Key points are not available for this paper at this time.
Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis, and hepatocellular carcinoma. Since there are several reports indicating that some viruses influence the tumor suppressor p53 function, we determined the effects of HCV proteins on p53 function and its mechanism determined by use of a reporter assay. Among seven HCV proteins investigated (core, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), only core protein augmented the transcriptional activity of p53 and increased the expression of p21 waf1 protein, which is a major target of p53. Core protein increased both DNA-binding affinity of p53 in electrophoretic morbidity shift assay and transcriptional ability of p53 itself in a reporter assay. The direct interaction between core protein and C terminus of p53 was also shown by glutathioneS-transferase fusion protein binding assay. In addition, core protein interacted with hTAFII28, a component of the transcriptional factor complex in vivo and in vitro. These results suggest that HCV core protein interacts with p53 and modulates p53-dependent promoter activities during HCV infection. Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis, and hepatocellular carcinoma. Since there are several reports indicating that some viruses influence the tumor suppressor p53 function, we determined the effects of HCV proteins on p53 function and its mechanism determined by use of a reporter assay. Among seven HCV proteins investigated (core, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), only core protein augmented the transcriptional activity of p53 and increased the expression of p21 waf1 protein, which is a major target of p53. Core protein increased both DNA-binding affinity of p53 in electrophoretic morbidity shift assay and transcriptional ability of p53 itself in a reporter assay. The direct interaction between core protein and C terminus of p53 was also shown by glutathioneS-transferase fusion protein binding assay. In addition, core protein interacted with hTAFII28, a component of the transcriptional factor complex in vivo and in vitro. These results suggest that HCV core protein interacts with p53 and modulates p53-dependent promoter activities during HCV infection. hepatitis C virus hepatocellular carcinoma polymerase chain reaction reverse transcription- polymerase chain reaction amino acids hemagglutinin TATA box-binding protein TATA box-binding protein-associated factors murine double minute 2 glutathione S-transferase Hepatitis C virus (HCV),1 a positive-stranded RNA virus, acts as a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) throughout the world (1Kuo G. Choo Q.-L. Alter H.J. Gitnick G.L. Redeker A.G. Purcell R.H. Miyamura T. Dienstag J.L. Alter M.J. Stevens C.E. Tegtmeier G.E. Bonino F. Colombo M. Lee A.S. Kuo C. Berger K. Shuster J.R. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 344: 362-364Crossref Scopus (3025) Google Scholar, 2Saito I. Miyamura T. Ohbayashi A. Harada H. Katayama T. Kikuchi S. Watanabe Y. Koi S. Onji M. Ohta Y. Choo Q.-L. Houghton M. Kuo G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6547-6549Crossref PubMed Scopus (1077) Google Scholar, 3Takano S. Yokosuka O. Imazeki F. Tagawa M. Omata M. Hepatology. 1995; 21: 650-655Crossref PubMed Google Scholar, 4Shiratori Y. Shiina S. Imamura M. Kato N. Kanai F. Okudaira T. Teratani T. Tohgo G. Toda N. Ohashi M. Ogura K. Niwa Y. Kawabe T. Omata M. Hepatology. 1995; 22: 1027-1033Crossref PubMed Google Scholar). More than 170 million persons are reported to be chronically infected worldwide (5World Health Organization 1998 press releases (1998), http://www.who.int/inf-pr-1998/en/pr98-36.html.Google Scholar). HCV, distantly related to the flaviviruses and the pestiviruses of the flavivirus family (6Choo Q.-L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Crossref PubMed Scopus (6184) Google Scholar, 7Miller R.H. Purcell R.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2057-2061Crossref PubMed Scopus (527) Google Scholar, 8Kato N. Hijikata M. Ootsuyama Y. Nakagawa N. Ohkoshi S. Sugimura T. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9524-9528Crossref PubMed Scopus (1087) Google Scholar), consists of an approximately 10-kilobase genome containing a large open reading frame encoding a polyprotein precursor of 3010–3033 amino acids and an untranslated region at the 5′ and 3′ ends of the genome. The putative organization of the HCV genome includes in order from the 5′ end the 5′-untranslated region, 3–4 structural proteins (core, E1, E2/p7), 6 nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), and the 3′-untranslated region (9Choo Q.-L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coito D. Medina-Selby R. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2451-2455Crossref PubMed Scopus (1521) Google Scholar, 10Hijikata M. Kato N. Ootsuyama Y. Nakagawa M. