1887

Abstract

Non-structural protein 4A (NS4A) of (HCV) functions as a cofactor for NS3 by forming a complex with it to augment its enzymic activities. NS4A also forms a complex with other HCV proteins, such as NS4B/NS5A, to facilitate the formation of the viral RNA replication complex on the endoplasmic reticulum (ER) membrane. In addition to its essential role in HCV replication, NS4A is thought to be involved in viral pathogenesis by affecting cellular functions. In this study, it was demonstrated that NS4A was localized not only on the ER, but also on mitochondria when expressed either alone or together with NS3 in the form of the NS3/4A polyprotein and in the context of HCV RNA replication in Huh7 cells harbouring an HCV RNA replicon. Moreover, NS4A expression altered the intracellular distribution of mitochondria significantly and caused mitochondrial damage, as evidenced by the collapsed mitochondrial transmembrane potential and release of cytochrome into the cytoplasm, which led ultimately to induction of apoptosis through activation of caspase-3, but not caspase-8. Consistently, Huh7 cells expressing NS3/4A and those harbouring an HCV RNA replicon were shown to be more prone to undergoing actinomycin D-induced, mitochondria-mediated apoptosis, compared with the control Huh7 cells. Taken together, these results suggest the possibility that HCV exerts cytopathic effect (CPE) on the infected cells under certain conditions and that NS4A is responsible, at least in part, for the conditional CPE in HCV-infected cells.

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2006-07-01
2024-03-28
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References

  1. Aizaki H., Lee K.-J., Sung V. M.-H., Ishiko H., Lai M. M. C. 2004; Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology 324:450–461 [CrossRef]
    [Google Scholar]
  2. Ashkenazi A., Dixit V. M. 1998; Death receptors: signaling and modulation. Science 281:1305–1308 [CrossRef]
    [Google Scholar]
  3. Bantel H., Schulze-Osthoff K. 2003; Apoptosis in hepatitis C virus infection. Cell Death Differ 10:S48–S58 [CrossRef]
    [Google Scholar]
  4. Bantel H., Lügering A., Poremba C., Lügering N., Held J., Domschke W., Schulze-Osthoff K. 2001; Caspase activation correlates with the degree of inflammatory liver injury in chronic hepatitis C virus infection. Hepatology 34:758–767 [CrossRef]
    [Google Scholar]
  5. Barbaro G., Di Lorenzo G., Asti A., Ribersani M., Belloni G., Grisorio B., Filice G., Barbarini G. 1999; Hepatocellular mitochondrial alterations in patients with chronic hepatitis C: ultrastructural and biochemical findings. Am J Gastroenterol 94:2198–2205 [CrossRef]
    [Google Scholar]
  6. Chen W., Calvo P. A., Malide D. & 10 other authors 2001; A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 7:1306–1312 [CrossRef]
    [Google Scholar]
  7. Ciccaglione A. R., Marcantonio C., Costantino A., Equestre M., Rapicetta M. 2003; Expression of HCV E1 protein in baculovirus-infected cells: effects on cell viability and apoptosis induction. Intervirology 46:121–126 [CrossRef]
    [Google Scholar]
  8. Ciccaglione A. R., Marcantonio C., Tritarelli E., Equestre M., Magurano F., Costantino A., Nicoletti L., Rapicetta M. 2004; The transmembrane domain of hepatitis C virus E1 glycoprotein induces cell death. Virus Res 104:1–9 [CrossRef]
    [Google Scholar]
