Modulation of the Transforming Growth Factor-β Signal Transduction Pathway by Hepatitis C Virus Nonstructural 5A Protein

Abstract

Transforming growth factor-β (TGF-β) is implicated in the pathogenesis of liver disease. TGF-β is involved both in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis C virus (HCV) infection often leads to cirrhosis and hepatocellular carcinoma. HCV nonstructural 5A (NS5A) protein is a multifunctional protein that modulates cytokine-mediated signal transduction pathways. To elucidate the molecular mechanism of HCV pathogenesis, we examined the effect of NS5A protein on TGF-β-stimulated signaling cascades. We show that NS5A protein inhibited the TGF-β-mediated signaling pathway in hepatoma cell lines as determined by reporter gene assay. To further investigate the role of NS5A, we examined the protein/protein interaction between NS5A and TGF-β signal transducers. Both in vitro and in vivo binding data showed that NS5A protein directly interacted with TGF-β receptor I (TβR-I) in hepatoma cell lines. This interaction was mapped to amino acids 148-238 of NS5A. We also found that NS5A protein co-localized with TβR-I in the cytoplasm of Huh7 cells and inhibited TGF-β-mediated nuclear translocation of Smad2. Furthermore, we demonstrate that NS5A protein abrogated the phosphorylation of Smad2 and the heterodimerization of Smad3 and Smad4. To further explore the relevance to viral infection, we examined the effect of the HCV subgenomic replicon on the TGF-β signaling pathway. We show that the HCV subgenomic replicon also inhibited TGF-β-induced signaling cascades. These results indicate that HCV NS5A modulates TGF-β signaling through interaction with TβR-I and that NS5A may be an important risk factor in HCV-associated liver pathogenesis. Transforming growth factor-β (TGF-β) is implicated in the pathogenesis of liver disease. TGF-β is involved both in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis C virus (HCV) infection often leads to cirrhosis and hepatocellular carcinoma. HCV nonstructural 5A (NS5A) protein is a multifunctional protein that modulates cytokine-mediated signal transduction pathways. To elucidate the molecular mechanism of HCV pathogenesis, we examined the effect of NS5A protein on TGF-β-stimulated signaling cascades. We show that NS5A protein inhibited the TGF-β-mediated signaling pathway in hepatoma cell lines as determined by reporter gene assay. To further investigate the role of NS5A, we examined the protein/protein interaction between NS5A and TGF-β signal transducers. Both in vitro and in vivo binding data showed that NS5A protein directly interacted with TGF-β receptor I (TβR-I) in hepatoma cell lines. This interaction was mapped to amino acids 148-238 of NS5A. We also found that NS5A protein co-localized with TβR-I in the cytoplasm of Huh7 cells and inhibited TGF-β-mediated nuclear translocation of Smad2. Furthermore, we demonstrate that NS5A protein abrogated the phosphorylation of Smad2 and the heterodimerization of Smad3 and Smad4. To further explore the relevance to viral infection, we examined the effect of the HCV subgenomic replicon on the TGF-β signaling pathway. We show that the HCV subgenomic replicon also inhibited TGF-β-induced signaling cascades. These results indicate that HCV NS5A modulates TGF-β signaling through interaction with TβR-I and that NS5A may be an important risk factor in HCV-associated liver pathogenesis. Hepatitis C virus (HCV) 2The abbreviations used are: HCV, hepatitis C virus; NS5A, nonstructural 5A; IFN, interferon; TGF-β, transforming growth factor-β; TβR, transforming growth factor-β receptor; R-Smad, receptor-specific Smad; Co-Smad, common partner Smad; HA, hemagglutinin; Luc, luciferase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; EMSA, electrophoretic mobility shift assay.2The abbreviations used are: HCV, hepatitis C virus; NS5A, nonstructural 5A; IFN, interferon; TGF-β, transforming growth factor-β; TβR, transforming growth factor-β receptor; R-Smad, receptor-specific Smad; Co-Smad, common partner Smad; HA, hemagglutinin; Luc, luciferase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; EMSA, electrophoretic mobility shift assay. is a causative agent of non-A/non-B hepatitis (1Houghton M. Weiner A. Han J. Kuo G. Choo Q.-L. 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Lippincott-Raven Publishers, Philadelphia1996: 1035-1058Google Scholar). The viral genome is ∼9.6 kb in length and encodes a single polyprotein precursor of a little more than 3000 amino acid residues. This polyprotein is processed by a combination of host and viral proteases into 10 individual mature proteins (6Reed K.E. Rice C.M. Curr. Top. Microbiol. Immunol. 