Citation: Susan L. Uprichard. Hepatitis C Virus Experimental Model Systems and Antiviral drug Research* .VIROLOGICA SINICA, 2010, 25(4) : 227-245.  http://dx.doi.org/10.1007/s12250-010-3134-0

Hepatitis C Virus Experimental Model Systems and Antiviral drug Research*

  • Corresponding author: Susan L. Uprichard, sluprich@uic.edu
  • Received Date: 17 March 2010
    Accepted Date: 18 April 2010
    Available online: 01 August 2010

    Fund Project: National Institutes of Health grants AI070827American Cancer Society grant RSG-09-076-01National Institutes of Health grants CA33266

  • An estimated 130 million people worldwide are chronically infected with hepatitis C virus (HCV) making it a leading cause of liver disease worldwide. Because the currently available therapy of pegylated interferon-alpha and ribavirin is only effective in a subset of patients, the development of new HCV antivirals is a healthcare imperative. This review discusses the experimental models available for HCV antiviral drug research, recent advances in HCV antiviral drug development, as well as active research being pursued to facilitate development of new HCV-specific therapeutics.

  • 加载中
    1. Abe K, Kurata T, Teramoto Y C. 1993. Lack of susceptibility of various primates and woodchucks to hepatitis C virus. J Med Primatol, 22 (7-8): 433-434.

    2. Afdhal N H. 2004. The natural history of hepatitis C. Semin Liver Dis, 24 Suppl 2: 3-8.

    3. Ahmed A, Keeffe E B. 1999. Treatment strategies for chronic hepatitis C: update since the 1997 National Institutes of Health Consensus Development Conference. J Gastroenterol Hepatol, 14 Suppl: S12-18.

    4. Alter H J, Seeff L B. 2000. Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome. Semin Liver Dis, 20 (1): 17-35.
        doi: 10.1055/s-2000-9505

    5. Alter M J, Margolis H S, Krawczynski K, et al. 1992. The natural history of community-acquired hepatitis C in the United States. The Sentinel Counties Chronic non-A, non-B Hepatitis Study Team. N Engl J Med, 327 (27): 1899-1905.
        doi: 10.1056/NEJM199212313272702

    6. Aly H H, Shimotohno K, Hijikata M. 2009. 3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV. Biochem Biophys Res Commun, 379 (2): 330-334.
        doi: 10.1016/j.bbrc.2008.12.054

    7. Azuma H, Paulk N, Ranade A, et al. 2007. Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/-mice. Nat Biotechnol, 25 (8): 903-910.
        doi: 10.1038/nbt1326

    8. Bartenschlager R. 2006. Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture. Curr Opin Microbiol, 9 (4): 416-422.
        doi: 10.1016/j.mib.2006.06.012

    9. Bartenschlager R, Lohmann V. 2000. Replication of hepatitis C virus. J Gen Virol, 81 Pt 7: 1631-1648.

    10. Bartosch B, Dubuisson J, Cosset F L. 2003. Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med, 197 (5): 633-642.
        doi: 10.1084/jem.20021756

    11. Bartosch B, Vitelli A, Granier C, et al. 2003. Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem, 278 (43): 41624-41630.
        doi: 10.1074/jbc.M305289200

    12. Beames B, Chavez D, Lanford R E. 2001. GB virus B as a model for hepatitis C virus. Ilar J, 42 (2): 152-160.
        doi: 10.1093/ilar.42.2.152

    13. Beard M R, Abell G, Honda M, et al. 1999. An infectious molecular clone of a Japanese genotype 1b hepatitis C virus. Hepatology, 30 (1): 316-324.
        doi: 10.1002/(ISSN)1527-3350

    14. Behrens S E, Grassmann C W, Thiel H J, et al. 1998. Characterization of an autonomous subgenomic pestivirus RNA replicon. J Virol, 72 (3): 2364-2372.

    15. Benedicto I, Molina-Jimenez F, Bartosch B, et al. 2009. The tight junction-associated protein occludin is required for a postbinding step in hepatitis C virus entry and infection. J Virol, 83 (16): 8012-8020.
        doi: 10.1128/JVI.00038-09

    16. Berger K L, Cooper J D, Heaton N S, et al. 2009. Roles for endocytic trafficking and phosphatidylinositol 4-kinase Ⅲ alpha in hepatitis C virus replication. Proc Natl Acad Sci USA, 106 (18): 7577-7582.
        doi: 10.1073/pnas.0902693106

    17. Berger K L, Randall G. 2009. Potential roles for cellular cofactors in hepatitis C virus replication complex formation. Commun Integr Biol, 2 (6): 471-473.
        doi: 10.4161/cib.2.6.9261

    18. Bissig K D, Le T T, Woods N B, et al. 2007. Repopulation of adult and neonatal mice with human hepatocytes: a chimeric animal model. Proc Natl Acad Sci U S A, 104 (51): 20507-20511.
        doi: 10.1073/pnas.0710528105

    19. Bissig K D, Wieland S F, Tran P, et al. 2010. Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment. J Clin Invest.

    20. Blanchard E, Belouzard S, Goueslain L, et al. 2006. Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol, 80 (14): 6964-6972.
        doi: 10.1128/JVI.00024-06

    21. Blight K J, Kolykhalov A A, Rice C M. 2000. Efficient initiation of HCV RNA replication in cell culture. Science, 290 (5498): 1972-1975.
        doi: 10.1126/science.290.5498.1972

    22. Blight K J, McKeating J A, Marcotrigiano J, et al. 2003. Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture. J Virol, 77 (5): 3181-3190.
        doi: 10.1128/JVI.77.5.3181-3190.2003

    23. Bode J G, Brenndorfer E D, Karthe J, et al. 2009. Interplay between host cell and hepatitis C virus in regulating viral replication. Biol Chem, 390 (10): 1013-1032.

    24. Boriskin Y S, Leneva I A, Pecheur E I, et al. 2008. Arbidol: a broad-spectrum antiviral compound that blocks viral fusion. Curr Med Chem, 15 (10): 997-1005.
        doi: 10.2174/092986708784049658

    25. Boriskin Y S, Pecheur E I, Polyak S J. 2006. Arbidol: a broad-spectrum antiviral that inhibits acute and chronic HCV infection. Virol J, 3: 56.
        doi: 10.1186/1743-422X-3-56

    26. Broering T J, Garrity K A, Boatright N K, et al. 2009. Identification and characterization of broadly neutralizing human monoclonal antibodies directed against the E2 envelope glycoprotein of hepatitis C virus. J Virol, 83 (23): 12473-12482.
        doi: 10.1128/JVI.01138-09

    27. Buck M. 2008. Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures. PLoS One, 3 (7): e2660.
        doi: 10.1371/journal.pone.0002660

    28. Buckwold V E, Beer B E, Donis R O. 2003. Bovine viral diarrhea virus as a surrogate model of hepatitis C virus for the evaluation of antiviral agents. Antiviral Res, 60 (1): 1-15.
        doi: 10.1016/S0166-3542(03)00174-8

    29. Bukh J. 2004. A critical role for the chimpanzee model in the study of hepatitis C. Hepatology, 39 (6): 1469-1475.
        doi: 10.1002/(ISSN)1527-3350

    30. Bukh J, Pietschmann T, Lohmann V, et al. 2002. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc Natl Acad Sci USA, 99 (22): 14416-14421.
        doi: 10.1073/pnas.212532699

    31. Burioni R, Perotti M, Mancini N, et al. 2008. Perspectives for the utilization of neutralizing human monoclonal antibodies as anti-HCV drugs. J Hepatol, 49 (2): 299-300.
        doi: 10.1016/j.jhep.2008.05.008

    32. Cai Z, Cai L, Jiang J, et al. 2007. Human serum amyloid A protein inhibits hepatitis C virus entry into cells. J Virol, 81 (11): 6128-6133.
        doi: 10.1128/JVI.02627-06

    33. Chang K, Wang T, Luo G. 2009. Proteomics study of the hepatitis C virus replication complex. Methods Mol Biol, 510: 185-193.
        doi: 10.1007/978-1-59745-394-3

    34. Chang K S, Cai Z, Zhang C, et al. 2006. Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities. J Virol, 80 (15): 7364-7374.
        doi: 10.1128/JVI.00586-06

    35. Chockalingam K, Simeon R L, Rice C M, et al. 2010. A cell protection screen reveals potent inhibitors of multiple stages of the hepatitis C virus life cycle. Proc Natl Acad Sci U S A, 107 (8): 3764-3769.
        doi: 10.1073/pnas.0915117107

    36. Choi S, Sainz B Jr., Corcoran P, et al. 2009. Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells. Xenobiotica, 39 (3): 205-217.
        doi: 10.1080/00498250802613620

    37. Choo Q-L, Kuo G, Weiner A J, et al. 1989. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science, 244: 359-362.
        doi: 10.1126/science.2523562

    38. Chung R, He W, Saquib A, et al. 2001. Hepatitis C virus replication is directly inhibited by IFN-alpha in a full-length binary expression system. Proc Natl Acad Sci USA, 98 (17): 9847-9852.
        doi: 10.1073/pnas.171319698

    39. Davis G L, Nelson D R, Terrault N, et al. 2005. A randomized, open-label study to evaluate the safety and pharmacokinetics of human hepatitis C immune globulin (Civacir) in liver transplant recipients. Liver Transpl, 11 (8): 941-949.
        doi: 10.1002/(ISSN)1527-6473

    40. Durantel D. 2009. Celgosivir, an alpha-glucosidase Ⅰ inhibitor for the potential treatment of HCV infection. Curr Opin Investig Drugs, 10 (8): 860-870.

