. A. and Peersen, O. B. (2004) Structural basis for proteolysis-dependent activation of the poliovirus RNAdependent RNA polymerase. EMBO J. 23, 3462-3471
  31. Tomei, L., Vitale, R. L., Incitti, I., Serafini, S., Altamura, S., et al. (2000) Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the Cterminal hydrophobic sequence. J. Gen. Virol. 81, 759-767 https://doi.org/10.1099/0022-1317-81-3-759
  32. Tomei, L., Altamura, S., Bartholomew, L., Bisbocci, M., Bailey, C., 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
  33. Vincent, S. and Settleman, J. (1997) The PRK2 kinase is a potential effector target of both Rho and Rac GTPases and regulates actin cytoskeletal organization. Mol. Cell. Biol. 17, 2247-2256 https://doi.org/10.1128/MCB.17.4.2247
  34. Wang, M., Ng, K. K., Cherney, M. M., Chan, L., Yannopoulos, C. G., 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
  35. Zhong, W., Uss, A. S., Ferrari, E., Lau, J. Y., and Hong, Z. (2000) De novo initiation of RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase. J. Virol. 74, 2017-2022 https://doi.org/10.1128/JVI.74.4.2017-2022.2000
  36. Blight, K. J. and Rice, C. M. (1997) Secondary structure determination of the conserved 98-base sequence at the 3' terminus of hepatitis C virus genome RNA. J. Virol. 71, 7345-7352
  37. Johnson, R. B., Sun, X. L., Hockman, M. A., Villarreal, E. C., Wakulchik, M., et al. (2000) Specificity and mechanism analysis of hepatitis C virus RNA-dependent RNA polymerase. Arch. Biochem. Biophys. 377, 129-134 https://doi.org/10.1006/abbi.2000.1749
  38. Shi, S. T., Lee, K. J., Aizaki, H., Hwang, S. B., and Lai, M. M. (2003) Hepatitis C virus RNA replication occurs on a detergent- resistant membrane that cofractionates with caveolin-2. J. Virol. 77, 4160-4168 https://doi.org/10.1128/JVI.77.7.4160-4168.2003
  39. Harms, G., Kraft, R., Grelle, G., Volz, B., Dernedde, J., et al. (2001) Identification of nucleolin as a new L-selectin ligand. Biochem. J. 360, 531−538 https://doi.org/10.1042/0264-6021:3600531
  40. Takeda, K., Kaisho, T., and Akira, S. (2003) Toll-like receptors. Annu. Rev. Immunol. 21, 335-376 https://doi.org/10.1146/annurev.immunol.21.120601.141126
  41. Flynn, P., Mellor, H., Casamassima, A., and Parker, P. J. (2000) Rho GTPase control of protein kinase C-related protein kinase activation by 3-phosphoinositide-dependent protein kinase. J. Biol. Chem. 275, 11064-11070 https://doi.org/10.1074/jbc.275.15.11064
  42. Butcher, S. J., Grimes, J. M., Makeyev, E. V., Bamford, D. H., and Stuart, D. I. (2001) A mechanism for initiating RNA-dependent RNA polymerization. Nature 410, 235-240 https://doi.org/10.1038/35065653
  43. Ishii, K., Tanaka, Y., Yap, C. C., Aizaki, H., Matsuura, Y., et al. (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
  44. Luo, G., Hamatake, R. K., Mathis, D. M., Racela, J., Rigat, K. L., et al. (2000) De novo initiation of RNA synthesis by the RNAdependent RNA polymerase (NS5B) of hepatitis C virus. J. Virol. 74, 851-863 https://doi.org/10.1128/JVI.74.2.851-863.2000
  45. Storer, R. (2001) Method for the treatment or prevention of Flaviviridae viral infection using nucleoside analogs. WO 20010-32153
  46. Butcher, S. J., Makeyev, E. V., Grimes, J. M., Stuart, D. I., and Bamford, D. H. (2000) Crystallization and preliminary X-ray crystallographic studies on the bacteriophage phi6 RNAdependent RNA polymerase. Acta Crystallogr. D Biol. Crystallogr. 56, 1473-1475 https://doi.org/10.1107/S0907444900010702
  47. Gao, L., Tu, H., Shi, S. T., Lee, K. J., Asanaka, M., et al. (2003) Interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis C virus RNA-dependent RNA polymerase. J. Virol. 77, 4149-4159 https://doi.org/10.1128/JVI.77.7.4149-4159.2003
