Hepatitis C Virus Core Protein Enhances p53 Function through Augmentation of DNA Binding Affinity and Transcriptional Ability*

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), 1 a positive-stranded RNA virus, acts as a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) throughout the world (1)(2)(3)(4). More than 170 million persons are reported to be chronically infected worldwide (5). HCV, distantly related to the flaviviruses and the pestiviruses of the flavivirus family (6 -8), 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 5Ј and 3Ј ends of the genome. The putative organization of the HCV genome includes in order from the 5Ј end the 5Ј-untranslated region, 3-4 structural proteins (core, E1, E2/p7), 6 nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), and the 3Ј-untranslated region (9 -11). However, the effect of these proteins on the function of HCV-infected cells is still not fully understood.
Mammalian Expression Plasmids for HCV Proteins-HCV RNA was extracted from the serum of a 50-year-old male patient with chronic hepatitis C type 1b using SepaGene-RV (Sankyo Junyaku, Tokyo, Japan). HCV genotyping was performed using reverse transcriptionpolymerase chain reaction (RT-PCR) with type-specific primers (39). HCV core, NS2, NS3, NS4B, and NS5B regions were amplified by RT-PCR with HCV region-specific primers using the extracted HCV RNA as a template. RT-PCR was performed as described previously (40,41). Type 1b HCV NS4A region was amplified by PCR with NS4Aspecific primers using pCMV/N1658 -1711 (encoding NS4) (42) (kindly provided by K. Shimotohno, Kyoto University) as a template. Type 1b HCV NS5A region was amplified by PCR with NS5A-specific primers using pM-NS5A/F5-R5 (encoding NS5A) (43) as a template. All synthetic primers had a XhoI restriction site for subcloning PCR products, except primers for the NS3 region that had a SalI restriction site.
Mammalian Expression Plasmids for p53, VP16, and TFIID-The pCXN2-p53, a human wild type p53 expression vector was constructed by subcloning the p53 cDNA fragment with a XhoI site into pCXN2, which was amplified by PCR using AdCAp53, a human wild type p53 expression adenovirus described previously (45), as a template. To generate pCXN2-Gal4BD-p53, p53 cDNA was subcloned into EcoRI and PstI sites of the pM vector (CLONTECH Laboratories, Palo Alto, CA), a mammalian expression vector for generating Gal4 DNA-binding domain fused protein. The Gal4BD-p53 cDNA fragment with a XhoI site was then amplified by PCR and subcloned into pCXN2. To generate pCXN2-Gal4BD-VP16, a Gal4BD fused activation domain of herpesvirus VP16 protein expression vector, Gal4BD-VP16 cDNA fragment with a XhoI site amplified using pM3-VP16 (CLONTECH) was subcloned into pCXN2.
All cloned plasmids were purified using the Endofree plasmid kit (Qiagen, Hilden, Germany). Nucleotide sequencing of constructed plasmids was performed using an autosequencer (PE Applied Biosystems, Foster City, CA) and the dye termination method as described previously (46) to confirm gene integration. Expression of the cloned genes was confirmed by immunoblotting the cell extracts of COS-7 cells transfected with each plasmid using the corresponding antibody.
In Vitro Transcription and Translation-For in vitro transcription and translation, the HCV core RNA was synthesized from PCR product with an extension at its 5Ј-end consisting of a T7 promoter using pCXN2-core as a template. In vitro transcription and translation was carried out using Protein Truncation Test Kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions. The translated core protein was biotinylated and confirmed by immunoblotting with ECL streptavidin conjugated with horseradish peroxidase kit (Amersham Pharmacia Biotech).
Reporter Plasmids for Luciferase Assay-The following vectors containing the Photinus pyralis (firefly) luciferase repoter gene driven by a basic promoter element (TATA box), plus an inducible cis-enhancer element were utilized as reporter plasmids: WWP-luc, containing a 2.4-kilobase pair genomic fragment of the human p21 promoter (37), and PG13-luc, containing 13 copies of the p53 binding sequences (47), both kindly provided by B. Vogelstein (Johns Hopkins University, Baltimore, MD); MDM2-luc, containing human MDM-2 promoter kindly provided by C. Prives (Columbia University, New York) (48), and pFR-luc, containing five Gal4 DNA binding sequences linked with the luciferase gene (Stratagene, La Jolla, CA), which was used as a reporter for transcription mediated by Gal4BD fusion proteins. To check transfection efficiency, pRL-TK, a control plasmid to express Renilla reniformis (seapansy) luciferase driven by herpes simplex virus thymidine kinase (Toyo Ink, Tokyo, Japan), was used.
