, Volume 38, Issue 5, pp 1823–1834 | Cite as

Potential Role of Hepatitis C Virus Alternate Reading Frame Protein in Negative Regulation of T-Bet Gene Expression

  • Dan Yan Zhu
  • Xiao Zhao Deng
  • Long Feng Jiang
  • Wen Xiao
  • Jia Ping Pei
  • Bing Jun Li
  • Chang Jun Wang
  • Jin Hai Zhang
  • Qi Zhang
  • Zhen Xian Zhou
  • Wei Liang Ding
  • Xiao Dong Xu
  • Ming Yue


Hepatitis C virus (HCV) is a major cause of chronic liver disease and has led to cirrhosis or hepatocellular carcinoma in a majority of infected individuals. We have previously demonstrated that the HCV alternate reading frame protein (F protein) is related to Th1/Th2 bias in chronic hepatitis C (CHC) patients, and we aimed to explore the relative molecular mechanisms here. A total of 104 cases including CHC patients and healthy donors were enrolled. T-bet and GATA-3 expression levels were analyzed in peripheral blood mononuclear cells (PBMCs). The levels of signal transducer and activator of transcription-1/-6(STAT1/6) and phosphorylated STAT1/6(pSTAT1/6) in PBMCs were measured by Western blotting. Our results showed that the levels of T-bet in PBMCs, as well as the levels of gamma interferon (IFN-γ) in sera, were decreased in anti-F protein antibody seropositive patients compared with anti-F protein antibody seronegative patients, whereas the levels of GATA-3 did not show difference between the two groups. Moreover, the decreased pSTAT1 and increased pSTAT6 were observed in PBMCs by HCV core/F protein stimulation with constant STAT1/6 expression. Taken together, it suggested that T-bet may be involved in Th1/Th2 bias induced by HCV F protein, and the disruption of STAT phosphorylation may participate in this mediation.


F protein hepatitis C virus Th1 Th2 T-bet 



This study was supported by grants from the National Natural Science Foundation of China (grant nos. 81172724 and 30972628), the National Major Scientific and Technological Projects (grant no. 2011ZX10004‑902), and the Natural Science Foundation of Jiangsu Province, China (grant no. BK2011840).

Conflicts of Interest

The authors declare no conflict of interest.