2000; 242: 55-84Crossref PubMed Scopus (476) Google Scholar). The three N-terminal structural proteins (core, E1, and E2) are required for nucleocapsid formation and assembly of viral particles. The remainders are the nonstructural proteins with various enzymatic activities, including serine protease, RNA helicase, and RNA-dependent RNA polymerase. Nonstructural 5A (NS5A) protein is a multifunctional phosphoprotein consisting of 447 amino acid residues. NS5A protein exists in two different sizes of polypeptide (p56 and p58), which is phosphorylated mainly at serine residues by cellular kinase (7Ide Y. Tanimoto A. Sasaguri Y. Padmanabhan R. Gene (Amst.). 1997; 201: 151-158Crossref PubMed Scopus (63) Google Scholar). NS5A protein is implicated in antiviral resistance to interferon (IFN). NS5A from HCV genotype 1b interacts with the IFN-inducible double-stranded RNA-activated protein kinase PKR through the IFN sensitivity-determining region in the middle domain of the protein and functions as a repressor of PKR (8Gale Jr., M. Korth M.J. Tang N.M. Tan S.L. Hopkins D.A. Dever T.E. Polyak S.J. Gretch D.R. Katz M.G. Virology. 1997; 230: 217-227Crossref PubMed Scopus (719) Google Scholar). NS5A protein has a nuclear localization signal at its C terminus that can direct a heterologous protein to the nucleus, although native NS5A is localized in the cytoplasm of HCV-infected patients and mammalian cells (9Ide Y. Zhang L. Chen M. Inchauspe G. Bahl C. Sasaguri Y. Padmanabhan R. Gene (Amst.). 1996; 182: 203-211Crossref PubMed Scopus (96) Google Scholar). 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Virology. 2002; 294: 94-105Crossref PubMed Scopus (103) Google Scholar, 15Lan K.H. Sheu M.L. Hwang S.J. Yen S.H. Chen S.Y. Wu J.C. Wang Y.J. Kato N. Omata M. Chang F.Y. Lee S.D. Oncogene. 2002; 21: 4801-4811Crossref PubMed Scopus (196) Google Scholar). Transforming growth factor-β (TGF-β) is one of the multifunctional cytokines that are involved in the regulation of cell proliferation and differentiation, apoptosis, and matrix production (16Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3972) Google Scholar, 17Derynck R. Akhurst R.J. Balmain A. Nat. Genet. 2001; 29: 117-129Crossref PubMed Scopus (1951) Google Scholar). The TGF-β-dependent signaling pathway involves two transmembrane serine/threonine kinases, viz. TGF-β receptor (TβR) types I and II (18Attisano L. Wrana J.L. Lopez-Casillas F. Massagué J. Biochim. Biophys. Acta. 1994; 1222: 71-80Crossref PubMed Scopus (311) Google Scholar, 19Derynck R. Trends Biochem. Sci. 1994; 19: 548-553Abstract Full Text PDF PubMed Scopus (277) Google Scholar). Upon ligand binding, TβR-II phosphorylates TβR-I at the Gly/Ser-rich domain. Phosphorylated TβR-I initiates the intracellular signal transduction pathway through the activation of receptor-specific Smad (R-Smad) proteins and common partner Smad (Co-Smad). An R- and Co-Smad complex translocates to the nucleus and regulates the transcription of target genes either positively or negatively (20Macias-Silva M. Abdollah S. Hoodless P.A. Pirone R. Attisano L. Wrana J.L. Cell. 1996; 87: 1215-1224Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 21Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. A. K. J. K. P. J. 1997; PubMed Scopus Google Scholar). TGF-β has a regulatory role in and cirrhosis A. J. N. N. Engl. J. Med. 1991; PubMed Scopus Google Scholar). TGF-β is also a growth in cell including carcinoma and Jr., R.J. N.J. R. Cell. PubMed Scopus Google Scholar, M. J.W. D.M. Massagué J. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar, Cell. 1990; Full Text PDF PubMed Scopus Google Scholar, M.J. Rev. PubMed Scopus Google Scholar, Rev. 1994; PubMed Scopus Google Scholar). we examined the of HCV NS5A protein on the TGF-β-dependent signaling pathway. We demonstrate that NS5A with TβR-I and inhibited the TGF-β-mediated signaling pathway in hepatoma cells and in HCV subgenomic replicon These data that the of NS5A with TβR-I can negatively TGF-β-stimulated of target which may be involved in HCV pathogenesis. NS5A of HCV was by the of HCV J.W. C.M. Cell. as a and into the and and by Kawabata and into the NS5A by and into the and cell lines in with and cells on with either or the as Lee Lee Chang Lee J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). To cell lines NS5A, Huh7 cells with the in the of in by Huh7 cells with as and used as a Huh7 cells HCV subgenomic by C. and in with amino acids and To HCV replicon cells with for of HCV replicon RNA was by and of resistance to Gene or Huh7 cells by the with of of consisting of in L. J.L. P. Zhou S. B. S.E. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), of reporter of in the of the S. S. P. S. 