Transient Transfection and Luciferase Assay-Approximately 4 ϫ 10 5 cells were plated onto a 6-well tissue culture plate (Iwaki Glass, Chiba, Japan) 24 h before transfection. Transfection was performed using FuGene6 Transfection Reagent (Roche Molecular Biochemicals) according to the manufacturer's instructions. Unless otherwise specified, the transfection complexes containing a total of 0.6 g of plasmids (0.39 g of firefly luciferase reporter plasmid, 0.01 g of pRL-TK, and 0.2 g of pCXN2 or various types of expression plasmids) were added to each well of the 6-well plate. Using SAOS-2 cells and Hep3B cells, 0.01 g of PCXN2-p53 was added to the transfection complexes when necessary.
Cells were harvested 36 h after transfection and luciferase assays were carried out with the PicaGene Dual Seapansy System (Toyo Ink).
Firefly luciferase activity and seapansy luciferase activity were measured as relative light unit with a luminometer (Lumat LB9507, EG&G Berthold, Bad Wildbad, Germany). Firefly luciferase activity was normalized for transfection efficiency based on seapansy luciferase activity. All assays were performed at least in triplicate.
Western Blotting Analysis-To determine the expression levels of p53 and p21, immunoblotting was performed. Cell extracts were adjusted to the same protein concentration by Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, IL) and resolved by SDS-polyacrylamide gel. The proteins were transferred to polyvinylidene difluoride membranes (Hybond-P; Amersham Pharmacia Biotech). Rabbit anti-p53 and goat anti-p21 IgG polyclonal antibodies were used for the first antibody, both of which were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). As for the second antibody, anti-rabbit antibody and anti-goat antibody conjugated with horseradish peroxidase were purchased from Amersham Pharmacia Biotech and Santa Cruz, respectively. The bound antigen was detected by ECL-plus (Amersham Pharmacia Biotech). To determine the expression levels of core protein, mouse anti-core monoclonal antibody (Austral Biologicals, San Ramon, CA) and anti-mouse antibody conjugated with horseradish peroxidase (Amersham Pharmacia Biotech) were used.
Preparation of Nuclear Extracts and DNA Binding Assays-Approximately 2.5 ϫ 10 6 SAOS-2 cells were plated onto a 10-cm dish (Iwaki Glass). Twenty-four hours later, a total of 3 g of plasmids (1.5 g of pCXN2-p53 and 1.5 g of pCXN2 or pCXN2-core) were transfected into SAOS-2 cells using FuGene6. The cells were harvested 36 h after transfection, and nuclear extracts were prepared according to mininuclear extraction methods (49). The concentration of the nuclear extracts was determined and adjusted to give equal concentrations. In addition, p53 was immunoblotted to confirm that there was the same amount of expression in pCXN2-p53 transfected cells. DNA binding assay was performed by electrophoretic mobility shift assay using p53 NuShift kit (GENEKA, Montréal, Canada) according to the manufacturer's protocol with a minor modification. Briefly, a synthetic doublestranded oligonucleotide containing two p53 consensus binding sites (5Ј-GGACATGCCCGGGCATGTC-3Ј) was end-labeled with [ 32 P]ATP using T4 polynucleotide kinase and then incubated with either 3 g of nuclear extracts or recombinant proteins and binding buffer (4% glycerol, 1 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.05 mg/ml poly(dI-dC)) for 40 min at 4°C. In order to visualize p53-DNA complexes, 200 ng of p53 monoclonal antibody pAb 421 (Calbiochem, La Jolla, CA) was required (50,51) and added to all mixtures. The specificity of p53 in this assay was tested by adding 100-fold excess of either wild type or mutant competitors (5Ј-GGATCGCCCCGGGCATGTC-3Ј), as well as the supershift caused by addition of 300 ng of the anti-p53 antibody (pAb 1801, Calbiochem). Reaction mixtures were loaded onto a 4% polyacrylamide gel using buffer containing 50 mM Tris, 200 mM glycine, pH 8.5, and 1 mM EDTA and then separated by electrophoresis in the same buffer.