  9. D'Agostino D. M., Ranzato L., Arrigoni G. & 10 other authors 2002; Mitochondrial alterations induced by the p13II protein of human T-cell leukemia virus type 1. J Biol Chem 277:34424–34433 [CrossRef]
    [Google Scholar]
  10. Davis S., Weiss M. J., Wong J. R., Lampidis T. J., Chen L. B. 1985; Mitochondrial and plasma membrane potentials cause unusual accumulation and retention of rhodamine 123 by human breast adenocarcinoma-derived MCF-7 cells. J Biol Chem 260:13844–13850
    [Google Scholar]
  11. Deng L., Nagano-Fujii M., Tanaka M., Nomura-Takigawa Y., Ikeda M., Kato N., Sada K., Hotta H. 2006; NS3 protein of Hepatitis C virus associates with the tumour suppressor p53 and inhibits its function in an NS3 sequence-dependent manner. J Gen Virol 87:1703–1713 [CrossRef]
    [Google Scholar]
  12. Deveraux Q. L., Roy N., Stennicke H. R., Van Arsdale T., Zhou Q., Srinivasula S. M., Alnemri E. S., Salvesen G. S., Reed J. C. 1998; IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J 17:2215–2223 [CrossRef]
    [Google Scholar]
  13. Egger D., Wölk B., Gosert R., Bianchi L., Blum H. E., Moradpour D., Bienz K. 2002; Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76:5974–5984 [CrossRef]
    [Google Scholar]
  14. Eguchi Y., Shimizu S., Tsujimoto Y. 1997; Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res 57:1835–1840
    [Google Scholar]
  15. Erdtmann L., Franck N., Lerat H., Seyec J. L., Gilot D., Cannie I., Gripon P., Hibner U., Guguen-Guillouzo C. 2003; The hepatitis C virus NS2 protein is an inhibitor of CIDE-B-induced apoptosis. J Biol Chem 278:18256–18264 [CrossRef]
    [Google Scholar]
  16. Everett H., Barry M., Sun X., Lee S. F., Frantz C., Berthiaume L. G., McFadden G., Bleackley R. C. 2002; The myxoma poxvirus protein, M11L, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore. J Exp Med 196:1127–1139 [CrossRef]
    [Google Scholar]
  17. Failla C., Tomei L., De Francesco R. 1995; An amino-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A. J Virol 69:1769–1777
    [Google Scholar]
  18. Fearnhead H. O., Rodriguez J., Govek E.-E., Guo W., Kobayashi R., Hannon G., Lazebnik Y. A. 1998; Oncogene-dependent apoptosis is mediated by caspase-9. Proc Natl Acad Sci U S A 95:13664–13669 [CrossRef]
    [Google Scholar]
  19. Feng P., Park J., Lee B.-S., Lee S.-H., Bram R. J., Jung J. U. 2002; Kaposi's sarcoma-associated herpesvirus mitochondrial K7 protein targets a cellular calcium-modulating cyclophilin ligand to modulate intracellular calcium concentration and inhibit apoptosis. J Virol 76:11491–11504 [CrossRef]
    [Google Scholar]
  20. Florese R. H., Nagano-Fujii M., Iwanaga Y., Hidajat R., Hotta H. 2002; Inhibition of protein synthesis by the nonstructural proteins NS4A and NS4B of hepatitis C virus. Virus Res 90:119–131 [CrossRef]
    [Google Scholar]
  21. Fujita T., Ishido S., Muramatsu S., Itoh M., Hotta H. 1996; Suppression of actinomycin D-induced apoptosis by the NS3 protein of hepatitis C virus. Biochem Biophys Res Commun 229:825–831 [CrossRef]
    [Google Scholar]
  22. Gewies A., Rokhlin O. W., Cohen M. B. 2000; Cytochrome c is involved in Fas-mediated apoptosis of prostatic carcinoma cell lines. Cancer Res 60:2163–2168
    [Google Scholar]
  23. Goldmacher V. S., Bartle L. M., Skaletskaya A. & 10 other authors 1999; A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc Natl Acad Sci U S A 96:12536–12541 [CrossRef]
    [Google Scholar]