    41. Einav S, Gerber D, Bryson P D, et al. 2008. Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis. Nat Biotechnol, 26 (9): 1019-1027.
        doi: 10.1038/nbt.1490

    42. Evans M J, von Hahn T, Tscherne D M, et al. 2007. Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature, 446 (7137): 801-805.
        doi: 10.1038/nature05654

    43. Fartoux L, Poujol-Robert A, Guechot J, et al. 2005. Insulin resistance is a cause of steatosis and fibrosis progression in chronic hepatitis C. Gut, 54 (7): 1003-1008.
        doi: 10.1136/gut.2004.050302

    44. Feinstone S, Alter H, Dienes H, et al. 1982. Non-A, non-B hepatitis in chimpanzees and marmosets. J Infect Dis, 144 (6): 588-598.

    45. Feld J J, Hoofnagle J H. 2005. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature, 436 (7053): 967-972.
        doi: 10.1038/nature04082

    46. Ferreon J C, Ferreon A C, Li K, et al. 2005. Molecular determinants of TRIF proteolysis mediated by the hepatitis C virus NS3/4A protease. J Biol Chem, 280 (21): 20483-20492.
        doi: 10.1074/jbc.M500422200

    47. Flisiak R, Feinman S V, Jablkowski M, et al. 2009. The cyclophilin inhibitor Debio 025 combined with PEG IFNalpha2a significantly reduces viral load in treatment-naive hepatitis C patients. Hepatology, 49 (5): 1460-1468.
        doi: 10.1002/hep.22835

    48. Foy E, Li K, Wang C, et al. 2003. Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science, 300 (5622): 1145-1148.
        doi: 10.1126/science.1082604

    49. Galun E, Burakova T, Ketzinel M, et al. 1995. Hepatitis C virus viremia in SCID-- > BNX mouse chimera. J Infect Dis, 172 (1): 25-30.
        doi: 10.1093/infdis/172.1.25

    50. Garry R F, Dash S. 2003. Proteomics computational analyses suggest that hepatitis C virus E1 and pestivirus E2 envelope glycoproteins are truncated class Ⅱ fusion proteins. Virology, 307 (2): 255-265.
        doi: 10.1016/S0042-6822(02)00065-X

    51. Gastaminza P, Whitten-Bauer C, Chisari F V. 2010. Unbiased probing of the entire hepatitis C virus life cycle identifies clinical compounds that target multiple aspects of the infection. Proc Natl Acad Sci USA, 107 (1): 291-296.
        doi: 10.1073/pnas.0912966107

    52. Glue P, Fang J W, Rouzier-Panis R, et al. 2000. Pegylated interferon-alpha2b: pharmacokinetics, phar-macodynamics, safety, and preliminary efficacy data. Hepatitis C Intervention Therapy Group. Clin Pharmacol Ther, 68 (5): 556-567.

    53. Goergen B, Jakobs S, Symmons P, et al. 1994. Quantitation of HCV-replication using one-step competitive reverse transcription-polymerase chain reaction and a solid phase, colorimetric detection method. J Hepatol, 21 (4): 678-682.
        doi: 10.1016/S0168-8278(94)80118-5

    54. Gosert R, Egger D, Lohmann V, et al. 2003. Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol, 77 (9): 5487-5492.
        doi: 10.1128/JVI.77.9.5487-5492.2003

    55. Gottwein J M, Scheel T K, Hoegh A M, et al. 2007. Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology, 133 (5): 1614-1626.
        doi: 10.1053/j.gastro.2007.08.005

    56. Gottwein J M, Scheel T K, Jensen T B, et al. 2009. Development and characterization of hepatitis C virus genotype 1-7 cell culture systems: role of CD81 and scavenger receptor class B type Ⅰ and effect of antiviral drugs. Hepatology, 49 (2): 364-377.
        doi: 10.1002/hep.22673

    57. Gouttenoire J, Penin F, Moradpour D. 2010. Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory. Rev Med Virol, 20 (2): 117-129.
        doi: 10.1002/rmv.v20:2

    58. Griffin S. 2010. Inhibition of HCV p7 as a therapeutic target. Curr Opin Investig Drugs, 11 (2): 175-181.

    59. Grompe M, Laconi E, Shafritz D. 1999. Principles of therapeutic liver repopulation. Semin Liver Dis, 19 (1): 7-14.
        doi: 10.1055/s-2007-1007093

    60. Grove J, Huby T, Stamataki Z, et al. 2007. Scavenger receptor BI and BII expression levels modulate hepatitis C virus infectivity. J Virol, 81 (7): 3162-3169.
        doi: 10.1128/JVI.02356-06

    61. Haid S, Pietschmann T, Pecheur E I. 2009. Low pH-dependent Hepatitis C Virus Membrane Fusion Depends on E2 Integrity, Target Lipid Composition, and Density of Virus Particles. J Biol Chem, 284 (26): 17657-17667.
        doi: 10.1074/jbc.M109.014647

    62. Heckel J L, Sandgren E P, Degen J L, et al. 1990. Neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator. Cell, 62 (3): 447-456.
        doi: 10.1016/0092-8674(90)90010-C

    63. Helle F, Dubuisson J. 2008. Hepatitis C virus entry into host cells. Cell Mol Life Sci, 65 (1): 100-112.
        doi: 10.1007/s00018-007-7291-8

    64. Hezode C, Forestier N, Dusheiko G, et al. 2009. Telaprevir and peginterferon with or without ribavirin for chronic HCV infection. N Engl J Med, 360 (18): 1839-1850.
        doi: 10.1056/NEJMoa0807650

    65. Hiraga N, Imamura M, Tsuge M, et al. 2007. Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis C virus and its susceptibility to interferon. FEBS Lett, 581 (10): 1983-1987.
        doi: 10.1016/j.febslet.2007.04.021

    66. Hiramatsu N, Dash S, Gerber M. 1997. HCV cDNA transfection to HepG2 cells. J Viral Hepat, 4 (Suppl 1): 61-67.

    67. Hirowatari Y, Hijikata M, Shimotohno K. 1995. A novel method for analysis of viral proteinase activity encoded by hepatitis C virus in cultured cells. Anal Biochem, 225 (1): 113-120.
        doi: 10.1006/abio.1995.1116

    68. Holzer M, Ziegler S, Albrecht B, et al. 2008. Identification of terfenadine as an inhibitor of human CD81-receptor HCV-E2 interaction: synthesis and structure optimization. Molecules, 13 (5): 1081-1110.
        doi: 10.3390/molecules13051081

    69. Honda A, Arai Y, Hirota N, et al. 1999. Hepatitis C virus structural proteins induce liver cell injury in transgenic mice. J Med Virol, 59 (3): 281-289.
        doi: 10.1002/(ISSN)1096-9071

    70. Hong Z, Beaudet-Miller M, Lanford R E, et al. 1999. Generation of transmissible hepatitis C virions from a molecular clone in chimpanzees. Virology, 256 (1): 36-44.
        doi: 10.1006/viro.1999.9603

    71. Hoofnagle J H. 2002. Course and outcome of hepatitis C. Hepatology, 36 (5 Suppl 1): S21-29.

    72. Hsu M, Zhang J, Flint M, et al. 2003. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci USA, 100 (12): 7271-7276.
        doi: 10.1073/pnas.0832180100

    73. Huang Y, Staschke K, De Francesco R, et al. 2007. Phosphorylation of hepatitis C virus NS5A nonstructural protein: a new paradigm for phosphorylation-dependent viral RNA replication? Virology, 364 (1): 1-9.
        doi: 10.1016/j.virol.2007.01.042