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5547-5551Crossref PubMed Scopus (579) Google Scholar, 11Grakoui A. Wychowski C. Lin C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 1385-1395Crossref PubMed Google Scholar). However, the effect of these proteins on the function of HCV-infected cells is still not fully understood. The tumor suppressor p53 protein monitors various cellular stresses such as DNA damage, while activated p53 functions via its ability to transactivate gene expression and mediates its downstream events mainly by two major pathways: cell cycle arrest and apoptosis (12Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6673) Google Scholar). Previous studies showed that p53 protein functionally interacts with several viral proteins variously. That is, some of these proteins were reported to suppress p53 function (13–25), but other viral proteins were reported to enhance p53 function (26Murono S. Yoshizaki T. Park C.S. Furusawa M. Histopathology. 1999; 34: 432-438Crossref PubMed Scopus (44) Google Scholar, 27Muganda P. Carrasco R. Qian Q. Cell. Mol. Biol. 1998; 44: 321-331PubMed Google Scholar, 28Roger J.A.G. Darerca O. Rookes S.M. Gallimore P.H. Virology. 1996; 218: 23-34Crossref PubMed Scopus (31) Google Scholar, 29McCornack S.J. Brazinski S.E. Moore J.L. Werness B.A. Goldstein D.J. Oncogene. 1997; 15: 265-274Crossref PubMed Scopus (88) Google Scholar, 30Chirillo P. Pagano S. Natoli G. Puri P.L. Burgio V.L. Balsano C. Levrero M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8162-8167Crossref PubMed Scopus (192) Google Scholar, 31Megyeri K. Berencsi K. Halazonetis T.D. Prendergast G.C. Gri G. Plotkin S.A. Rovera G. Gonczol E. Virology. 1999; 259: 74-84Crossref PubMed Scopus (43) Google Scholar, 32Devireddy L.R. Jones C.J. J. Virol. 1999; 73: 3778-3788Crossref PubMed Google Scholar). Recently HCV proteins have been found to modulate various intracellular signal transductin pathways (33Ray R.B. Lagging L.M. Meyer K. Steele R. Ray R. Virus Res. 1995; 37: 209-220Crossref PubMed Scopus (245) Google Scholar, 34Tsuchihara K. Hijikata M. Fukuda K. Kuroki T. Yamamoto N. Shimotohno K. Virology. 1999; 258: 100-107Crossref PubMed Scopus (102) Google Scholar, 35Matsumoto M. Hsieh T.Y. Zhu N.L. VanArsdale T. Hwang S.B. Jeng K.S. Gorbalenya A.E. Lo S.Y. Ou J.H. Ware C.F. Lai M.M.C. J. Virol. 1997; 71: 1301-1309Crossref PubMed Google Scholar, 36Taylor D.R. Shi S.T. Romano P.R. Barber G.N. Lai M.M.C. Science. 1999; 285: 107-110Crossref PubMed Scopus (659) Google Scholar). Then, in this study, we investigated whether HCV proteins (core, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) influence p53 function, with special attention paid to its transcriptional regulation of p21 (37el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7869) Google Scholar), a main downstream protein of the p53-dependent pathway that participates in cell cycle arrest (38el-Deiry W.S. Harper J.W. O'Connor P.M. Velculescu V.E. Canman C.E. Jackman J. Pietenpol J.A. Burrell M. Hill D.E. Wang Y. Wiman K.G. Mercer W.E. Kastan M.B. Kohn K.G. S.J. Kinzler K.W. Vogelstein B. Res. Google Scholar). cells cells cells carcinoma cells and murine were from cell and cells were in with cells were in containing cells from were in with and amino HCV RNA was from the of a with chronic hepatitis C HCV was reverse chain reaction with H. Y. S. K. Y. Y. T. K. F. Y. M. J. Virol. 73: PubMed Scopus Google Scholar). HCV NS2, NS3, NS4B, and were by with HCV the HCV RNA as a was as N. Yokosuka O. Omata M. K. M. J. 1990; PubMed Scopus Google Scholar, N. Yokosuka O. K. Y. M. Omata M. Res. 1993; PubMed Scopus Google Scholar). HCV region was by with Y. Hijikata M. S. T. Shimotohno K. J. Virol. 1995; PubMed Google by K. as a HCV region was by with N. Y. Omata M. J. Virol. 1997; 71: PubMed Google as a the region that a were with and the of by J. a expression a promoter H. K. J. 1991; PubMed Scopus Google Scholar), to NS2, NS3, NS4A, NS4B, NS5A, and which the amino acids core protein, was also from of core gene hemagglutinin core protein expression core and were by the of and core the of and which the core protein and were also by the The a p53 expression was by the p53 with a which was by a p53 expression M. Kanai F. H. T. K. T. Y. T. Y. H. Omata M. 1999; 44: PubMed Scopus Google Scholar), as a p53 was and of the a expression DNA-binding The with a was by and a of protein expression with was of core protein and protein and factors expression were was by M. of and was by expression a promoter was by R. of was by also by R. was the of hTAFII28, and by I. were also the of hTAFII28, and were the of was an and the as G. Toda N. Kanai F. Kato N. Y. K. Imazeki F. M. Omata M. Res. 1996; Google to gene of the was by the cell of cells with the in and the HCV core RNA was from with an at its of a promoter as a In and was to the The core protein was and by with