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Exploiting hepatitis C virus activation of NFκB to deliver HCV-responsive expression of interferons α and γ

Gene Therapy volume 10, pages 1861–1873 (2003)Cite this article

Abstract

Chronic infection with hepatitis C virus (HCV) may lead to liver failure and hepatocellular carcinoma. Current treatment for HCV includes high systemic doses of interferonα (IFNα), which is effective in less than half of patients and may have severe side effects. We designed conditional IFNα and IFNγ expression constructs to be triggered by HCV-induced activation of NFκB, and delivered these using highly efficient recombinant Tag-deleted SV40-derived vectors. NFκB activates the HIV-1NL4-3 long terminal repeat (HIVLTR) as a promoter, which accounts for the conditional transgene expression. Human hepatocyte lines and primary rat hepatocytes (PRH) were transduced with SV[HIVLTR](IFN) vectors, and transfected with HCV cDNA. Production of human and murine IFNα and IFNγ in cytosol and culture supernatants was measured. HCV activated the HIVLTR to produce and secrete IFNs, and did so largely through the NFκB binding sites of the HIVLTR. Levels of IFNs secreted, and the magnitude of induction in response to HCV, were greater in hepatocyte lines than in primary cultured hepatocytes. However, even in the latter, supernatant IFNα concentrations achieved by this approach were similar to therapeutic serum concentrations sought in systemic IFNα-treated patients. In coculture studies, secreted IFNα activated its cognate response elements in untransduced cells, suggesting that its potential inhibitory effects on HCV may not be limited to transduced cells. Although HCV replication in culture is difficult to assess, HCV-induced IFNα production demonstrably reduced HCV transcription. Conditional expression of IFNs within the liver may represent an attractive approach to therapy of severe chronic HCV infection that could avoid the side effects of systemic treatment regimens.

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References

  1. EASL International Consensus Conference on hepatitis C. Paris February 26–28, 1999; consensus statement. J Hepatol 1999; 30: 956–961.

  2. McHutchinson JG et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 1998; 339: 1485–1492.

    Article  Google Scholar 

  3. Lauer GM, Walker BD . Medical progress: hepatitis C virus infection. N Engl J Med 2001; 345: 41–52.

    Article  CAS  Google Scholar 

  4. Gale Jr M, Katze MG . Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmocol Ther 1998; 78: 29–46.

    Article  CAS  Google Scholar 

  5. Zern M et al. A novel SV40-based vector successfully transduces and expresses an α1-antitrypsin ribozyme in a human hepatoma-derived cell line. Gene Therapy 1999; 6: 114–120.

    Article  CAS  Google Scholar 

  6. Sauter BV et al. Gene transfer to the liver using a replication-deficient recombinant SV40 vector results in long-term amelioration of jaundice in Gunn rats. Gastroenterology 2000; 119: 1348–1357.

    Article  CAS  Google Scholar 

  7. Gong G, Waris G, Tanveer R, Siddiqui A . Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NFκB. Proc Natl Acad Sci 2001; 98: 9599–9604.

    Article  CAS  Google Scholar 

  8. Li Y, Mak G, Franza Jr BR . In vitro study of functional involvement of Sp1, NFκB/Rel, and AP1 in phorbol 12-myristate 13-acetate-mediated HIV-1 long terminal repeat activation. J Biol Chem 1994; 269: 30616–30619.

    CAS  PubMed  Google Scholar 

  9. Cordelier P, Calarota S, Strayer DS . Trans-activated IFNα2 delivered to T cells by SV40 inhibits early stages in the HIV-1 replicative cycle. J Hematother Stem Cell Res 2002; 11: 817–828.

    Article  CAS  Google Scholar 

  10. Lindenbach BD, Rice CM . Flaviviridae: the viruses and their replication. In: Knipe DM, Howley PM (eds). Fields' Virology. Lippincott, Williams & Wilkins: Philadelphia, 2001.

    Google Scholar 

  11. Iordanov MS, Wong J, Bell JC, Magun BE . Activation of NFκB by double-stranded RNA (dsRNA) in the absence of protein kinase R and RNase L demonstrates the existence of two separate dsRNA-triggered antiviral programs. Mol Cell Biol 2001; 21: 61–72.

    Article  CAS  Google Scholar 

  12. Chu WM et al. JNK2 and IKκβ are required for activating the innate response to viral infection. Immunity 1999; 11: 721–731.

    Article  CAS  Google Scholar 

  13. Zamanian-Daryoush M, Mogensen TH, DiDonato JA, Williams BR . NFκB activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NFκB-inducing kinase and IκB kinase. Mol Cell Biol 2000; 20: 1278–1290.

    Article  CAS  Google Scholar 

  14. Malmgaard L, Salazar-Mather TP, Lewis CA, Biron CA . Promotion of α/β interferon induction during in vivo viral infection through α/β interferon receptor/STAT1 system-dependent and -independent pathways. J Virol 2002; 76: 4520–4525.