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PubMed Scopus Google Scholar). infection, cells with of the at for cells and in 10 and The cell by 10 through a on and at for 10 The was at for with either or The further with of protein for The with cell and the proteins by to a and by either or the in vivo binding between NS5A and Huh7 cells with the Huh7 cells either or with TGF-β for The cell was as and with for at The further with of protein for These proteins by and by Huh7 or cells on with either the and or the and with phosphate-buffered and in and for at in for at and with or for at three with PBS, cells further with for at two with in and three with PBS, cells the of the cells on with the and cells in and to with protein was by and cells on with the cells with PBS, in 10 and for on for and at for The was as a The was with C and and in C. The for 10 with for and at for 10 at The was as a nuclear The protein was determined by the a protein of protein to or and Huh7 cells with the cells with TGF-β for the and in A. of proteins to and to a The was in for and at with and in and two in the was with either or in for at an cells NS5A, the HCV subgenomic and at a of in at cells with TGF-β for and nuclear as Lee Hwang J. Full Text Full Text PDF PubMed Scopus (63) Google Scholar). cells by in and in of for on by at for at The with and in of C. The was further with in a for and at for 10 at The protein in nuclear was determined by the was with an J. 2002; Full Text Full Text PDF PubMed Scopus Google with in of binding and at for The by on a native and by the was with nuclear in binding for the of HCV NS5A TGF-β-induced and has a role in and cirrhosis in liver A. J. N. N. Engl. J. Med. 1991; PubMed Scopus Google Scholar). we found that TGF-β was in hepatocellular carcinoma of NS5A and NS5A regulates cell signaling we the role of NS5A in TGF-β-dependent activation a reporter gene assay. hepatoma cell lines with the NS5A and either the reporter S. S. P. S. J. 1998; PubMed Scopus Google or the reporter L. J.L. P. Zhou S. B. S.E. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). cells either or with TGF-β for and the was in TGF-β a of the in Huh7 was at a and was by NS5A. of NS5A protein inhibited TGF-β-induced activation by with the in both reporter is that cells are to reporter with Huh7 We found that of NS5A protein inhibited TGF-β-induced activation in a of protein showed effect on TGF-β-induced activation These results that NS5A protein the TGF-β-induced signal transduction pathway at the in hepatoma cell lines. cell TGF-β is a of and cell cycle at R. Akhurst R.J. Balmain A. Nat. Genet. 2001; 29: 117-129Crossref PubMed Scopus (1951) Google Scholar, J. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). is a kinase and with cell proliferation and differentiation, apoptosis, and transformation J.W. S.J. Curr. Genet. 1996; PubMed Scopus Google Scholar, T. R. B. Cell. 75: Full Text PDF PubMed Scopus Google Scholar). Majumder M. Ghosh A.K. Steele R. Ray R. Ray R.B. J. Virol. 2001; 75: 1401-1407Crossref PubMed Scopus (223) Google that HCV NS5A protein in a To investigate the direct effect of NS5A protein on we a reporter gene the Huh7 cells with the NS5A the and the reporter TGF-β-induced was in of NS5A protein inhibited TGF-β-induced in a is that the of was by NS5A protein and we the effect of NS5A protein on TGF-β-induced cell Huh7 cells either or NS5A with TGF-β for and a was Upon TGF-β of the cells with the of Huh7 cells NS5A that cells are to TGF-β-induced cell with These results that HCV NS5A protein TGF-β-induced cell through of NS5A protein TGF-β-mediated HCV NS5A protein TGF-β-stimulated Both Huh7 and cells with either the or reporter with or the NS5A cells either or with TGF-β for and on The data NS5A TGF-β-mediated activation in a Huh7 cells with the with and of HCV NS5A cells and was of cell to with and and NS5A TGF-β-mediated Huh7 cells with the with and of the HCV NS5A The was at by with cells and was of cell to with and and NS5A protein TGF-β-induced cell Huh7 cells and NS5A with TGF-β for and a was as NS5A with TβR-I in and in further investigate NS5A protein the TGF-β-mediated signaling we examined NS5A protein interacts with TGF-β signal transducers. viral proteins TGF-β signaling through Smad we examined the interaction between NS5A and Smad proteins a assay. The NS5A was into Huh7 cells with and with and the proteins cell with and the proteins either NS5A protein to with Smad proteins We examined the interaction between NS5A and TβR-I in We a and protein in with either or for at in selectively to protein to with To further in vitro interaction between NS5A and we a assay. was with NS5A in cells with a virus cell with either or and the proteins by NS5A interacted with TβR-I in vivo We by a for we NS5A interacts with Huh7 cells with of the cells either or with TGF-β for to with and the proteins by NS5A protein interacted with TβR-I of TGF-β We also that NS5A interacted with TβR-I in Huh7 cells NS5A protein Both in vitro and in vivo protein/protein interaction data that NS5A protein may with To Huh7 cells with the and and we examined the localization by was that NS5A protein is localized to the and in Huh7 cells Polyak S.J. H. Taylor D.R. Gretch D.R. Virology. 