with The containing the gene by a promoter an were as reporter containing a of the p21 promoter (37el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7869) Google Scholar), and containing of the p53 binding S.E. Pietenpol J.A. S. A. Kinzler K.W. Vogelstein B. Science. PubMed Scopus Google Scholar), both by B. Vogelstein containing promoter by C. P. Y. C. M. Mol. Cell. Biol. 1996; Scopus Google Scholar), and containing DNA binding with the gene which was as a reporter by fusion a to by virus was cells were a was to the the containing a of of of reporter of and of various of expression were to of the cells and of was to the were and were with the activity and activity were as with a activity was on were at in the expression of p53 and was were to the protein by and by The proteins were to and were the both of which were from the and with were from and The was by the expression of core protein, and with were cells were a a of of of and of were cells The cells were and were to E. P. Res. 1989; PubMed Scopus Google Scholar). The of the was determined and to In addition, p53 was to that there was the of expression in DNA binding assay was by electrophoretic shift assay p53 to the with a a containing two p53 binding was with and with of proteins and binding at In order to of p53 was K.G. D.E. Lee 1998; PubMed Scopus Google Scholar, K.G. Lee J. 1998; PubMed Scopus Google and to The of p53 in this assay was by of as as the by of of the were a containing and and by in the and which of p53 protein to glutathioneS-transferase in were by containing the of the of These were and with as by the of was approximately and the cell were in of containing and of fusion protein was to and with of the cell 2 of in core protein, at were with the and the proteins were by core with The expression of in E. was the as was by and in of the fusion protein was to and with of containing core the were determined by with cells were with of with of were with of and by with of by of of with the proteins were by of the was as The results from of activity were of with The the from with a of were the effect of HCV proteins on p53 function, containing the promoter of a p53 target gene (37el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7869) Google Scholar), was with a of p53 expression and of seven HCV protein expression the cells were shown in the activity of cell was than that of cell The other HCV not influence p21 promoter the effect of HCV core protein on transcriptional in of the p53 expression were in cells p53. activity of cell was than that of cell only core protein HCV proteins p21 promoter activity the of p53 whether core protein also the effect in other cell two cell with p53 and and cell B. J.R. M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: PubMed Scopus Google were In the was by while other cell were in the of p53 The of the p21 promoter activity by core protein was in but in and cells whether the of p21 promoter activity by core protein in a was with various of and a of Core protein activity from the p21 promoter in a in the of p53 2 However, the was not in the of p53. the effect of core protein on transcriptional were Core protein also the transcriptional of the p21 promoter in a in cells 2 the region of core protein the of p21 promoter we the and as fusion proteins expression and and These core protein expression were the assay with as a reporter The results showed that core protein effect on p21 promoter the core protein p21 promoter activity in cells 2 p21 promoter activity was by core protein in the assay cellular p21 protein expression was of p21 protein were determined by cell shown in the of p21 expression increased in to the of core whether this in p21 expression was to a in the of the expression of p53 was by the of the of p53 protein from a of was this These results that the of the p21 protein was not to an in p53 protein of the effects by core protein on the p21 promoter cells were with of of and of with of of the were by to activities are by the activity of cell as are as the of at cells were as in but and activities were activities are by the activity of cell as are as the of at cells were with of of of and the of various of HCV core protein expression of the were by to activities were as are by the activity of cell as and are as the of at of p21 protein by core cells and cells were as in the to cells were and the protein was and of and core protein with results of the are a was in cells with p53. In the of p21 protein increased and the of p53 protein was with an of core protein whether the of p21 promoter activity by core protein on p53 a reporter containing only of the was the assay. In both cells and core protein the transcriptional ability of p53 in a and The of activity was not in cells p53 results were in and in cells not whether core protein the other p53 a reporter containing promoter with was The activity of was