  48. Ismaili, H. M. A., Cheng, Y. X., Lavallee, J. F., Siddiqui, A., and Storer, R. (2001) Method for the treatment or prevention of flavivirus infections using nucleoside analogues. WO 20010- 60315
  49. Medzhitov, R. (2001) Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1, 135-145 https://doi.org/10.1038/35100529
  50. Appleby, T. C., Luecke, H., Shim, J. H., Wu, J. Z., Cheney, I. W., et al. (2005) Crystal structure of complete rhinovirus RNA polymerase suggests front loading of protein primer. J. Virol. 79, 277-288 https://doi.org/10.1128/JVI.79.1.277-288.2005
  51. Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., et al. (2000) The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. Nat. Med. 6, 1375-1379 https://doi.org/10.1038/82191
  52. Ng, K. K., Pendas-Franco, N., Rojo, J., Boga, J. A., Machin, A., et al. (2004) Crystal structure of norwalk virus polymerase reveals the carboxyl terminus in the active site cleft. J. Biol. Chem. 279, 16638-16645 https://doi.org/10.1074/jbc.M400584200
  53. Ray, R. B., Lagging, L. M., Meyer, K., and Ray, R. (1996) Hepatitis C virus core protein cooperates with Ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype. J. Virol. 70, 4438-4443
  54. Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., et al. (1998) RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region. J. Biol. Chem. 273, 15479-15486 https://doi.org/10.1074/jbc.273.25.15479
  55. Lan, S., Wang, H., Jiang, H., Mao, H., Zhang, X., et al. (2003) Direct interaction between $\alpha$-actinin and hepatitis C virus NS5B. FEBS Lett. 554, 289-294 https://doi.org/10.1016/S0014-5793(03)01163-3
  56. Behrens, S. E., Tomei, L., and De Francesco, R. (1996) Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J. 15, 12-22
  57. Pace, P., Nizi, E., Pacini, B., Pesci, S., Matassa, V., et al. (2004) The monoethyl ester of meconic acid is an active site inhibitor of HCV NS5B RNA-dependent RNA polymerase. Bioorg. Med. Chem. Lett. 14, 3257-3261 https://doi.org/10.1016/j.bmcl.2004.03.087
  58. Dhanak, D., Duffy, K. J., Johnston, V. K., Lin-Goerke, J., Darcy, M., et al. (2002) Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNAdependent RNA polymerase. J. Biol. Chem. 277, 38322-38327 https://doi.org/10.1074/jbc.M205566200
  59. Nguyen, T. T., Gates, A. T., Gutshall, L. L., Johnston, V. K., Gu, B., et al. (2003) Resistance profile of a hepatitis C virus RNAdependent RNA polymerase benzothiadiazine inhibitor. Antimicrob. Agents Chemother. 47, 3525-3530 https://doi.org/10.1128/AAC.47.11.3525-3530.2003
  60. Ago, H., Adachi, T., Yoshida, A., Yamamoto, M., Habuka, N., et al. (1999) Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure 7, 1417-1426 https://doi.org/10.1016/S0969-2126(00)80031-3
  61. Hamamoto, I., Nishimura, Y., Okamoto, T., Aizaki, H., Liu, M., et al. (2005) HumanVAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B. J. Virol. 79, 13473-13482 https://doi.org/10.1128/JVI.79.21.13473-13482.2005
  62. Kao, C. C., Yang, X., Kline, A., Wang, Q. M., Barket, D., et al. (2000) Template requirements for RNA synthesis by a recombinant hepatitis C virus RNA-dependent RNA polymerase. 74, 11121-11128 https://doi.org/10.1128/JVI.74.23.11121-11128.2000
  63. Lin, C., Wu, J. W., Hsiano, K., and Su, M. S. (1997) The hepatitis C virus NS4A: interactions with the NS4B and NS5A proteins. J. Viol. 71, 6465-6471
  64. Stuyver, L. J., Whitaker, T., McBrayer, T. R., Hernandez- Santiago, B. I., Lostia, S., et al. (2003) Ribonucleoside analogue that blocks replicaton of bovine viral diarrhea and hepatitis C viruses in culture. Antimicrob. Agents Chemother. 47, 244-254 https://doi.org/10.1128/AAC.47.1.244-254.2003
  65. Uchida, M., Hino, N., Yamanaka, T., Fukushima, H., Imanishi, T., et al. (2002) Hepatitis C virus core protein binds to a Cterminal region of NS5B RNA polymerase. Hepatol. Res. 22, 297-306 https://doi.org/10.1016/S1386-6346(02)00005-0