    Article  CAS  Google Scholar 

  15. Mogensen TH, Paludan SR . Molecular pathways in virus-induced cytokine production. Microbiol Mol Biol Rev 2001; 65: 131–150.

    Article  CAS  Google Scholar 

  16. Iordanov MS et al. Activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways. Mol Cell Biol 2000; 20: 617–627.

    Article  CAS  Google Scholar 

  17. Williams BRG . PKR; a sentinel kinase for cellular stress. Oncogene 1999; 18: 6112–6120.

    Article  CAS  Google Scholar 

  18. De Haro C, Mendez R, Santoyo J . The eIF-2α kinases and the control of protein synthesis. FASEB J 1996; 10: 1378–1387.

    Article  CAS  Google Scholar 

  19. Boehm U, Groot M, Howard JC, Klamp T . Cellular responses to interferon-γ. Annu Rev Immunol 1997; 15: 749–794.

    Article  CAS  Google Scholar 

  20. Corssmit EPM, De Metz J, Saurwein HP, Romijn JA . Biologic responses to IFN-α administration in humans. J Interferon Cytokine Res 2000; 20: 1039–1047.

    Article  CAS  Google Scholar 

  21. Domanski P et al. A region of the β subunit of the interferon α receptor different from box 1 interacts with Jak1 and is sufficient to activate the Jak-Stat pathway and induce an antiviral state. J Biol Chem 1997; 272: 26388–26393.

    Article  CAS  Google Scholar 

  22. Barnes BJ, Moore PA, Pitha PM . Virus-specific activation of a novel interferon regulatory factor, IRF-5, results in the induction of distinct interferon α genes. J Biol Chem 2001; 276: 23382–23390.

    Article  CAS  Google Scholar 

  23. Decker T . Double-stranded RNA and interferon-α induce transcription through different molecular mechanisms. J Interferon Res 1992; 12: 445–448.

    Article  CAS  Google Scholar 

  24. Harcourt JL, Offerman MK . Multiple signaling cascades are differntially involved in gene induction by double stranded RNA in interferon-α-primed cells. Eur J Biochem 2001; 268: 1373–1381.

    Article  CAS  Google Scholar 

  25. Melen K, Keskinen P, Lehtonen A, Julkuen I . Interferon-induced gene expression and signaling in human hepatoma cell lines. J Hepatol 2000; 33: 764–772.

    Article  CAS  Google Scholar 

  26. Biron CA . Initial and innate responses to viral infections-pattern setting in immunity or disease. Curr Opin Microbiol 1999; 2: 374–381.

    Article  CAS  Google Scholar 

  27. Sareneva T, Julkunen I, Matikainen S . IFN-α and IL-I2 induce IL-I8 receptor gene expression in human NK and T cells. J Immunol 2000; 165: 1933–1938.

    Article  CAS  Google Scholar 

  28. Mosmann TR, Coffman RL . TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 1989; 7: 145–173.

    Article  CAS  Google Scholar 

  29. Perussia B . Lymphokine-activated killer cells, natural killer cells and cytokines. Curr Opin Immunol 1991; 3: 49–55.

    Article  CAS  Google Scholar 

  30. Sad S, Marcotte R, Mosmann TR . Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or T2 cytokines. Immunity 1995; 2: 271–279.

    Article  CAS  Google Scholar 

  31. Valente G et al. Distribution of interferon-γ receptor in human tissues. Eur J Immunol 1992; 22: 2403–2412.

    Article  CAS  Google Scholar 

  32. Schindler C, Darnell Jr JE . Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem 1995; 64: 621–651.

    Article  CAS  Google Scholar 

  33. Mannering GJ, Deloria LB . The pharmacology and toxicology of the interferons: an overview. Annu Rev Pharmacol Toxicol 1986; 26: 455–515.

    Article  CAS  Google Scholar 

  34. Brassard DL, Grace MJ, Bordens RW . Interferon-α as an immunotherapeutic protein. J Leukoc Biol 2002; 71: 565–581.

    CAS  PubMed  Google Scholar 

  35. Strayer DS et al. Durability of transgene expression and vector integration: recombinant SV40-derived gene therapy vectors. Mol Ther 2002; 6: 227–237.

    Article  CAS  Google Scholar 

  36. Jones KA, Peterlin BM . Control of RNA initiation and elongation at the HIV-1 promoter. Annu Rev Biochem 1994; 63: 717–743.

    Article  CAS  Google Scholar 

  37. Alcami J et al. Absolute dependence on kappa-B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes. EMBO J 1995; 14: 1552–1560.

    Article  CAS  Google Scholar 

  38. Chen BK, Feinberg MB, Baltimore D . The κB sites in the human immunodeficiency virus type 1 long terminal repeat enhance virus replication yet are not absolutely required for viral growth. J Virol 1997; 71: 5495–5504.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Ghosh S, May MJ, Koop EB . NFκB and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998; 16: 225–240.