    74. Ikeda M, Abe K, Yamada M, et al.2006. Different anti-HCV profiles of statins and their potential for combination therapy with interferon. Hepatology, 44 (1): 117-125.
        doi: 10.1002/(ISSN)1527-3350

    75. Ikeda M, Yi M, Li K, et al. 2002. Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol, 76 (6): 2997-3006.
        doi: 10.1128/JVI.76.6.2997-3006.2002

    76. Ilan E, Arazi J, Nussbaum O, et al. 2002. The hepatitis C virus (HCV)-Trimera mouse: a model for evaluation of agents against HCV. J Infect Dis, 185 (2): 153-161.
        doi: 10.1086/jid.2002.185.issue-2

    77. Iro M, Witteveldt J, Angus A G, et al. 2009. A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication. Antiviral Res, 83 (2): 148-155.
        doi: 10.1016/j.antiviral.2009.04.007

    78. Jensen T B, Gottwein J M, Scheel T K, et al. 2008. Highly efficient JFH1-based cell-culture system for hepatitis C virus genotype 5a: failure of homologous neutralizing-antibody treatment to control infection. J Infect Dis, 198 (12): 1756-1765.
        doi: 10.1086/596514

    79. Jirasko V, Montserret R, Appel N, et al. 2008. Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly. J Biol Chem, 283 (42): 28546-28562.
        doi: 10.1074/jbc.M803981200

    80. Jones C T, Murray CL, Eastman D K, et al. 2007. Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol, 81 (16): 8374-8383.
        doi: 10.1128/JVI.00690-07

    81. Jopling C L, Yi M, Lancaster A M, et al. 2005. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science, 309 (5740): 1577-1581.
        doi: 10.1126/science.1113329

    82. Kapadia S B, Barth H, Baumert T, et al. 2007. Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I. J Virol, 81 (1): 374-383.
        doi: 10.1128/JVI.01134-06

    83. Karayiannis P, Scheuer P J, Bamber M, et al. 1983. Experimental infection of Tamarins with human non-A, non-B hepatitis virus. JMedVirol, 11: 251-256.

    84. Kato T, Date T, Miyamoto M, et al. 2003. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology, 125 (6): 1808-1817.
        doi: 10.1053/j.gastro.2003.09.023

    85. Kato T, Furusaka A, Miyamoto M, et al. 2001. Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient. J Med Virol, 64 (3): 334-339.
        doi: 10.1002/(ISSN)1096-9071

    86. Kawamura T, Furusaka A, Koziel M J, et al. 1997. Transgenic expression of hepatitis C virus structural proteins in the mouse. Hepatology, 25 (4): 1014-1021.
        doi: 10.1002/(ISSN)1527-3350

    87. Keck Z Y, Machida K, Lai M M, et al. 2008. Therapeutic control of hepatitis C virus: the role of neutralizing monoclonal antibodies. Curr Top Microbiol Immunol, 317: 1-38.

    88. Khromykh A A, Westaway E G. 1997. Subgenomic replicons of the flavivirus Kunjin: construction and applications. J Virol, 71 (2): 1497-1505.

    89. Kneteman N M, Mercer D F. 2005. Mice with chimeric human livers: who says supermodels have to be tall? Hepatology, 41 (4): 703-706.
        doi: 10.1002/(ISSN)1527-3350

    90. Kneteman N M, Toso C. 2009. In vivo study of HCV in mice with chimeric human livers. Methods Mol Biol, 510: 383-399.
        doi: 10.1007/978-1-59745-394-3

    91. Kneteman N M, Weiner A J, O'Connell J, et al. 2006. Anti-HCV therapies in chimeric scid-Alb/uPA mice parallel outcomes in human clinical application. Hepatology, 43 (6): 1346-1353.
        doi: 10.1002/(ISSN)1527-3350

    92. Koike K, Moriya K, Ishibashi K, et al. 1995. Expression of hepatitis C virus envelope proteins in transgenic mice. JGenVirol, 76: 3031-3038.

    93. Kolykhalov A, Mihalik K, Feinstone S, et al. 2000. Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo. J Virol, 74 (4): 2046-2051.
        doi: 10.1128/JVI.74.4.2046-2051.2000

    94. Kolykhalov A A, Agapov E V, Blight K J, et al. 1997. Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science, 277 (5325): 570-574.
        doi: 10.1126/science.277.5325.570

    95. Kota S, Scampavia L, Spicer T, et al. 2009. A Time-Resolved Fluorescence-Resonance Energy Transfer Assay for Identifying Inhibitors of Hepatitis C Virus Core Dimerization. Assay Drug Dev Technol.

    96. Koutsoudakis G, Herrmann E, Kallis S, et al. 2007. The level of CD81 cell surface expression is a key determinant for productive entry of hepatitis C virus into host cells. J Virol, 81 (2): 588-598.
        doi: 10.1128/JVI.01534-06

    97. Koutsoudakis G, Kaul A, Steinmann E, et al. 2006. Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses. J Virol, 80 (11): 5308-5320.
        doi: 10.1128/JVI.02460-05

    98. Krieger N, Lohmann V, Bartenschlager R. 2001. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 2001 May; 75 (10): 4614-24, 75 (10): 4614-4624.

    99. Kwo P, Lawitz E J, McCone J, et al. 2008. HCV SPRINT-1: Boceprevir plus Peginterferon alfa-2b/ Ribavirin for Treatment of Genotype 1 Chronic Hepatitis C in Previously Untreated Patients: 59th Annual Meeting of the American Association for the Study of Liver Diseases, San Francisco, CA. Hepatology, 48, Suppl 1 (4): 1027A.

    100. Lanford R E, Bigger C, Bassett S, et al. 2001. The chimpanzee model of hepatitis C virus infections. Ilar J, 42 (2): 117-126.
        doi: 10.1093/ilar.42.2.117

    101. Lanford R E, Hildebrandt-Eriksen E S, Petri A, et al. 2010. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science, 327 (5962): 198-201.
        doi: 10.1126/science.1178178

    102. Lavie M, Voisset C, Vu-Dac N, et al. 2006. Serum amyloid A has antiviral activity against hepatitis C virus by inhibiting virus entry in a cell culture system. Hepatology, 44 (6): 1626-1634.
        doi: 10.1002/hep.v44:6

    103. Lavillette D, Bartosch B, Nourrisson D, et al. 2006. Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes. J Biol Chem, 281 (7): 3909-3917.
        doi: 10.1074/jbc.M509747200

    104. Lavillette D, Pecheur E I, Donot P, et al. 2007. Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus. J Virol, 81 (16): 8752-8765.
        doi: 10.1128/JVI.02642-06

    105. Law M, Maruyama T, Lewis J, et al. 2008. Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med, 14 (1): 25-27.
        doi: 10.1038/nm1698

    106. Lemm J A, O'Boyle D, 2nd, Liu M, et al. 2010. Identification of hepatitis C virus NS5A inhibitors. J Virol, 84 (1): 482-491.
        doi: 10.1128/JVI.01360-09

    107. Lerat H, Honda M, Beard M R, et al. 2002. Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus. Gastroenterology, 122 (2): 352-365.
        doi: 10.1053/gast.2002.31001

    108. Li K, Foy E, Ferreon J C, et al. 2005. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci USA, 102 (8): 2992-2997.
        doi: 10.1073/pnas.0408824102

    109. Li Q, Brass A L, Ng A, et al. 2009. A genome-wide genetic screen for host factors required for hepatitis C virus propagation. Proc Natl Acad Sci USA, 106 (38): 16410-16415.
        doi: 10.1073/pnas.0907439106

    110. Li X D, Sun L, Seth R B, et al. 2005. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc Natl Acad Sci U S A, 102 (49): 17717-17722.
        doi: 10.1073/pnas.0508531102

    111. Liang T J, Rehermann B, Seeff L B, et al. 2000. Pathogenesis, natural history, treatment, and prevention of hepatitis C. Ann Intern Med, 132 (4): 296-305.
        doi: 10.7326/0003-4819-132-4-200002150-00008

    112. Lim S P, Soo H M, Tan Y H, et al. 2002. Inducible system in human hepatoma cell lines for hepatitis C virus production. Virology, 303 (1): 79-99.
        doi: 10.1006/viro.2002.1687

    113. Lindenbach B D, Evans M J, Syder A J, et al. 2005. Complete replication of hepatitis C virus in cell culture. Science, 309 (5734): 623-626.
        doi: 10.1126/science.1114016

    114. Lindenbach B D, Meuleman P, Ploss A, et al. 2006. Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc Natl Acad Sci USA.