  66. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., et al. (1989) Isolation of a cDNA clone derived from a bloodborne non-A, non-B viral hepatitis genome. Science 244, 359-362 https://doi.org/10.1126/science.2523562
  67. Sun, X. L., Johnson, R. B., Hockman, M. A., and Wang, Q. M. (2000a) De novo RNA synthesis catalyzed by HCV RNAdependent RNA polymerase. Biochem. Biophys. Res. Commun. 268, 798-803 https://doi.org/10.1006/bbrc.2000.2120
  68. Sun, W., Vincent, S., Settleman, J., and Johnson, G. L. (2000b) MEK kinase 2 binds and activates protein kinase C-related kinase2. Bifurcation of kinase regulatory pathways at the level of an MAPK kinase kinase. J. Biol. Chem. 275, 24421-24428 https://doi.org/10.1074/jbc.M003148200
  69. Wasley, A. and Alter, M. J. (2000) Epidemiology of hepatitis C: geographic differences and temporal trends. Semin. Liver Dis. 20, 1-16 https://doi.org/10.1055/s-2000-9506
  70. Bond, A. T., Mangus, D. A., He, F., and Jacobson, A. (2001) Absence of Dbp2p alters both nonsense-mediated mRNA decay and rRNA processing. Mol. Cell. Biol. 21, 7366-7379 https://doi.org/10.1128/MCB.21.21.7366-7379.2001
  71. Carroll, S. S., Tomassini, J. E., Bosserman, M., Getty, K., Stahlhut, M. W., et al. (2003) Inhibition of hepatitis C virus RNA replication by 2′-modified nucleoside analogs. J. Biol. Chem. 278, 11979-11984 https://doi.org/10.1074/jbc.M210914200
  72. Lin, C., Lindenbach, B. D., Pragai, B. M., McCourt, D. W., and Rice, C. M. (1994) Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J. Virol. 67, 3835-3844
  73. Laurila, M. R., Makeyev, E. V., and Bamford, D. H. (2002) Bacteriophage phi 6 RNA-dependent RNA polymerase: molecular details of initiating nucleic acid synthesis without primer. J. Biol. Chem. 277, 17117-17124 https://doi.org/10.1074/jbc.M111220200
  74. Maag, D., Castro, C., Hong, Z., and Cameron, C. E. (2001) Hepatitis C virus RNA-dependent RNA polymerase (NS5B) as a mediator of the antiviral activity of ribavirin. J. Biol. Chem. 276, 46094-46098 https://doi.org/10.1074/jbc.C100349200
  75. De Francesco, R., Tomei, L., Altamura, S., Summa, V., and Migliaccio, G. (2003) Approaching a new era for hepatitis C virus therapy: inhibitors of the NS3-4A serine protease and the NS5B RNA-dependent RNA polymerase. Antiviral Res. 58, 1-16 https://doi.org/10.1016/S0166-3542(03)00028-7
  76. Lau, J. Y. N., Tam, R. C., Liang, T. J., and Hong, Z. (2002) Mechanism of action of ribavirin in the combination treatment of chronic HCV infection. Hepatology 35, 1002-1009 https://doi.org/10.1053/jhep.2002.32672
  77. Shimakami, T., Hijikata, M., Luo, H., Ma, Y. Y., Kaneko, S., et al. (2004) Effect of interaction between hepatitis C virus NS5A and NS5B on hepatitis C virus RNA replication with the hepatitis C virus replicon. J. Virol. 78, 2738-2748 https://doi.org/10.1128/JVI.78.6.2738-2748.2004
  78. Beaulieu, P. L. and Tsantrizos, Y. S. (2004) Inhibitors of the HCV NS5B polymerase: new hope for the treatment of hepatitis C infections. Curr. Opin. Investig. Drugs 5, 838-850
  79. Bonifacino, J. S. and Weissman, A. M. (1998) Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu. Rev. Cell. Dev. Biol. 14, 19-57
  80. Izumi, R. E., Valdez, B., Banerjee, R., Srivastava, M., and Dasgupta, A. (2001) Nucleolin stimulates viral internal ribosome entry site-mediated translation. Virus Res. 76, 17-29 https://doi.org/10.1016/S0168-1702(01)00240-4
  81. McKercher, G., Beaulieu, P. L., Lamarre, D., Laplante, S., Lefebvre, S., et al. (2004) Specific inhibitors of HCV polymerase identified using an NS5B with lower affinity for template/ primer substrate. Nucleic Acids Res. 32, 422-431 https://doi.org/10.1093/nar/gkh160
  82. Naka, K., Dansako, H., Kobayashi, N., Ikeda, M., and Kato, N. (2005) Hepatitis C virus NS5B delays cell cycle progression by inducing interferon-$\beta$ via toll-like receptor 3 signaling pathway without replicating viral genomes. Virology (in press)