also by core protein in a in both and cells core protein the of p53 the were p53 protein were not increased by core protein, the of whether not core protein modulates p53 DNA binding activity was shift assay was the of which been with and a shown in binding activity was by core protein cells with that in cells 2 and These were were by an of but not by the and in addition, were with the p53 showed that core protein binding activity 2 and the p53 expression were in by of binding was not to a in p53 expression were of p53 with in core protein in an in binding activity 2 and The were with an of the but not with of and showed that core protein binding activity 2 and These results showed that p53 DNA binding affinity was by the core whether core protein modulates p53 transcriptional ability itself of its DNA binding a DNA-binding fusion protein expression and DNA-binding with were Since DNA binding of was by in this the activity the transcriptional ability of p53 of with of and were cells were the shown in core protein the activity by in a this effect is on transcriptional to the affinity of a of expression was Core protein effect on the activity by not These results showed that core protein p53-dependent transcriptional ability and the of p53 function by core protein not only on of the binding affinity but also on of transcriptional ability of p53 the the of binding affinity and transcriptional ability of p53 by core protein, direct interaction between core protein and p53 was by binding assay. core protein the cell of which been with were with protein, and proteins were by shown in core protein to but not to which region of p53 was interaction with core protein, and of p53 were to protein and the binding assay. shown in core protein to but not to that of p53 was core protein binding these fusion proteins with in core protein not only an interaction with but that the of p53 was the the interaction between p53 and core protein was we whether core protein itself transcriptional the transcriptional activity of core protein, a protein expression by core gene the was and were cells and the cell were However, fusion protein not from that core protein itself not transcriptional activity not core protein itself transcriptional was that core protein as a transcriptional studies have that p53 interacts with C.J. J.L. R. R. Science. 1995; PubMed Scopus Google Scholar), and G. J. Y. J.L. C. Mol. Cell. Biol. 1996; PubMed Google in a transcriptional to the of of and to the of p53 transcriptional ability by core protein, the ability of core protein to with was by assay. cells were with and hTAFII28, and cell were with and the proteins were by with shown in core protein interacted with The was interaction was by in core and In core protein was with fusion protein protein and with the shown in the core protein be by that core protein to hTAFII28, a component of the transcriptional factor In this study, we showed investigated HCV only core protein augmented the promoter activity and the expression of p21 waf1 showed that this in a p53-dependent and from several in the of p53 in the of p21 promoter activity by the core protein was not 2 p53-dependent of p21 promoter activity by core the by core protein was also p53. However, the effect was in be by the that p53 is by protein in in and of p53 M. T. J.M. P.M. J. Virol. PubMed Google Scholar). the ability of by core protein was in was reported that p53 by T. G. F. C.J. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). Since is not in cells in of the in cells be than that in other cell These also suggest p53-dependent of p21 promoter activity by core reporter core protein the activity by but effect on the activity by showed that core protein by p53 These results suggest that core protein p21 promoter activity in a p53-dependent viral proteins have been reported to enhance p53 function (26Murono S. Yoshizaki T. Park C.S. Furusawa M. Histopathology. 1999; 34: 432-438Crossref PubMed Scopus (44) Google Scholar, 27Muganda P. Carrasco R. Qian Q. Cell. Mol. Biol. 1998; 44: 321-331PubMed Google Scholar, 28Roger J.A.G. Darerca O. Rookes S.M. Gallimore P.H. Virology. 1996; 218: 23-34Crossref PubMed Scopus (31) Google Scholar, 29McCornack S.J. Brazinski S.E. Moore J.L. Werness B.A. Goldstein D.J. Oncogene. 1997; 15: 265-274Crossref PubMed Scopus (88) Google Scholar, 30Chirillo P. Pagano S. Natoli G. Puri P.L. Burgio V.L. Balsano C. Levrero M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8162-8167Crossref PubMed Scopus (192) Google Scholar, 31Megyeri K. Berencsi K. Halazonetis T.D. Prendergast G.C. Gri G. Plotkin S.A. Rovera G. Gonczol E. Virology. 1999; 259: 74-84Crossref PubMed Scopus (43) Google Scholar, 32Devireddy L.R. Jones C.J. J. Virol. 