    115. Liu S, Yang W, Shen L, et al. 2009. Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection. J Virol, 83 (4): 2011-2014.
        doi: 10.1128/JVI.01888-08

    116. Lohmann V, Hoffmann S, Herian U, et al. 2003. Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol, 77 (5): 3007-3019.
        doi: 10.1128/JVI.77.5.3007-3019.2003

    117. Lohmann V, Korner F, Dobierzewska A, et al. 2001. Mutations in Hepatitis C Virus RNAs Conferring Cell Culture Adaptation. J Virol, 75 (3): 1437-1449.
        doi: 10.1128/JVI.75.3.1437-1449.2001

    118. Lohmann V, Korner F, Koch J, et al. 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science, 285 (5424): 110-113.
        doi: 10.1126/science.285.5424.110

    119. Maeda N, Watanabe M, Okamoto S, et al. 2004. Hepatitis C virus infection in human liver tissue engrafted in mice with an infectious molecular clone. Liver Int, 24 (3): 259-267.
        doi: 10.1111/liv.2004.24.issue-3

    120. Masson D, Koseki M, Ishibashi M, et al. 2009. Increased HDL cholesterol and apoA-I in humans and mice treated with a novel SR-BI inhibitor. Arterioscler Thromb Vasc Biol, 29 (12): 2054-2060.
        doi: 10.1161/ATVBAHA.109.191320

    121. Matsumura T, Hu Z, Kato T, et al. 2009. Amphipathic DNA polymers inhibit hepatitis C virus infection by blocking viral entry. Gastroenterology, 137 (2): 673-681.
        doi: 10.1053/j.gastro.2009.04.048

    122. McHutchison J G, Everson G T, Gordon S C, et al. 2009. Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection. N Engl J Med, 360 (18): 1827-1838.
        doi: 10.1056/NEJMoa0806104

    123. Meertens L, Bertaux C, Dragic T. 2006. Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles. J Virol, 80 (23): 11571-11578.
        doi: 10.1128/JVI.01717-06

    124. Mercer D, Schiller D, Elliott J, et al. 2001. Hepatitis C virus replication in mice with chimeric human livers. Nat Med, 7 (8): 927-933.
        doi: 10.1038/90968

    125. Meunier J C, Russell R S, Goossens V, et al. 2008. Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus. J Virol, 82 (2): 966-973.
        doi: 10.1128/JVI.01872-07

    126. Moradpour D, Penin F, Rice C M. 2007. Replication of hepatitis C virus. Nat Rev Microbiol, 5 (6): 453-463.
        doi: 10.1038/nrmicro1645

    127. Moriya K, Yotsuyanagi H, Shintani Y, et al. 1997. Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. J Gen Virol, 78 (Pt 7): 1527-1531.

    128. Murakami Y, Noguchi K, Yamagoe S, et al. 2009. Identification of bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication by tube-capture-RT-PCR. Antiviral Res, 83 (2): 112-117.
        doi: 10.1016/j.antiviral.2009.03.008

    129. Murray E M, Grobler J A, Markel E J, et al. 2003. Persistent replication of hepatitis C virus replicons expressing the betalactamase reporter in subpopulations of highly permissive Huh7 cells. J Virol, 77 (5): 2928-2935.
        doi: 10.1128/JVI.77.5.2928-2935.2003

    130. Myung J, Khalap N, Kalkeri G, et al. 2001. Inducible model to study negative strand RNA synthesis and assembly of hepatitis C virus from a full-length cDNA clone. J Virol Methods, 94 (1-2): 55-67.
        doi: 10.1016/S0166-0934(01)00278-6

    131. Nahmias Y, Goldwasser J, Casali M, et al. 2008. Apolipoprotein B-dependent hepatitis C virus secretion is inhibited by the grapefruit flavonoid naringenin. Hepatology, 47 (5): 1437-1445.
        doi: 10.1002/hep.22197

    132. Nakabayashi H, Taketa K, Miyano K, et al. 1982. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res, 42 (9): 3858-3863.

    133. Noto H, Raskin P. 2006. Hepatitis C infection and diabetes. J Diabetes Complications, 20 (2): 113-120.
        doi: 10.1016/j.jdiacomp.2006.01.001

    134. Paeshuyse J, Kaul A, De Clercq E, et al. 2006. The non-immunosuppressive cyclosporin DEBIO-025 is a potent inhibitor of hepatitis C virus replication in vitro. Hepatology, 43 (4): 761-770.
        doi: 10.1002/(ISSN)1527-3350

    135. Pan K L, Lee J C, Sung H W, et al. 2009. Development of NS3/4A protease-based reporter assay suitable for efficiently assessing hepatitis C virus infection. Antimicrob Agents Chemother, 53 (11): 4825-4834.
        doi: 10.1128/AAC.00601-09

    136. Pasquinelli C, Shoenberger J M, Chung J, et al. 1997. Hepatitis C virus core and E2 protein expression in transgenic mice. Hepatology, 25 (3): 719-727.
        doi: 10.1002/(ISSN)1527-3350

    137. Pecheur E I, Lavillette D, Alcaras F, et al. 2007. Biochemical mechanism of hepatitis C virus inhibition by the broad-spectrum antiviral arbidol. Biochemistry, 46 (20): 6050-6059.
        doi: 10.1021/bi700181j

    138. Pietschmann T, Kaul A, Koutsoudakis G, et al. 2006. Construction and characterization of infectious intra-genotypic and intergenotypic hepatitis C virus chimeras. Proc Natl Acad Sci USA, 103 (19): 7408-7413.
        doi: 10.1073/pnas.0504877103

    139. Pietschmann T, Zayas M, Meuleman P, et al. 2009. Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations. PLoS Pathog, 5 (6): e1000475.
        doi: 10.1371/journal.ppat.1000475

    140. Pileri P, Uematsu Y, Campagnoli S, et al. 1998. Binding of hepatitis C virus to CD81. Science, 282 (5390): 938-941.
        doi: 10.1126/science.282.5390.938

    141. Ploss A, Evans M J, Gaysinskaya V A, et al. 2009. Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature, 457 (7231): 882-886.
        doi: 10.1038/nature07684

    142. Ploss A, Khetani S R, Jones C T, et al. 2010. Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures. Proc Natl Acad Sci USA, 107 (7): 3141-3145.
        doi: 10.1073/pnas.0915130107

    143. Ploss A, Rice C M. 2009. Towards a small animal model for hepatitis C. EMBO Rep, 10 (11): 1220-1227.
        doi: 10.1038/embor.2009.223

    144. Popescu C I, Dubuisson J. 2009. Role of lipid metabolism in hepatitis C virus assembly and entry. Biol Cell, 102 (1): 63-74.

    145. Poynard T, Yuen M F, Ratziu V, et al. 2003. Viral hepatitis C. Lancet, 362 (9401): 2095-2100.
        doi: 10.1016/S0140-6736(03)15109-4

    146. Prabhu R, Joshi V, Garry R F, et al. 2004. Interferon alpha-2b inhibits negative-strand RNA and protein expression from full-length HCV1a infectious clone. Exp Mol Pathol, 76 (3): 242-252.
        doi: 10.1016/j.yexmp.2004.01.004

    147. Reisner Y, Dagan S. 1998. The Trimera mouse: generating human monoclonal antibodies and an animal model for human diseases. Trends Biotechnol, 16 (6): 242-246.
        doi: 10.1016/S0167-7799(98)01203-7

    148. Romero-Gomez M. 2006. Hepatitis C and insulin resistance: steatosis, fibrosis and non-response. Rev Esp Enferm Dig, 98 (8): 605-615.