  24. Gosert R., Egger D., Lohmann V., Bartenschlager R., Blum H. E., Bienz K., Moradpour D. 2003; Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 77:5487–5492 [CrossRef]
    [Google Scholar]
  25. Gross A., McDonnell J. M., Korsmeyer S. J. 1999; BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13:1899–1911 [CrossRef]
    [Google Scholar]
  26. Hahn C. S., Cho Y. G., Kang B.-S., Lester I. M., Hahn Y. S. 2000; The HCV core protein acts as a positive regulator of Fas-mediated apoptosis in a human lymphoblastoid T cell line. Virology 276:127–137 [CrossRef]
    [Google Scholar]
  27. Hayashi J., Kishihara Y., Yamaji K., Furusyo N., Yamamoto T., Pae Y., Etoh Y., Ikematsu H., Kashiwagi S. 1997; Hepatitis C viral quasispecies and liver damage in patients with chronic hepatitis C virus infection. Hepatology 25:697–701 [CrossRef]
    [Google Scholar]
  28. He Y., Nakao H., Tan S.-L., Polyak S. J., Neddermann P., Vijaysri S., Jacobs B. L., Katze M. G. 2002; Subversion of cell signaling pathways by hepatitis C virus nonstructural 5A protein via interaction with Grb2 and P85 phosphatidylinositol 3-kinase. J Virol 76:9207–9217 [CrossRef]
    [Google Scholar]
  29. Hidajat R., Nagano-Fujii M., Deng L., Tanaka M., Takigawa Y., Kitazawa S., Hotta H. 2005; Hepatitis C virus NS3 protein interacts with ELKS- δ and ELKS- α , members of a novel protein family involved in intracellular transport and secretory pathways. J Gen Virol 86:2197–2208 [CrossRef]
    [Google Scholar]
  30. Hiramatsu N., Hayashi N., Katayama K., Mochizuki K., Kawanishi Y., Kasahara A., Fusamoto H., Kamada T. 1994; Immunohistochemical detection of Fas antigen in liver tissue of patients with chronic hepatitis C. Hepatology 19:1354–1359 [CrossRef]
    [Google Scholar]
  31. Hsu E. C., Hsi B., Hirota-Tsuchihara M. & 8 other authors 2003; Modified apoptotic molecule (BID) reduces hepatitis C virus infection in mice with chimeric human livers. Nat Biotechnol 21:519–525 [CrossRef]
    [Google Scholar]
  32. Ishido S., Fujita T., Hotta H. 2000; Possible involvement of the NS3 protein of hepatitis C virus in hepatocarcinogenesis: its interaction with the p53 tumor suppressor. In Hepatocellular Carcinoma: Methods and Protocols pp  37–55 Edited by Habib N. A. Totowa: Humana Press;
    [Google Scholar]
  33. Jacotot E., Ravagnan L., Loeffler M. & 15 other authors 2000; The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore. J Exp Med 191:33–45 [CrossRef]
    [Google Scholar]
  34. Jacotot E., Ferri K. F., El Hamel C. & 17 other authors 2001; Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein R and Bcl-2. J Exp Med 193:509–520 [CrossRef]
    [Google Scholar]
  35. Kato J., Kato N., Yoshida H., Ono-Nita S. K., Shiratori Y., Omata M. 2002; Hepatitis C virus NS4A and NS4B proteins suppress translation in vivo. J Med Virol 66:187–199 [CrossRef]
    [Google Scholar]
  36. Kim J.-E., Song W. K., Chung K. M., Back S. H., Jang S. K. 1999; Subcellular localization of hepatitis C viral proteins in mammalian cells. Arch Virol 144:329–343 [CrossRef]
    [Google Scholar]
  37. Kiyosawa K., Sodeyama T., Tanaka E. & 8 other authors 1990; Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology 12:671–675 [CrossRef]
    [Google Scholar]
  38. Kuang W.-F., Lin Y.-C., Jean F., Huang Y.-W., Tai C.-L., Chen D.-S., Chen P.-J., Hwang L.-H. 2004; Hepatitis C virus NS3 RNA helicase activity is modulated by the two domains of NS3 and NS4A. Biochem Biophys Res Commun 317:211–217 [CrossRef]
    [Google Scholar]