  83. Ng, K. K., Cherney, M. M., Vazquez, A. L., Machin, A., Alonso, J. M., et al. (2002) Crystal structures of active and inactive conformations of a caliciviral RNA-dependent RNA polymerase. J. Biol. Chem. 277, 1381-1387 https://doi.org/10.1074/jbc.M109261200
  84. Quilliam, L. A., Lambert, Q. T., Mickelson-Young, L. A., Westwick, J. K., Sparks, A. B., et al. (1996) Isolation of a NCKassociated kinase, PRK2, an SH3-binding protein and potential effector of Rho protein signaling. J. Biol. Chem. 271, 28772-28776 https://doi.org/10.1074/jbc.271.46.28772
  85. Olsen, D. B., Eldrup, A. B., Bartholomew, L., Bhat, B., Bosserman, M. R., et al. (2004) A 7-deaza-adenosine analogue is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties. Antimicrob. Agents Chemother. 48, 3944-3953 https://doi.org/10.1128/AAC.48.10.3944-3953.2004
  86. Stansfield, I., Avolio, S., Colarusso, S., Gennari, N., Narjes, F., et al. (2004) Active site inhibitors of HCV NS5B polymerase. The development and pharmacophore of 2-thienyl-5,6-dihydroxypyrimidine- 4-carboxylic acid. Bioorg. Med. Chem. Lett. 14, 5085-5088 https://doi.org/10.1016/j.bmcl.2004.07.075
  87. Ferrer-Orta, C., Arias, A., Perez-Luque, R., Escarmis, C., Domingo, E., et al. (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 https://doi.org/10.1074/jbc.M405465200
  88. Lohmann, V., Korner, F., Herian, U., and Bartenschlager, R. (1997) Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J. Virol. 71, 8416-8428
  89. Suzich, J. A., Tamura, J. K., Palmer-Hill, F., Warrener, P., Grakoui, A., et al. (1993) Hepatitis Cvirus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes. J. Virol. 67, 6152-6158
  90. Tomei, L., Altamura, S., Bartholomew, L., Biroccio, A., Ceccacci, A., et al. (2003) Mechanism of action and antiviral activity of benzimidazole-based 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
  91. Tu, H., Gao, L., Shi, S. T., Taylor, D. R., Yang, T., et al. (1999) Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein. Virology 263, 30-41 https://doi.org/10.1006/viro.1999.9893
  92. Manns, M. P., McHutchison, J. G., Gordon, S. C., Rustgi, V. K., Shiffman, M., et al. (2001) Peginterferon $\alpha$-2b plus ribavirin compared with interferon $\alpha$-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial. Lancet 358, 958-965 https://doi.org/10.1016/S0140-6736(01)06102-5
  93. Ni, Z. J. and Wagman, A. S. (2004) Progress and development of small molecule HCV antivirals. Curr. Opin. Drug Discov. Dev. 7, 446-459
  94. Kamer, G. and Argos, P. (1984) Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res. 12, 7269-7282 https://doi.org/10.1093/nar/12.18.7269
  95. Kyono, K., Miyashiro, M., and Taguchi, I. (2002) Human eukaryotic initiation factor 4AII associates with hepatitis C virus NS5B protein in vitro. Biochem. Biophys. Res. Commun. 292, 659-666 https://doi.org/10.1006/bbrc.2002.6702
  96. Matsumoto, M., Kikkawa, S., Kohase, M., Miyake, K., and Seya, T. (2002) Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNAmediated signaling. Biochem. Biophys. Res. Commun. 293, 1364- 1369 https://doi.org/10.1016/S0006-291X(02)00380-7
  97. Cheng, J.-C., Chang, M.-F., and Chang, S. C. (1999) Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3′ end of the viral RNA. J. Virol. 73, 7044 - 7049
  98. Gopalsamy, A., Lim, K., Ciszewski, G., Park, K., Ellingboe, J. W., et al. (2004) Discovery of pyrano[3,4-b]indoles as potent and selective HCV NS5B polymerase inhibitors. J. Med. Chem. 47, 6603-6608 https://doi.org/10.1021/jm0401255
  99. Hirano, M., Kaneko, S., Yamashita, T., Luo, H., Qin, W., et al. (2003) Direct interaction between nucleolin and hepatitis C virus NS5B. J. Biol. Chem. 278, 5109-5115 https://doi.org/10.1074/jbc.M207629200