    149. Romero-Gomez M. 2006. Insulin resistance and hepatitis C. World J Gastroenterol, 12 (44): 7075-7080.
        doi: 10.3748/wjg.v12.i44.7075

    150. Sainz B Jr., Barretto N, Uprichard S L. 2009. Hepatitis C Virus infection in phenotypically distinct Huh7 cell lines. PLoS ONE, 4 (8): e6561.
        doi: 10.1371/journal.pone.0006561

    151. Sainz B, Jr., Chisari F V. 2006. Production of infectious hepatitis C virus by well-differentiated, growth-arrested human hepatoma-derived cells. J Virol, 80 (20): 10253-10257.
        doi: 10.1128/JVI.01059-06

    152. Sainz B, Jr., TenCate V, Uprichard S L. 2009. Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection. Virol J, 6: 103.
        doi: 10.1186/1743-422X-6-103

    153. Sarrazin C, Rouzier R, Wagner F, et al. 2007. SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders. Gastroenterology, 132 (4): 1270-1278.
        doi: 10.1053/j.gastro.2007.01.041

    154. Scarselli E, Ansuini H, Cerino R, et al. 2002. The human scavenger receptor class B type Ⅰ is a novel candidate receptor for the hepatitis C virus. Embo J, 21 (19): 5017-5025.
        doi: 10.1093/emboj/cdf529

    155. Schaller T, Appel N, Koutsoudakis G, et al. 2007. Analysis of hepatitis C virus superinfection exclusion by using novel fluorochrome gene-tagged viral genomes. J Virol, 81 (9): 4591-4603.
        doi: 10.1128/JVI.02144-06

    156. Scheel T K, Gottwein J M, Jensen T B, et al. 2008. Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization. Proc Natl Acad Sci USA, 105 (3): 997-1002.
        doi: 10.1073/pnas.0711044105

    157. Schinazi R, Ilan E, Black P, et al. 1999. Cell-based and animal models for hepatitis B and C viruses. Antivir Chem Chemother, 10 (3): 99-114.
        doi: 10.1177/095632029901000301

    158. Steinmann E, Whitfield T, Kallis S, et al. 2007. Antiviral effects of amantadine and iminosugar derivatives against hepatitis C virus. Hepatology, 46 (2): 330-338.

    159. Sulkowski M S. 2007. Specific targeted antiviral therapy for hepatitis C. Curr Gastroenterol Rep, 9 (1): 5-13.
        doi: 10.1007/s11894-008-0015-x

    160. Suzuki T, Aizaki H, Murakami K, et al. 2007. Molecular biology of hepatitis C virus. J Gastroenterol, 42 (6): 411-423.
        doi: 10.1007/s00535-007-2030-3

    161. Tai A W, Benita Y, Peng L F, et al. 2009. A functional genomic screen identifies cellular cofactors of hepatitis C virus replication. Cell Host Microbe, 5 (3): 298-307.
        doi: 10.1016/j.chom.2009.02.001

    162. Tencate V, Sainz B J, Cotler S, et al. 2010. Potential treatment options and future research to increase hepatitis C virus treatment response rate. Hepatic Medicine: Evidence and Research, in press.

    163. Trotard M, Lepere-Douard C, Regeard M, et al. 2009. Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening. Faseb J, 23 (11): 3780-3789.
        doi: 10.1096/fj.09-131920

    164. Tscherne D M, Jones C T, Evans M J, et al. 2006. Time-and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J Virol, 80 (4): 1734-1741.
        doi: 10.1128/JVI.80.4.1734-1741.2006

    165. Uprichard S L, Chung J, Chisari F V, et al. 2006. Replication of a hepatitis C virus replicon clone in mouse cells. Virol J, 3: 89.
        doi: 10.1186/1743-422X-3-89

    166. von Hahn T, Rice C M. 2008. Hepatitis C virus entry. J Biol Chem, 283 (7): 3689-3693.
        doi: 10.1074/jbc.R700024200

    167. Wakita T, Pietschmann T, Kato T, et al. 2005. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med, 11 (7): 791-796.
        doi: 10.1038/nm1268

    168. Wang C, Gale M Jr., Keller B C, et al. 2005. Identification of FBL2 as a geranylgeranylated cellular protein required for hepatitis C virus RNA replication. Mol Cell, 18 (4): 425-434.
        doi: 10.1016/j.molcel.2005.04.004

    169. Watanabe T, Katagiri J, Kojima H, et al. 1987. Studies on transmission of human non-A, non-B hepatitis to marmosets. J Med Virol, 22: 143-156.
        doi: 10.1002/(ISSN)1096-9071

    170. Welsch S, Miller S, Romero-Brey I, et al. 2009. Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe, 5 (4): 365-375.
        doi: 10.1016/j.chom.2009.03.007

    171. Williams R. 2006. Global challenges in liver disease. Hepatology, 44 (3): 521-526.
        doi: 10.1002/(ISSN)1527-3350

    172. Wolk B, Buchele B, Blum H E, et al. A dynamic view of hepatitis C virus replication complexes. Presented at the 11th International Symposium on HCV and related viruses; 2004, Heidelberg, Germany.

    173. Wu G Y, Konishi M, Walton C M, et al. 2005. A novel immunocompetent rat model of HCV infection and hepatitis. Gastroenterology, 128 (5): 1416-1423.
        doi: 10.1053/j.gastro.2005.03.015

    174. Wunschmann S, Medh J D, Klinzmann D, et al. 2000. Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor. J Virol, 74 (21): 10055-10062.
        doi: 10.1128/JVI.74.21.10055-10062.2000

    175. Xie Z, Riezu-Boj J, Lasarte J, et al. 1998. Transmission of hepatitis C virus infection to tree shrews. Virology, 244 (2): 513-520.
        doi: 10.1006/viro.1998.9127

    176. Yanagi M, Purcell R H, Emerson S U, et al. 1999. Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras. Virology, 262 (1): 250-263.
        doi: 10.1006/viro.1999.9889

    177. Yanagi M, Purcell R H, Emerson S U, et al. 1997. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci USA, 94 (16): 8738-8743.
        doi: 10.1073/pnas.94.16.8738

    178. Yanagi M, St Claire M, Emerson S U, et al. 1999. In vivo analysis of the 3' untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone. Proc Natl Acad Sci USA, 96 (5): 2291-2295.
        doi: 10.1073/pnas.96.5.2291

    179. Yanagi M, St Claire M, Shapiro M, et al. 1998. Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology, 244 (1): 161-172.
        doi: 10.1006/viro.1998.9092

    180. Yang J P, Zhou D, Wong-Staal F. 2009. Screening of small-molecule compounds as inhibitors of HCV entry. Methods Mol Biol, 510: 295-304.
        doi: 10.1007/978-1-59745-394-3

    181. Ye J, Wang C, Sumpter R Jr., et al. 2003. Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation. Proc Natl Acad Sci U S A, 100 (26): 15865-15870.
        doi: 10.1073/pnas.2237238100

    182. Yi M, Bodola F, Lemon S M. 2002. Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein. Virology, 304 (2): 197-210.
        doi: 10.1006/viro.2002.1652

    183. Yi M, Lemon S. 2002. Replication of subgenomic hepatitis A virus RNAs expressing firefly luciferase is enhanced by mutations associated with adaptation of virus to growth in cultured cells. J Virol, 76 (3): 1171-1180.
        doi: 10.1128/JVI.76.3.1171-1180.2002

    184. Yi M, Villanueva R A, Thomas D L, et al. 2006. Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci USA, 103 (7): 2310-2315.
        doi: 10.1073/pnas.0510727103

    185. Yoo B J, Selby M J, Choe J, et al. 1995. Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV. J Virol, 69 (1): 32-38.

    186. Yu X, Sainz B Jr., Uprichard S L. 2009. Development of a cell-based hepatitis C virus infection fluorescent resonance energy transfer assay for high-throughput antiviral compound screening. Antimicrob Agents Chemother, 53 (10): 4311-4319.
        doi: 10.1128/AAC.00495-09

    187. Zeisel M B, Barth H, Schuster C, et al. 2009. Hepatitis C virus entry: molecular mechanisms and targets for antiviral therapy. Front Biosci, 14: 3274-3285.

    188. Zeisel M B, Koutsoudakis G, Schnober E K, et al. 2007. Scavenger receptor class B type Ⅰ is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81. Hepatology, 46 (6): 1722-1731.
        doi: 10.1002/hep.v46:6

    189. Zhang Y, Weady P, Duggal R, et al. 2008. Novel chimeric genotype 1b/2a hepatitis C virus suitable for high-throughput screening. Antimicrob Agents Chemother, 52 (2): 666-674.
        doi: 10.1128/AAC.01133-07

    190. Zhao X, Tang Z, Klumpp B, et al. 2002. Primary hepatocytes of Tupaia belangeri as a potential model for hepatitis C virus infection. J Clin Invest, 109 (2): 221-232.
        doi: 10.1172/JCI0213011

    191. Zhong J, Gastaminza P, Cheng G, et al. 2005. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA, 102 (26): 9294-9299.
        doi: 10.1073/pnas.0503596102

    192. Zhu H, Elyar J, Foss R, et al. 2009. Primary human hepatocyte culture for HCV study. Methods Mol Biol, 510: 373-382.
        doi: 10.1007/978-1-59745-394-3

  • 加载中

Figures(3) / Tables(2)

Article Metrics

Article views(5098) PDF downloads(17) Cited by()

Related
Proportional views

    Hepatitis C Virus Experimental Model Systems and Antiviral drug Research*

      Corresponding author: Susan L. Uprichard, sluprich@uic.edu
    • Department of Medicine, Section of Hepatology and Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, USA
    Fund Project:  National Institutes of Health grants AI070827American Cancer Society grant RSG-09-076-01National Institutes of Health grants CA33266

    Abstract: An estimated 130 million people worldwide are chronically infected with hepatitis C virus (HCV) making it a leading cause of liver disease worldwide. Because the currently available therapy of pegylated interferon-alpha and ribavirin is only effective in a subset of patients, the development of new HCV antivirals is a healthcare imperative. This review discusses the experimental models available for HCV antiviral drug research, recent advances in HCV antiviral drug development, as well as active research being pursued to facilitate development of new HCV-specific therapeutics.