  1. 1.
    Choo, Q.L., G. Kuo, A.J. Weiner, L.R. Overby, D.W. Bradley, and M. Houghton. 1989. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244: 359–62.CrossRefPubMedGoogle Scholar
  2. 2.
    Morgan, R.L., B. Baack, B.D. Smith, A. Yartel, M. Pitasi, and Y. Falck-Ytter. 2013. Eradication of hepatitis C virus infection and the development of hepatocellular carcinoma: a meta-analysis of observational studies. Annals of Internal Medicine 158: 329–37.CrossRefPubMedGoogle Scholar
  3. 3.
    Lechner, F., D.K. Wong, P.R. Dunbar, R. Chapman, R.T. Chung, P. Dohrenwend, et al. 2000. Analysis of successful immune responses in persons infected with hepatitis C virus. The Journal of Experimental Medicine 191: 1499–512.PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Thimme, R., J. Bukh, H.C. Spangenberg, S. Wieland, J. Pemberton, C. Steiger, et al. 2002. Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proceedings of the National Academy of Sciences of the United States of America 99: 15661–8.PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Gruener, N.H., F. Lechner, M.C. Jung, H. Diepolder, T. Gerlach, G. Lauer, et al. 2001. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus. Journal of Virology 75: 5550–8.PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Lechmann, M., R.P. Woitas, B. Langhans, R. Kaiser, H.G. Ihlenfeldt, G. Jung, et al. 1999. Decreased frequency of HCV core-specific peripheral blood mononuclear cells with type 1 cytokine secretion in chronic hepatitis C. Journal of Hepatology 31: 971–8.CrossRefPubMedGoogle Scholar
  7. 7.
    Wedemeyer, H., X.S. He, M. Nascimbeni, A.R. Davis, H.B. Greenberg, J.H. Hoofnagle, et al. 2002. Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection. Journal of Immunology (Baltimore, Md. : 1950) 169: 3447–58.CrossRefGoogle Scholar
  8. 8.
    Moradpour, D., F. Penin, and C.M. Rice. 2007. Replication of hepatitis C virus. Nature Reviews. Microbiology 5: 453–63.CrossRefPubMedGoogle Scholar
  9. 9.
    Walewski, J.L., T.R. Keller, D.D. Stump, and A.D. Branch. 2001. Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame. RNA (New York, NY) 7: 710–21.CrossRefGoogle Scholar
  10. 10.
    Varaklioti, A., N. Vassilaki, U. Georgopoulou, and P. Mavromara. 2002. Alternate translation occurs within the core coding region of the hepatitis C viral genome. The Journal of Biological Chemistry 277: 17713–21.CrossRefPubMedGoogle Scholar
  11. 11.
    Xu, Z., J. Choi, T.S. Yen, W. Lu, A. Strohecker, S. Govindarajan, et al. 2001. Synthesis of a novel hepatitis C virus protein by ribosomal frameshift. The EMBO Journal 20: 3840–8.PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    McMullan, L.K., A. Grakoui, M.J. Evans, K. Mihalik, M. Puig, A.D. Branch, et al. 2007. Evidence for a functional RNA element in the hepatitis C virus core gene. Proceedings of the National Academy of Sciences of the United States of America 104: 2879–84.PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Kong, J., X. Deng, Z. Wang, J. Yang, Y. Zhang, and J. Yu. 2009. Hepatitis C virus F protein: a double-edged sword in the potential contribution of chronic inflammation to carcinogenesis. Molecular Medicine Reports 2: 461–9.PubMedGoogle Scholar
  14. 14.
    Yue, M., X. Deng, X. Zhai, K. Xu, J. Kong, J. Zhang, et al. 2013. Th1 and Th2 cytokine profiles induced by hepatitis C virus F protein in peripheral blood mononuclear cells from chronic hepatitis C patients. Immunology Letters 152: 89–95.CrossRefPubMedGoogle Scholar
  15. 15.
    Shin, H.J., J.B. Lee, S.H. Park, J. Chang, and C.W. Lee. 2009. T-bet expression is regulated by EGR1-mediated signaling in activated T cells. Clinical immunology (Orlando, Fla) 131: 385–94.CrossRefGoogle Scholar
  16. 16.