1999; 73: 3778-3788Crossref PubMed Google Scholar). These proteins are which are to be related to viral and as a of a of viral and cellular the mechanism of this is by the in p53 protein during the In this study, we showed that core protein, a structural protein, p53 function the of p53 two were core protein the of p53 DNA binding as shown in electrophoretic shift assay also showed the direct binding between core and the C terminus of the p53 are a of reports indicating that the C terminus of p53 protein its DNA binding affinity D.W. Cell. 71: Full Text PDF PubMed Scopus Google Scholar, A. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, Halazonetis T.D. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar), and that protein, a of a family of proteins that cellular activity by binding and several with the C terminus of p53 to suppress and its DNA binding affinity M.J. J.L. Halazonetis T.D. 1998; PubMed Scopus Google Scholar). is that binding of the core protein to p53 protein the C terminus of p53 and protein the p53 DNA binding core protein also the transcriptional ability of p53 of binding affinity from the results is the interaction between core protein and interaction was cell that core protein p53. p53 interacts with its transcriptional activity G. J. Y. J.L. C. Mol. Cell. Biol. 1996; PubMed Google Scholar), and was reported to with G. M. A. P. I. J. 1995; PubMed Scopus Google Scholar). core protein itself not transcriptional these to that core protein as a transcriptional in a complex of core protein, hTAFII28, and that the transcriptional ability of p53. p53 and transcriptional factors are to mainly in the be whether core protein the and with the transcriptional are in the of the core protein J.H. Res. PubMed Scopus Google Scholar, A. Natoli G. D. A. Levrero M. E. J. Full Text PDF PubMed Scopus Google Scholar). was reported that core protein in the but was also found in the K. T. K. M. T. D. J.R. M. J. Virol. 1998; PubMed Google Scholar, Lee J. Virol. 1995; PubMed Google Scholar). we whether core protein the ability of of p53 function, and showed that core protein effect on the p21 promoter 2 this core protein the ability to the the p53-dependent of p21 However, be that only a of the core protein the the we which are to in the not fully the of the of p53 function by core been that p53 protein is by and A.J. Kastan M. 1998; PubMed Scopus Google Scholar), whether such a by core protein be results are in to reports R.B. Steele R. Meyer K. Ray R. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, R.B. Steele R. Meyer K. Ray R. 1998; PubMed Scopus Google in which core protein was shown to suppress the activities of p53 and p21 by reporter In study, only core protein expression was and was the be that the core protein expression promoter activities to a In we seven HCV protein expression the promoter and showed that only core protein the p53 function in a p53-dependent and we showed not only the activities from p21 promoter but also the expression of the p21 protein by core is to the protein expression of the target of this Lo S. K. Ou Virology. 1999; PubMed Scopus Google reported that core protein p53 However, the mechanism of of p53 function by core protein showed in to the of p53 function by core protein, that this effect is from two the of binding affinity and transcriptional ability of p53 by core In addition, we showed that core protein interacts with hTAFII28, a of a transcriptional core protein been reported to other cellular (33Ray R.B. Lagging L.M. Meyer K. Steele R. Ray R. Virus Res. 1995; 37: 209-220Crossref PubMed Scopus (245) Google Scholar, 34Tsuchihara K. Hijikata M. Fukuda K. Kuroki T. Yamamoto N. Shimotohno K. Virology. 1999; 258: 100-107Crossref PubMed Scopus (102) Google Scholar, 35Matsumoto M. Hsieh T.Y. Zhu N.L. VanArsdale T. Hwang S.B. Jeng K.S. Gorbalenya A.E. Lo S.Y. Ou J.H. Ware C.F. Lai M.M.C. J. Virol. 1997; 71: 1301-1309Crossref PubMed Google Scholar, M. R. Hwang S.B. Lee A.S. Lai M.M.C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), the of regulation are not understood. interaction between core protein and be a to the the various effects of core protein on other HCV is to be a causative agent of The core protein was both in and Y. M. T. K. 1996; PubMed Google Scholar). was reported that the of hepatocellular carcinoma p53 p21 protein, while only a of hepatocellular carcinoma with p53 p21 expression S.T. M. J. 1998; PubMed Scopus Google Scholar). Previous of the p53 gene in from that of p53 is in but in H. Imazeki F. M. Omata M. 1993; PubMed Scopus Google Scholar, T. H. A. M. S. Res. Google Scholar). p53 was reported to be a in with K. E. A. C. S. M. S. A. A. J. 1998; PubMed Scopus Google Scholar). is that p53 is the of the of p53 ability by core protein and the In the showed HCV proteins only core protein augmented the promoter activity and the expression of p21 waf1 and to p53 in vitro. These results suggest that HCV core protein the various functions of p53 and to the of and are also to during the of this
Otsuka et al. (Wed,) studied this question.