    • Hepatitis C virus (HCV), a member of the Flaviviridae family of enveloped positive-strand RNA viruses, is a leading cause of liver disease worldwide. Although HCV infection is usually asymptomatic and 10-30% of infected individuals successfully clear the infection[2, 145], ~70% of infections persist with the risk of progressive liver complications, such as fibrosis, cirrhosis, steatosis, insulin resistance, and/or hepatocellular carcinoma (HCC)[4, 5, 43, 71, 111, 133, 148, 149], which can ultimately necessitate liver transplantation if HCV infection is not successfully treated (Fig. 1).

      Figure 1.  Progression of HCV infection. HCV infection is successfully cleared by 10%-30% of individuals after a transient acute infection. However, at least 70% of infections result in long-term chronic infection. Chronic HCV infection can led to progressive liver disease such as fibrosis, cirrhosis, steatosis, insulin resistance, or hepatocellular carcinoma unless successfully treated. Ultimately, HCV-associated liver disease can necessitate liver transplantation. Unfortunately, for those who receive a liver transplant, allograft infection with HCV is universal with disease progression in the new liver significantly accelerated compared to pre-transplant rates.

      To date, combination pegylated interferon-alpha (pIFN-a) and ribavirin[52] is the licensed standard of care (SOC) treatment for HCV; however, several limitations restrict its use and efficacy. First, because viral, host, and environmental factors significantly affect the success of SOC treatment (reviewed in [162]), numerous contraindications limit the number of patients eligible for therapy. Of those who are treated, sustained virological response (SVR) is only achieved in ~80% of individuals infected with genotypes 2 or 3 and 40%-50% of individuals infected with genotypes 1 or 4[3]. In addition, therapy itself has a spectrum of toxic side effects and complications, which severely limit patient compliance and thus treatment efficacy [45]. Hence, with an estimated 130 million people worldwide chronically infected[4], and the number of HCV patients needing medical care is expected to increase dramatically over the next decade[171], the development of new more specific HCV antivirals is a healthcare imperative. This review will discuss the experimental models available for HCV antiviral drug research, the most recent advances in HCV antiviral drug development, as well as future research directions focused on developing new HCV-specific therapeutics.

    • Since it's discovery in 1989 as the causative agent on non-A non-B hepatitis[37], the HCV lifecycle and host-virus interactions that determine infection outcome have been difficult to study because experimental HCV cell culture infection systems and suitable small animal models have not been readily available. Conse-quently, the development of preventive vaccines and anti-HCV therapeutics has been severely hampered. Notably however, different model systems have now been developed and successfully used to study isolated aspects of the HCV lifecycle (Table 1) and three models in particular have significantly advanced our understanding of HCV and accelerated HCV antiviral development. These include the development of HCV replicons, HCV pseudotyped particles (HCVpp), and most recently infectious HCV cell culture systems, each of which is discussed in detail below.

      Table 1.  HCV Experimental Models

    • Shortly after the 1989 cloning of the HCV genome [37], full length HCV RNA was synthesized and shown to be infectious in chimpanzees after intrahepatic inoculation[13, 70, 94, 176, 177, 179] providing insights into salient aspects of HCV infection and pathogenesis[93, 178] (reviewed in [29]and [100]); however, these consensus clones were found to be replication defective in cell culture, limiting in vitro HCV model development. This changed in 1999, when Lohman et al overcame the in vitro HCV replication barrier by devising a genotype 1b HCV replicon system[118] based upon previous autonomous replication models of other flaviviruses[14, 88]. Specifically, selectable subgenomic HCV RNAs were engineered in which the region encoding the HCV structural genes, with or without NS2, was replaced by the selectable antibiotic resistant marker, neomycin phosphotransferase (Neo). Upon transfection into mammalian cells, Neo is translated via the HCV internal ribosome entry site (IRES), while the viral nonstructural (NS) proteins required to replicate the input template RNA are synthesized from the encephalomyocarditis virus (EMCV) IRES (Fig. 2). Antibiotic-resistant cell clones, harboring autonomously replicating HCV replicon RNA can then be selectively expanded.

      Figure 2.  HCV genomic structure and polyprotein processing. A: Diagram of subgenomic and full length bicistronic HCV replicons. B: Diagram of the HCV RNA genome. The ~9.6kb RNA encodes a single open reading frame for translation of an ~3010 amino acid polyprotein. Both viral and host proteases cleave this polyprotein into at least 10 viral protein products (Amino acid numbers are shown above each protein for HCV H77 strain; genotype 1a; GenBank accession number AF011752). Dark blue arrows denote cleavage sites by the endoplasmic reticulum signal peptidase, purple arrow indicates C-terminal processing of the core protein by signal peptide peptidase and black and red arrows indicate cleavages by the HCV NS2/3 and NS3/4a proteases, respectively. The structural proteins (blue) are located at the 5' end of the ORF, followed by p7 (aqua) and the non-structural (NS) proteins (green). Descriptive titles and known activities for each viral protein are listed.

      Although it was discovered that robust HCV replicon replication required numerous cell culture adaptive mutations[21, 98, 116, 117] which when cloned back into a full length construct resulted in an HCV genome incapable of producing infectious virus[8] or causing infection in chimpanzees following intrahepatic inoculation[30, 139], HCV replicons have proven invaluable for the in vitro study of HCV replication and subgenomic replicons of several genotypes (e.g. 1a, 1b and 2a) as well as full-length replicons having been developed[21, 22, 75, 84, 167]. In addition to the identification of RNA elements and proteins involved in the viral replication process, HCV replicons have provided the means to characterize the viral replication complex at the biochemical and ultrastructural level with fluorescently-tagged viral proteins, such as a NS5A-GFP fusion, having allowed for visualization and tracking HCV replication complexes in living cells [155, 172]. Most important to anti-HCV drug discovery and the study of HCV drug resistance, numerous replicon constructs with exogenous reporters, such as luciferase, secreted alkaline phosphatase, chloramphenicol transferase, beta-lactamase, or beta-galactasidase have facilitated the development of reporter-based HCV replication screening assays[53, 67, 98, 129, 182, 183].

    • To allow for the study of HCV entry, researchers created infectious pseudoparticles incorporating the HCV E1-E2 glycoproteins on murine leukemia virus (MLV) or HIV-1 retroviral core particles[10, 72]. Although HCVpp only resemble HCV at the level of surface glycoprotein expression, their ability to mimic HCV entry has helped dissect the HCV entry process identifying numerous cellular entry factors and neutralizing epitopes on the viral glycoproteins [63, 87, 166, 187]. With pseudotype particles of all six HCV genotypes now available encoding for various reporter proteins (e.g. GFP, β-galactosidase, or luciferase) the HCVpp system represents a focused tool for studying HCV entry across many HCV genotypes and thus will undoubtedly continue to facilitate the development of viral entry inhibitors, as illustrated by a 2009 report describing a luciferase-based HCVpp high throughput screening approach for the identification of HCV entry inhibitors[180].

    • The ability to recapitulate the entire viral lifecycle in vitro was finally achieved in 2005 when several groups reported robust HCV infection and production of infectious progeny HCV (termed HCVcc) in Huh7 human hepatoma cell cultures using the HCV genotype 2a JFH-1 consensus genome cloned from a Japanese Fulminant Hepatitis patient or derivative thereof[85, 113, 167, 191]. These systems yield viral titers between 104-106infectious units/mL allowing infection to spread throughout a culture within days after inoculation at low multiplicities of infection (MOI) and the serial passage of virus without loss in infectivity. Importantly, HCVcc is infectious in chimpanzees and primary human hepatocytes transplanted into SCID-uPA mice, and virus recovered from these animals is infectious in vitro[65, 114].