  39. Lan K.-H., Sheu M.-L., Hwang S.-J. & 8 other authors 2002; HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis. Oncogene 21:4801–4811 [CrossRef]
    [Google Scholar]
  40. Leprat P., Ratinaud M. H., Maftah A., Petit J. M., Julien R. 1990; Use of nonyl acridine orange and rhodamine 123 to follow biosynthesis and functional assembly of mitochondrial membrane during L1210 cell cycle. Exp Cell Res 186:130–137 [CrossRef]
    [Google Scholar]
  41. Li L., Lorenzo P. S., Bogi K., Blumberg P. M., Yuspa S. H. 1999; Protein kinase C δ targets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector. Mol Cell Biol 19:8547–8558
    [Google Scholar]
  42. Li X.-D., Sun L., Seth R. B., Pineda G., Chen Z. J. 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 102:17717–17722 [CrossRef]
    [Google Scholar]
  43. Lin C.-F., Chen C.-L., Chang W.-T., Jan M.-S., Hsu L.-J., Wu R.-H., Tang M.-J., Chang W.-C., Lin Y.-S. 2004; Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramide- and etoposide-induced apoptosis. J Biol Chem 279:40755–40761 [CrossRef]
    [Google Scholar]
  44. Liu X., Kim C. N., Yang J., Jemmerson R., Wang X. 1996; Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86:147–157 [CrossRef]
    [Google Scholar]
  45. Lohmann V., Körner F., Dobierzewska A., Bartenschlager R. 2001; Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 75:1437–1449 [CrossRef]
    [Google Scholar]
  46. Moorman J. P., Prayther D., McVay D., Hahn Y. S., Hahn C. S. 2003; The C-terminal region of hepatitis C core protein is required for Fas-ligand independent apoptosis in Jurkat cells by facilitating Fas oligomerization. Virology 312:320–329 [CrossRef]
    [Google Scholar]
  47. Mottola G., Cardinali G., Ceccacci A., Trozzi C., Bartholomew L., Torrisi M. R., Pedrazzini E., Bonatti S., Migliaccio G. 2002; Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology 293:31–43 [CrossRef]
    [Google Scholar]
  48. Muramatsu S., Ishido S., Fujita T., Itoh M., Hotta H. 1997; Nuclear localization of the NS3 protein of hepatitis C virus and factors affecting the localization. J Virol 71:4954–4961
    [Google Scholar]
  49. Muzio M., Chinnaiyan A. M., Kischkel F. C. & 11 other authors 1996; FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85:817–827 [CrossRef]
    [Google Scholar]
  50. Pang P. S., Jankowsky E., Planet P. J., Pyle A. M. 2002; The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J 21:1168–1176 [CrossRef]
    [Google Scholar]
  51. Rahmani Z., Huh K.-W., Lasher R., Siddiqui A. 2000; Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential. J Virol 74:2840–2846 [CrossRef]
    [Google Scholar]
  52. Reed K. E., Rice C. M. 2000; Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol 242:55–84
    [Google Scholar]
  53. Ruggieri A., Harada T., Matsuura Y., Miyamura T. 1997; Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein. Virology 229:68–76 [CrossRef]
    [Google Scholar]
  54. Ruggieri A., Murdolo M., Rapicetta M. 2003; Induction of FAS ligand expression in a human hepatoblastoma cell line by HCV core protein. Virus Res 97:103–110 [CrossRef]
    [Google Scholar]
  55. Sacco R., Tsutsumi T., Suzuki R. & 8 other authors 2003; Antiapoptotic regulation by hepatitis C virus core protein through up-regulation of inhibitor of caspase-activated DNase. Virology 317:24–35 [CrossRef]
    [Google Scholar]
  56. Satoh S., Tanji Y., Hijikata M., Kimura K., Shimotohno K. 1995; The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A. J Virol 69:4255–4260