    Afkarian, M., J.R. Sedy, J. Yang, N.G. Jacobson, N. Cereb, S.Y. Yang, et al. 2002. T-bet is a STAT1-induced regulator of IL-12R expression in naive CD4+ T cells. Nature Immunology 3: 549–57.CrossRefPubMedGoogle Scholar
  17. 17.
    Avni, O., D. Lee, F. Macian, S.J. Szabo, L.H. Glimcher, and A. Rao. 2002. T(H) cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes. Nature Immunology 3: 643–51.PubMedGoogle Scholar
  18. 18.
    Murphy, K.M., and S.L. Reiner. 2002. The lineage decisions of helper T cells. Nature Reviews. Immunology 2: 933–44.CrossRefPubMedGoogle Scholar
  19. 19.
    Shi, M., T.H. Lin, K.C. Appell, and L.J. Berg. 2008. Janus-kinase-3-dependent signals induce chromatin remodeling at the Ifng locus during T helper 1 cell differentiation. Immunity 28: 763–73.PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Hwang, E.S., S.J. Szabo, P.L. Schwartzberg, and L.H. Glimcher. 2005. T helper cell fate specified by kinase-mediated interaction of T-bet with GATA-3. Science 307: 430–3.CrossRefPubMedGoogle Scholar
  21. 21.
    Finotto, S., M.F. Neurath, J.N. Glickman, S. Qin, H.A. Lehr, F.H. Green, et al. 2002. Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet. Science 295: 336–8.CrossRefPubMedGoogle Scholar
  22. 22.
    Solomou, E.E., K. Keyvanfar, and N.S. Young. 2006. T-bet, a Th1 transcription factor, is up-regulated in T cells from patients with aplastic anemia. Blood 107: 3983–91.PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Lugo-Villarino, G., S. Ito, D.M. Klinman, and L.H. Glimcher. 2005. The adjuvant activity of CpG DNA requires T-bet expression in dendritic cells. Proceedings of the National Academy of Sciences of the United States of America 102: 13248–53.PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Sullivan, B.M., O. Jobe, V. Lazarevic, K. Vasquez, R. Bronson, L.H. Glimcher, et al. 2005. Increased susceptibility of mice lacking T-bet to infection with Mycobacterium tuberculosis correlates with increased IL-10 and decreased IFN-gamma production. Journal of Immunology (Baltimore, Md. : 1950) 175: 4593–602.CrossRefGoogle Scholar
  25. 25.
    Tantisira, K.G., E.S. Hwang, B.A. Raby, E.S. Silverman, S.L. Lake, B.G. Richter, et al. 2004. TBX21: a functional variant predicts improvement in asthma with the use of inhaled corticosteroids. Proceedings of the National Academy of Sciences of the United States of America 101: 18099–104.PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Yao, Z.Q., S.N. Waggoner, M.W. Cruise, C. Hall, X. Xie, D.W. Oldach, et al. 2005. SOCS1 and SOCS3 are targeted by hepatitis C virus core/gC1qR ligation to inhibit T-cell function. Journal of Virology 79: 15417–29.PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Kondo, Y., V.M. Sung, K. Machida, M. Liu, and M.M. Lai. 2007. Hepatitis C virus infects T cells and affects interferon-gamma signaling in T cell lines. Virology 361: 161–73.CrossRefPubMedGoogle Scholar
  28. 28.
    Simmonds, P., F. McOmish, P.L. Yap, S.W. Chan, C.K. Lin, G. Dusheiko, et al. 1993. Sequence variability in the 5′ non-coding region of hepatitis C virus: identification of a new virus type and restrictions on sequence diversity. The Journal of General Virology 74(Pt 4): 661–8.CrossRefPubMedGoogle Scholar
  29. 29.
    Xu, X., X. Yu, X. Deng, M. Yue, J. Zhang, D. Zhu, et al. 2014. Hepatitis C virus alternate reading frame protein decreases interferon-alpha secretion in peripheral blood mononuclear cells. Molecular Medicine Reports 9: 730–6.PubMedGoogle Scholar
  30. 30.
    Schmittgen, T.D., and K.J. Livak. 2008. Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols 3: 1101–8.CrossRefPubMedGoogle Scholar
  31. 31.
    Anthony, D.D., A.B. Post, H. Valdez, D.L. Peterson, M. Murphy, and P.S. Heeger. 2001. ELISPOT analysis of hepatitis C virus protein-specific IFN-gamma-producing peripheral blood lymphocytes in infected humans with and without cirrhosis. Clinical immunology (Orlando, Fla) 99: 232–40.Google Scholar