      With this tool to dissect the complete viral life cycle now available, high throughput screening (HTS) assays utilizing the infectious HCV cell culture system are rapidly being developed as a means to identify new HCV-specific therapeutics targeting all aspects of infection. In 2008, Zhang et al published an HCVcc HTS assay, utilizing an HCVcc construct encoding a luciferase reporter gene[189]. Although HTS assays based on exogenous reporters are common, inherent issues such as specificity (i.e. effect of compound on reporter expression/function) and effects of foreign sequences on viral infection (e.g. decreases in HCVcc infection efficiency) can be problematic. To this end, several groups have recently developed HCVcc-based HTS systems, which do not depend on insertion of a foreign reporter into the viral constructs[77, 128, 135, 186]. For example, Yu et al, (2009) recently developed a simple mix-and-measure cell-based HCV infection HTS assay based on an HCV NS3 protease FRET assay[186]. Unlike typical viral HTS assays that infect at a high MOI and are thus focused on a single cycle of virus replication, the assay described by Yu et al. (2009) incorporates a low MOI approach allowing inhibitors that target any aspect of the HCV lifecycle (e.g. entry, replication, assembly, egress and spread) to be detected over the course of several rounds of viral replication and spread[186]. Following this low MOI approach, Gastaminza et al, recently developed an HCVcc colorimetric assay based on immuno-staining with an anti-E2 antibody to identify 33 inhibitors of HCV at multiple lifecycle stages[51]; however, this approach is not as readily amenable to HTS as it requires numerous antibody incubations and washing steps. To circumvent the need for secondary toxicity screening, Chockalingam et al, (2010) very recently reported the development of an HCVcc cell protection assay, which measures cell viability as a readout of anti-HCV compound activity. As such, viability indicates not only effective blocking of HCV infection, but also the lack of drug-induced cytotoxicity[35]. Using this approach, the authors successfully screened a 1280 compound library and identified 55 compounds targeting HCV at the level of entry, replication and virus production[35].

    • HCV replicons, HCVpp, and most recently infectious HCVcc systems have advanced our understanding of the viral lifecycle highlighting numerous potential antiviral drug targets (Fig. 3), and each step of the HCV life cycle continues to be rigorously studied to identify and test additional HCV-specific therapeutics. While the term "Specifically Targeted Antiviral Therapy for Hepatitis C" (STAT-C) was originally coined to describe inhibitors that directly target HCV proteins[159], inhibitors directed against host proteins involved in HCV infection are also proving to be valuable drug targets and an unprecedented number of putative inhibitors specifically targeting each step of the viral lifecycle have already been identified and are currently being evaluated clinically (Table 2). The two types of STAT-C molecules that have progressed furthest in the pipeline are NS3/4a protease inhibitors [Telapravir (TVR; VX-950; Vertex) [64, 122] and Boceprevir (BVR; SCH503034; Schering) [99, 153]] and NS5B polymerase inhibitors (reviewed in [162]). The use of these compounds in combination with current HCV SOC have yielded promising results, and their clinical availability in the immediate future will certainly mark the beginning of an exciting period that is anticipated to revolutionize HCV therapy. Further improvements in treatment efficacy in the future will likely also be achieved by increasing the repertoire of specifically targeted HCV therapies to include compounds that target additional viral and viral-host interactions. Thus, each step of the viral lifecycle (Fig. 3), each of the 10 viral proteins, and the viral RNA genome itself (Fig. 2B) represent potential drug targets for exploitation.

      Figure 3.  The HCV Lifecycle. 1. Binding of the virus to cell surface receptors; 2. HCV entry into the cell via clathrin-mediated endocytosis; 3. Endocytic vesicle acidification and release of the viral genome into cytoplasm (i.e. fusion and uncoating); 4. IRES-mediated polyprotein translation; 5. Polyprotein processing; 6. Formation of viral replication complexes (i.e. the membranous web); 7. Viral RNA replication; 8. Packaging and assembly of progeny virions; 9. Virion maturation and release via the cellular secretory system. Although depicted separately, many of these viral functions likely occur concurrently and within close proximity. In particular, we show here lipid droplets (LD), which are thought to be the site of HCV particle assembly, closely associated with the membranous web; however the spatial relationship of these two viral activities remains to be determined. GAG, glycosaminoglycan; LDLR, low density lipoprotein receptor; SR-B1, scavenger receptor class B member I, CLDN1, claudin-1; OCLN, occludin; IRES, internal ribosome entry site; LD, lipid droplets.

      Table 2.  Specifically Targeted HCV Inhibitors (March 2010)

    • HCV infection begins with the binding of the viral particle to receptors on the host cell surface. Four cellular receptors have been shown to be necessary, but not sufficient for HCV entry, the tetraspanin protein CD81[10, 72, 140, 174, 191], the scavenger receptor class B member Ⅰ (SR-B1)[10, 60, 82, 154, 188], and the tight junction proteins CLDN1[42] and OCLN[15, 115, 141] (Fig. 3, Step 1). Following a multi-step binding event, the viral particle is taken up into the cell via clathrin-coated mediated endocytosis[20, 123] (Fig. 3, Step 2). Within the acidified endosomal compartment E1/E2-mediated class Ⅱ fusion occurs between the virion envelope and the endosomal membrane[11, 50, 61, 72, 97, 103, 104, 164] resulting in nucleocapsid release into the cell cytoplasm (Fig. 3, Step 3). Although HCV entry is still incompletely defined, the identification of required entry factors and development of neutralizing antibodies against the virus[26, 31, 105, 125] has permitted the in vitro discovery of a number of novel HCV entry inhibitors which may prove to be effective clinically. For example, the compound Terfenadine has been shown to interfere with the HCV-E2-CD81 receptor interaction[68]. Likewise, serum amyloid A may similarly function by binding to SR-B1 preventing HCV-SR-B1 interactions[32, 102]. Post binding inhibition of HCV membrane fusion in vitro has been reported with the broad-spectrum antiviral compound, Arbidol [24, 25, 137]. More recently, post-binding inhibition was demonstrated both in vitro and in vivo in the chimeric mouse model using amphipathic DNA polymers, which are hypothesized to block viral entry at the level of virion internalization and/or fusion[121]. Notably, however, several entry inhibitors are already at the clinical trial phase including the human hepatitis C immune globulin Civacir (Nabi Biopharmacueticals)[39] and the SR-B1 inhibitor ITX5061 (iTherX Pharma-ceuticals)[120], both of which inhibit HCV entry at the level of virion-receptor interactions.

    • The ~9.6 kb viral RNA genome released into the cell cytoplasm encodes a single open reading frame flanked by highly structured 5' and 3' untranslated regions (UTRs). The 5' UTR contains an IRES that is required for translation of a ~3010 amino acid viral polyprotein (Fig. 2B and Fig. 3, Step 4), which is co-and post-translationally cleaved by cellular and viral proteases into mature structural and NS proteins (Fig. 2B and Fig. 3, Step 5). Past efforts to block translation of the HCV polyprotein involved targeting the viral IRES with antisense oligonucleotides, ribozymes, and siRNAs, but the compounds that entered clinical trials were halted due to lack of response and/or toxicity. However, the positive clinical performance of NS3/4a protease inhibitors highlights viral protein production as an effective therapeutic target. Compounds that block the viral NS3/4a protease inhibit polyprotein cleavage and thus HCV infection by prevent the processing needed to generate the individual viral proteins required for viral replication. In addition, inhibition of NS3/4a activity reduces NS3/4a-mediated cleavage of the host IFN simulating proteins Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) and IFNαpromoter stimulator (IPS-1, CARDIF, VISA), and thus also restores certain aspects of the host cell innate immune response which is otherwise suppressed by NS3/4a[46, 48, 108, 110]. As such, protease inhibitor development has progressed with two promising protease inhibitors currently in Phase Ⅲ clinical trials.