    [Google Scholar]
  57. Schwer B., Ren S., Pietschmann T., Kartenbeck J., Kaehlcke K., Bartenschlager R., Yen T. S. B., Ott M. 2004; Targeting of hepatitis C virus core protein to mitochondria through a novel C-terminal localization motif. J Virol 78:7958–7968 [CrossRef]
    [Google Scholar]
  58. Shu H.-B., Halpin D. R., Goeddel D. V. 1997; Casper is a FADD- and caspase-related inducer of apoptosis. Immunity 6:751–763 [CrossRef]
    [Google Scholar]
  59. Siavoshian S., Abraham J. D., Thumann C., Kieny M. P., Schuster C. 2005; Hepatitis C virus core, NS3, NS5A, NS5B proteins induce apoptosis in mature dendritic cells. J Med Virol 75:402–411 [CrossRef]
    [Google Scholar]
  60. Skulachev V. P. 1998; Cytochrome c in the apoptotic and antioxidant cascades. FEBS Lett 423:275–280 [CrossRef]
    [Google Scholar]
  61. Soguero C., Joo M., Chianese-Bullock K. A., Nguyen D. T., Tung K., Hahn Y. S. 2002; Hepatitis C virus core protein leads to immune suppression and liver damage in a transgenic murine model. J Virol 76:9345–9354 [CrossRef]
    [Google Scholar]
  62. Song J., Fujii M., Wang F., Itoh M., Hotta H. 1999; The NS5A protein of hepatitis C virus partially inhibits the antiviral activity of interferon. J Gen Virol 80:879–886
    [Google Scholar]
  63. Sung V. M.-H., Shimodaira S., Doughty A. L., Picchio G. R., Can H., Yen T. S. B., Lindsay K. L., Levine A. M., Lai M. M. C. 2003; Establishment of B-cell lymphoma cell lines persistently infected with hepatitis C virus in vivo and in vitro: the apoptotic effects of virus infection. J Virol 77:2134–2146 [CrossRef]
    [Google Scholar]
  64. Taguchi T., Nagano-Fujii M., Akutsu M., Kadoya H., Ohgimoto S., Ishido S., Hotta H. 2004; Hepatitis C virus NS5A protein interacts with 2′,5′-oligoadenylate synthetase and inhibits antiviral activity of IFN in an IFN sensitivity-determining region-independent manner. J Gen Virol 85:959–969 [CrossRef]
    [Google Scholar]
  65. Takigawa Y., Nagano-Fujii M., Deng L., Hidajat R., Tanaka M., Mizuta H., Hotta H. 2004; Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome. Microbiol Immunol 48:591–598 [CrossRef]
    [Google Scholar]
  66. Tong M. J., El-Farra N. S., Reikes A. R., Co R. L. 1995; Clinical outcomes after transfusion-associated hepatitis C. N Engl J Med 332:1463–1466 [CrossRef]
    [Google Scholar]
  67. Wang F., Yoshida I., Takamatsu M., Ishido S., Fujita T., Oka K., Hotta H. 2000; Complex formation between hepatitis C virus core protein and p21Waf1/Cip1/Sdi1. Biochem Biophys Res Commun 273:479–484 [CrossRef]
    [Google Scholar]
  68. Wang H.-W., Sharp T. V., Koumi A., Koentges G., Boshoff C. 2002; Characterization of an anti-apoptotic glycoprotein encoded by Kaposi's sarcoma-associated herpesvirus which resembles a spliced variant of human survivin. EMBO J 21:2602–2615 [CrossRef]
    [Google Scholar]
  69. Wölk B., Sansonno D., Kräusslich H.-G., Dammacco F., Rice C. M., Blum H. E., Moradpour D. 2000; Subcellular localization, stability, and trans -cleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated cell lines. J Virol 74:2293–2304 [CrossRef]
    [Google Scholar]
  70. Zhu N., Ware C. F., Lai M. M. 2001; Hepatitis C virus core protein enhances FADD-mediated apoptosis and suppresses TRADD signaling of tumor necrosis factor receptor. Virology 283:178–187 [CrossRef]
    [Google Scholar]
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