  32. 32.
    Farrar, J.D., W. Ouyang, M. Lohning, M. Assenmacher, A. Radbruch, O. Kanagawa, et al. 2001. An instructive component in T helper cell type 2 (Th2) development mediated by GATA-3. The Journal of Experimental Medicine 193: 643–50.PubMedCentralCrossRefPubMedGoogle Scholar
  33. 33.
    Nawijn, M.C., G.M. Dingjan, R. Ferreira, B.N. Lambrecht, A. Karis, F. Grosveld, et al. 2001. Enforced expression of GATA-3 in transgenic mice inhibits Th1 differentiation and induces the formation of a T1/ST2-expressing Th2-committed T cell compartment in vivo. Journal of Immunology (Baltimore, Md. : 1950) 167: 724–32.CrossRefGoogle Scholar
  34. 34.
    Forbes, E., N. van Panhuys, B. Min, and G. Le Gros. 2010. Differential requirements for IL-4/STAT6 signalling in CD4 T-cell fate determination and Th2-immune effector responses. Immunology and Cell Biology 88: 240–3.CrossRefPubMedGoogle Scholar
  35. 35.
    Bergqvist, A., S. Sundstrom, L.Y. Dimberg, E. Gylfe, and M.G. Masucci. 2003. The hepatitis C virus core protein modulates T cell responses by inducing spontaneous and altering T-cell receptor-triggered Ca2+ oscillations. The Journal of Biological Chemistry 278: 18877–83.CrossRefPubMedGoogle Scholar
  36. 36.
    Large, M.K., D.J. Kittlesen, and Y.S. Hahn. 1999. Suppression of host immune response by the core protein of hepatitis C virus: possible implications for hepatitis C virus persistence. Journal of Immunology (Baltimore, Md. : 1950) 162: 931–8.Google Scholar
  37. 37.
    Ray, R.B., R. Steele, K. Meyer, and R. Ray. 1998. Hepatitis C virus core protein represses p21WAF1/Cip1/Sid1 promoter activity. Gene 208: 331–6.CrossRefPubMedGoogle Scholar
  38. 38.
    Pichlmair, A., K. Kandasamy, G. Alvisi, O. Mulhern, R. Sacco, M. Habjan, et al. 2012. Viral immune modulators perturb the human molecular network by common and unique strategies. Nature 487: 486–90.CrossRefPubMedGoogle Scholar
  39. 39.
    Szabo, S.J., S.T. Kim, G.L. Costa, X. Zhang, C.G. Fathman, and L.H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100: 655–69.CrossRefPubMedGoogle Scholar
  40. 40.
    Kitamura, N., F. Kitamura, O. Kaminuma, S. Miyatake, H. Tatsumi, S. Nemoto, et al. 2007. IL-4 gene transcription in human T cells is suppressed by T-bet. International Archives of Allergy and Immunology 143(Suppl 1): 68–70.CrossRefPubMedGoogle Scholar
  41. 41.
    Maillard, P., K. Krawczynski, J. Nitkiewicz, C. Bronnert, M. Sidorkiewicz, P. Gounon, et al. 2001. Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients. Journal of Virology 75: 8240–50.PubMedCentralCrossRefPubMedGoogle Scholar
  42. 42.
    Masalova, O.V., S.N. Atanadze, E.I. Samokhvalov, N.V. Petrakova, T.I. Kalinina, V.D. Smirnov, et al. 1998. Detection of hepatitis C virus core protein circulating within different virus particle populations. Journal of Medical Virology 55: 1–6.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Dan Yan Zhu
    • 1
  • Xiao Zhao Deng
    • 1
    • 2
  • Long Feng Jiang
    • 3
  • Wen Xiao
    • 4
  • Jia Ping Pei
    • 1
  • Bing Jun Li
    • 2
  • Chang Jun Wang
    • 2
  • Jin Hai Zhang
    • 2
  • Qi Zhang
    • 2
  • Zhen Xian Zhou
    • 5
  • Wei Liang Ding
    • 6
  • Xiao Dong Xu
    • 2
  • Ming Yue
    • 4
  1. 1.Department of Biochemistry and Molecular Biology, School of Basic MedicineNanjing Medical UniversityNanjingChina
  2. 2.Huadong Research Institute for Medicine and BiotechnicsNanjingChina
  3. 3.Department of Infectious DiseasesThe First Affiliated Hospital with Nanjing Medical UniversityNanjingChina
  4. 4.School of Life Science and TechnologyChina Pharmaceutical UniversityNanjingChina
  5. 5.Department of Clinical LaboratoryNanjing Second HospitalNanjingChina
  6. 6.Department of Clinical LaboratoryYixing People’s HospitalYixingChina

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