    • Once expressed, the viral proteins NS3 to NS5B, directed at least in part by NS4B[57], assemble on and remodel cytoplasmic ER membranes to form viral RNA replication complexes, which have been termed the membranous web[54] (Fig. 3, Step 6-7). Within this complex, the viral NS5B RNA-dependent RNA polymerase first functions to synthesize com-plementary negative-strand RNA from the positive RNA genome template. The newly synthesized negative-strand RNA then provides the template for NS5B amplification of ~10-fold excess positive-strand genomic RNA. Currently, six NS5B inhibitors are in Phase Ⅱ clinical trials (Table 2). Meanwhile, accumulating data continues to reveal that HCV RNA replication is a highly complex process, dependent on both viral and host proteins (reviewed in [23]), all of which could potentially serve as antiviral targets. In fact, the viral hydrophilic phosphoprotein, NS5A is required for HCV RNA replication, most likely via association with host cell proteins[73]. Notably, with the expansion of siRNA and proteomics technologies the list of host cell proteins involved in HCV replication is rapidly expanding[16, 17, 33, 109, 161, 163]. As such, compounds which block viral replication by inhibiting other HCV NS proteins or targeting host cell proteins shown to be essential for viral replication are also in development. Those currently in clinical trial (Table 2) include NS5A inhibitors (Bristol Myers Squibb)[106], the NS4B inhibitor Clemizole (Eiger BioPharmaceuticals)[41], HMG Co A reductase inhibitors[74, 168, 181], cyclophilin inhibitors [e.g. DeBIO-025 (DebioPharm), SCY-635 (ScyNexis) and NIM 811 (Novartis)][47, 134], and the liver-specific microRNA miR-122 inhibitor LNA-antimiR/SPC3649 (Santaris Pharma)[81, 101], but it is anticipated many more will follow.

    • The details of HCV virion assembly, maturation and egress are the least under stood, as they have only recently become amenable to systematic study with the development of the HCVcc infection system. Nonetheless, based on other flavivirus systems[170], encapsidated HCV virions are believed to become enveloped with E1/E2 bearing cellular membranes upon budding into the ER. Progeny virions are believed to then exit the cell via the secretory pathway, during which glycosylation of viral glycoproteins and association with cellular lipoproteins (e.g. very-low density lipoproteins (vLDL)) occurs. (Fig. 2, Steps 8-9). As the viral proteins and viral-host interactions that mediate capsid assembly, envelopment, and virion maturation are elucidated a plethora of promising drug targets are being identified. In terms of viral targets, a screen to identify compounds that inhibit HCV core dimerization was recently reported[95] and other viral proteins, such as p7[58] and NS2[79, 80], are also emerged as essential factors in viral assembly and release. In terms of cellular targets, host factors involved in lipoprotein secretion are prime candidates[144] and it has been shown that infectious HCVcc particle egress is blocked by the vLDL secretion inhibitor naringenin found in grapefruit[131]. One type of HCV virion maturation inhibitor in clinical trials are compounds targeting the cellular α-glucosidase Ⅰ and Ⅱ enzymes involved in glycoprotein processing. Because viruses tend to be more sensitive to decreases in the activity of these enzymes than the host cell, a-glucosidase inhibition can be employed to induce misfolding of the HCV envelope glycoproteins preventing virion maturation and release. For example, iminosugars have been shown to inhibit α-glucosidases, resulting in anti-HCV activity in vitro[158]. Celgosivir (Mignex), a prodrug of castanospermine, has also been shown to inhibit α-glucosidase I[40] and has reached Phase Ⅱ clinical trials.

    • Although the development of useful experimental model systems has ushered in a new productive era of HCV molecular virology research and drug discovery, there are still challenges to overcome.

    • Robust HCV infection in cell culture has only been achieved with genotype 2a derivative clones. While attempts to propagate the infectious genotype 1a H77 clone[184] and an infectious genotype 1b virus in cell culture[139] have resulted in detectable HCV levels, de novo virus production was low, limiting the utility of the systems. We do not fully understand the restriction that prevent efficient propagation other HCV genotypes in vitro, but studies have mapped the 3' end of the JFH-1 clone as sufficient to confer replication permissiveness to other HCV clones, therefore chimeric HCV genomes containing these necessary 3' region of the JFH-1 clone recombined with the corresponding 5' regions from genotypes 1-6 have been developed[55, 56, 78, 138, 156].

    • In addition to expanding the repertoire of available infectious HCV clones, improvements in hepatocyte cell culture is required as well. To date, robust HCV infection has only been published in one continuous human hepatoma cell line, Huh7[96, 113, 132, 150]. Studying HCV infection in Huh7 cells has certainly expanded our ability investigate the viral life cycle; however, Huh7 cells are transformed and only marginally mimic the state of hepatocytes in vivo[126, 160], which limits our ability to elucidate how HCV interacts with and induces alterations in hepatocytes in vivo to produce clinically observed HCV-associated liver disease. To address this issue, novel two-dimensional[151] and three-dimensional[152] culture systems have been developed to coax Huh7 cells to up-regulate hepatocyte-specific transcripts, become Phase Ⅰ and Phase Ⅱ drug metabolism competent[36], and exhibit more specific localization of tight junction, cell adhesion, and polarity markers[152]. While these systems may prove useful in understanding how HCV interacts with polarized cells or disrupts specific aspects of hepatocyte physiology, Huh7 cells by nature remain transformed. As such, primary liver cell cultures remain the most physiologically relevant in vitro model for the study of HCV, and numerous laboratories are trying to develop ways to adapt primary human hepatocytes for HCV research[6, 27, 192]. One recent publication of note describes a human hepatocyte cell culture system permissible to infection with patient serum of HCV genotypes 1, 2, 3 and 4 mimicking the kinetics of HCV infection in humans and producing infectious virions that can infect na ve human hepatocytes[27]. More recently, HCV infection was established in micropatterned co-cultures (MPCCs) of primary human hepatocytes with supportive stroma[142], which is relevant to antiviral screening because MPCCs can be readily scaled down to a multi-well format for HTS when coupled with appropriate fluorescence-and/or luminescence-based reporter systems. Together, these systems represent important advances in the use of primary human hepatocytes in HCV research and should complement the existing Huh7-based HCV systems by facilitating the understanding of the effects HCV has on it natural host cell, but it remains to be determined if widespread use of these systems will be possible.

    • The most significant obstacle impeding the preclinical testing of new HCV therapeutics and development of vaccines is the lack of HCV small animal models (reviewed in [143]). Efforts to develop small animal infection models have included trying to transmit HCV to tree shrews [175, 190], marmosets/ tamarins[44, 83, 169], and other primates[1], but only the chimpanzee model of HCV infection, has proven efficacious[93, 178]. The species barriers that prevent HCV infection in non-human hosts are not completely defined, but include blocks in viral entry and non-permissiveness for HCV RNA replication. To avoid issues of infectivity and simply study the effects of HCV proteins in vivo, several non-replicating transgenic mouse lineages that express one or more of the HCV proteins have been created[69, 86, 92, 107, 127, 136]. Although there is a great deal of variation in the HCV protein expression levels observed in these models and in the resulting pathology, these mice have demonstrated that expression of specific HCV proteins at least under some circumstances can induce disturbances in lipid metabolism and possibly contribute to the development of HCC, but it remains unclear how the effects observed in these expression systems relate to clinically observed HCV pathology.

      To eliminate issues of transgene expression levels and create a more authentic small animal model of HCV infection, hepatic xenorepopulation approaches have been used to study HCV infection in vivo [19, 49, 59, 76, 90, 91, 124, 147, 157] The basis of these models involves transplanting primary human hepatocytes into immunodeficient mice that loose their endogenous hepatocytes due to either a lethal hepatocyte transgene [49, 62, 76, 147, 157] or a hepatotoxic defect in an essential liver enzyme [7, 18] allowing the transplanted HCV-permissive human hepatocytes to repopulate the mouse liver. While this approach has been invaluable for HCV antiviral drug development as a means of confirming in vivo efficacy in a true infection model, creating and working with these chimeric mice is technically challenging (reviewed in [89]) making the model impractical for widespread use.

      To try and develop an HCV-permissive infectious mouse model, we and others have shown that HCV JFH-1 subgenomic replicons and full length genomes can replicate in mouse cells[34, 165] indicating that a species block at the level of HCV RNA replication can be overcome, but that additional blocks in post-replication steps involved in progeny virus production may exist[165]. In terms of HCV entry, Ploss et al., recently determined the species-specific determinants for HCVpp entry into mouse cells to be human CD81 and human occludin, as expression of these two human receptors in murine fibroblast cell lines resulted in permissiveness for HCVpp[141]. Notably however, infectious HCVcc entry could not be demonstrated suggesting additional entry factors are required for HCVcc entry into mouse cells. Hence, although we have made advancements towards the development of an HCV-permissive mouse model, this goal has yet to be achieved.

    • The lack of targeted HCV-specific treatments to date has hindered the success of HCV treatment strategies. However, the HCV experimental models and HTS assays described in this review have already led to the discovery of numerous potential HCV therapeutics, and ongoing efforts to improve and expand upon these experimental models will continue to benefit the HCV antiviral drug development effort. As such, new HCV therapeutics are on the horizon and promise a future in which effective HCV treatment may be achieved with a "cocktail" approach comprised of compounds that specifically target the virus and critical viral-host interactions.

    Figure (3)  Table (2) Reference (192) Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return