    Article  CAS  Google Scholar 

  40. Berkhout B, Jeang KT . Functional roles for the TATA promoter and enhancers in basal and Tat-induced expression of the human immunodeficiency virus type 1 long terminal repeat. J Virol 1992; 66: 139–149.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Parrott C et al. Variable role of the long terminal repeat Sp1-binding sites in human immunodeficiency virus replication in T lymphocytes. J Virol 1991; 65: 1414–1419.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Jones KA . Tat and the HIV-1 promoter. Curr Opin Cell Biol 1993; 5: 461–468.

    Article  CAS  Google Scholar 

  43. Kinoshita S et al. The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells. Immunity 1997; 6: 235–240.

    Article  CAS  Google Scholar 

  44. Schafer SL, Vlach J, Pitha PM . Cooperation between Herpes simplex virus type 1-encoded ICP0 and Tat to support transcription of immunodeficiency virus type 1 long terminal repeat in vivo can occur in the absence of the TAT binding site. J Virol 1996; 70: 6937–6946.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Dal Monte P et al. TAR and Sp1-independent transactivation of HIV long terminal repeat by the Tat protein in the presence of human cytomegalovirus IE1/IE2. AIDS 1997; 11: 297–303.

    Article  CAS  Google Scholar 

  46. Cheng H, Tarnok J, Parks WP . Human immunodeficiency virus type 1 genome activation induced by human T-cell leukemia virus type 1 Tax protein is through cooperation of NFκB and Tat. J Virol 2001; 72: 13427–13432.

    Google Scholar 

  47. Huang L-M et al. Reciprocal regulatory interaction between human herpes virus 8 and human immunodeficiency virus type 1. J Biol Chem 2001; 276: 13427–13432.

    Article  CAS  Google Scholar 

  48. Engellman GL, Richardson A, Katz A, Fierer JA . Age-related changes in isolated rat hepatocytes. Comparison of size, morphology, binucleation, and protein content. Mech Ageing Dev 1981; 16: 385–395.

    Article  Google Scholar 

  49. McKee HJ, Strayer DS . Immune responses against SIV envelope glycoprotein, using recombinant SV40 as a vaccine delivery vector. Vaccine 2002; 20: 3613–3625.

    Article  CAS  Google Scholar 

  50. Kondo R, Feitelson MA, Strayer DS . Use of SV40 to immunize against hepatitis B surface antigen: implications for the use of SV40 for gene transduction and its use as an immunizing agent. Gene Therapy 1998; 5: 575–582.

    Article  CAS  Google Scholar 

  51. Strayer DS, Zern MA, Chowdhury JR . What can SV40-derived vectors do for gene therapy? Curr Opin Mol Ther 2002; 4: 313–323.

    CAS  PubMed  Google Scholar 

  52. Strayer DS . SV40-based gene transfer vectors: turning an adversary into a friend. Curr Opin Mol Ther 2000; 2: 570–578.

    CAS  PubMed  Google Scholar 

  53. Strayer DS . Gene Therapy using SV40-derived vectors: what does the future hold? J Cell Physiol 1999; 181: 375–384.

    Article  CAS  Google Scholar 

  54. Strayer DS et al. Generation of recombinant SV40 vectors for gene transfer. In: Raptis L (ed) SV40 Protocols, vol. 165, Methods in Molecular Biology Walker JM (ed). Humana Press: Totowa, NJ, 2001.

    Google Scholar 

  55. Watanabe H et al. Number and position of mutations in the interferon (IFN) sensitivity-determining region of the gene for nonstructural protein 5A correlate with IFN efficacy in hepatitis C virus genotype 1b infection. J Infect Dis 2001; 183: 1195–1203.

    Article  CAS  Google Scholar 

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Acknowledgements

We greatly appreciate the input of Drs J Roy Chowdhury, Pierre Cordelier, Mark Feitelson, Geetha Jayan, and Aleem Siddiqui. The assistance of Dr Jan Hoek's lab was helpful in establishing primary hepatocyte cultures. Dr Mark Feitelson (TJU) kindly supplied us with pRC/CMV-HCV; Dr Henry Wu (Immune Response Corp) provided us with the cDNA for hIFNα 2b; Dr Janet S Butel (Baylor College of Medicine) gave us the plasmids carrying the original wtSV40 genome, from which rSV40 vectors were made. The current work was supported by NIH Grants AI41399, AI48244, and RR13156.

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  1. Department of Pathology and Cell Biology, Jefferson Medical College, Philadelphia, PA, USA

    A A Matskevich & D S Strayer

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Matskevich, A., Strayer, D. Exploiting hepatitis C virus activation of NFκB to deliver HCV-responsive expression of interferons α and γ. Gene Ther 10, 1861–1873 (2003). https://doi.org/10.1038/sj.gt.3302091

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