2002; PubMed Scopus Google Scholar). that NS5A was localized in the and showed of NS5A and in the cytoplasm as We further that NS5A and co-localized in cells results indicate that NS5A protein interacts with TβR-I both in vitro and in The of NS5A the the region in NS5A that is for TβR-I binding, the interaction of TβR-I with various of NS5A was determined by a in cells with the virus in interacted with an N-terminal of NS5A acids and a of NS5A acids the interacted with that the middle region acids of NS5A is required for NS5A TGF-β-mediated and of activation the phosphorylation and nuclear translocation of Smad Huh7 Smad2 protein was localized in the as in To the molecular mechanism of the effect of NS5A on TGF-β-mediated we examined NS5A nuclear translocation of Smad2 Huh7 cells with and in the or of NS5A. into and nuclear and the nuclear translocation of Smad2 protein was of the nuclear translocation of Smad2 protein NS5A protein in the nuclear translocation of Smad2 protein was inhibited by NS5A protein To further in we examined the effect of NS5A protein on TGF-β-induced nuclear translocation of Smad2 by K. M. F. Y. M. Y. N. T. T. M. J. K. Hepatology. 2000; PubMed Scopus Google Scholar), was in the cytoplasm and nucleus of Huh7 Upon TGF-β in the nucleus TGF-β-induced nuclear translocation of Smad2 was inhibited by NS5A protein middle and These results indicate that TGF-β-dependent nuclear of Smad proteins are inhibited by NS5A protein in hepatoma cell lines. NS5A interacts with TβR-I and TβR-I an important role in the TGF-β-dependent signaling pathway through the phosphorylation of we examined the role of NS5A protein in Smad2 To investigate NS5A TGF-β-mediated Smad2 Huh7 cells with either or NS5A with TGF-β for the that NS5A inhibited TGF-β-mediated phosphorylation of Smad2 in a These data indicate that NS5A TGF-β-mediated phosphorylation of Smad2 protein through interaction with NS5A of Smad3 and TβR-I phosphorylates proteins at the (20Macias-Silva M. Abdollah S. Hoodless P.A. Pirone R. Attisano L. Wrana J.L. Cell. 1996; 87: 1215-1224Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, Y. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar), which a in heterodimerization with Co-Smad A. Imamura T. Souchelnytskyi S. Kawabata M. A. K. J. K. P. J. 1997; PubMed Scopus Google Scholar). To the effect of NS5A on heterodimerization of Smad3 and Huh7 cells with and in the or of NS5A. cell with and the complex was by TβR-I formation between Smad3 and of NS5A inhibited complex formation between Smad3 and the protein of Smad3 and by NS5A, may NS5A modulates the TGF-β-dependent signaling pathway. NS5A TGF-β-induced Smad the effect of NS5A protein on Smad protein results in TGF-β-induced was Huh7 cells NS5A or with TGF-β for and nuclear was the in the to Upon TGF-β of in cells in cells NS5A protein is that TGF-β-induced as J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the of an further the of binding and HCV the TGF-β-induced further the effect of NS5A on TGF-β-induced in the of viral infection or viral RNA we an in vitro that production of HCV particles. we TGF-β-induced signaling in an HCV subgenomic replicon in which the HCV subgenomic RNA in Huh7 cells C. J. Virol. 2001; 75: PubMed Scopus Google Scholar). Both and HCV subgenomic replicon cells with reporter cells either or with TGF-β for reporter gene was in TGF-β-induced in HCV subgenomic replicon cells was by with Huh7 We examined NS5A protein can with TβR-I in the of viral RNA both and HCV subgenomic replicon cell with and proteins with in NS5A protein in HCV subgenomic replicon cells interacted with We further TGF-β-mediated Smad2 phosphorylation is by the HCV subgenomic Both and HCV subgenomic replicon cells with TGF-β for the and Smad2 phosphorylation was determined TGF-β Smad2 phosphorylation in cells the phosphorylation of Smad2 was inhibited by in HCV subgenomic replicon cells and We also examined Smad in and HCV subgenomic replicon cells by TGF-β-stimulated also in HCV subgenomic replicon cells These data that NS5A TGF-β-mediated signaling through interaction with TGF-β is one of the multifunctional cytokines that are implicated in cell including cell growth cell and of cell production and of matrix and apoptosis of cell lines. 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