GST Binding Assay-pGEX4T1-p53, p53N, and p53C, which express three types of p53 protein fused to glutathione S-transferase (GST) in Escherichia coli, were constructed by subcloning PCR amplified XhoI fragments containing cDNA sequences for the 1-393, 1-71, or 72-393 aa of p53, respectively, into the XhoI site of pGEX4T1 (Amersham Pharmacia Biotech, Uppsala, Sweden). These constructs were expressed in E. coli BL21 (DE3) (Stratagene) and purified with glutathione-Sepharose 4B beads as specified by the manufacturer (Amersham Pharmacia Biotech). 0.6 g of pCXN2-core was transfected into approximately 4 ϫ 10 5 COS-7 cells, and the cell lysates were prepared in 200 l of immunoprecipitation buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA, 0.25% gelatin, 0.02% sodium azide, 100 g/ml phenylmethylsulfonyl fluoride, and 1 g/ml aprotinin. Approximately 10 g of each fusion protein was bound to glutathione-Sepharose and incubated with 150 l of the cell lysates or 2 l of in vitro translated core protein, respectively, at 4°C overnight. Beads were washed four times with the same buffer and the bound proteins were blotted by either anti-HCV core monoclonal antibody or streptavidin conjugated with horseradish peroxidase.
The expression of GST-hTAF II 28 in E. coli was conducted using the same procedures as for GST-p53. pGEX4T1-hTAF II 28 was constructed by subcloning hTAF II 28 cDNA into pGEX4T1 using PCR amplification and expressed in BL21. Approximately 10 g of the fusion protein was bound to glutathione-Sepharose and incubated with 3 l of the in vitro translational mixture containing biotinylated core protein. After washing, the precipitated products were determined by streptavidin conjugated with horseradish peroxidase.
Immunoprecipitation-4 ϫ 10 5 COS-7 or SAOS-2 cells were transfected with 0.4 g of pCXN2-core or pCXN2-HA-core together with 0.4 g of HA-tagged hTAF II vectors (pCXN2-HA-hTAF II 18,20,28,70,TBP) or pCXN2-TAF II 31/32. Cell extracts were prepared with 200 l of immunoprecipitation buffer and precipitated by incubation with 0.2 g of anti-HA polyclonal antibody or anti-core antibody followed by addition of 20 l of Protein A-Sepharose (Amersham Pharmacia Biotech). After being washed 4 times with immunoprecipitation buffer, the precipitated proteins were revealed by immunoblotting using anti-core antibody or anti-hTAF II 31/32 antibody (Santa Cruz). For reference, 10 l (1/20) of the lysate was used as "input." Statistical Analysis-The results from assays of luciferase activity were analyzed using analysis of variance (ANOVA) with post-hoc Scheffe testing (StatView J, Abacus Concepts Inc., Barkeley, CA). The data represent the mean Ϯ S.D. calculated from three independent experiments. Differences with a value of p Ͻ 0.05 were considered significant.

RESULTS
Enhancement of p21 waf1 Promoter Activity by Core Protein-To determine the effect of HCV proteins on p53 function, WWP-luc containing the promoter of p21, a well known p53 target gene (37), was co-transfected with a fixed level of p53 expression plasmid and each of seven HCV protein expression plasmids into p53-null SAOS-2 cells. Thirty-six hours later, the cells were assayed for luciferase activity. As shown in Fig. 1A, the relative firefly luciferase activity of WWP-luc/pCXN2-core transfected cell lysates was 5.4 Ϯ 0.7 (mean Ϯ S.D.) times higher than that of WWP-luc/pCXN2-transfected cell lysates. The other HCV proteins, however, did not influence p21 promoter activity.
To analyze the effect of HCV core protein on endogenous p53-mediated transcriptional activation in hepatocytes, similar experiments without transfection of the p53 expression plasmid were conducted in HepG2 cells having wild type p53. Relative firefly luciferase activity of WWP-luc/pCXN2-core transfected cell lysates was 3.2 Ϯ 0.5 (mean Ϯ S.D.) times higher than that of WWP-luc/pCXN2 transfected cell lysates (Fig. 1B). Thus, only core protein among HCV proteins significantly enhanced p21 promoter activity under the presence of either endogenous or exogenous p53 protein.
To examine whether core protein also demonstrated the same effect in other cell lines, two cell lines with supposedly intact p53 (HeLa and NIH3T3) and one p53-deficient cell line (Hep3B) (52) were used. In Hep3B, the experiment was performed by adding pCXN2-p53, while other cell lines were examined in the absence of exogenous p53 protein. The enhancement of the p21 promoter activity by core protein was observed in Hep3B but weakly in NIH3T3 and HeLa cells (Fig. 1C).
Enhancement of p21 waf1 Promoter Activity by Core Protein in a Dose-dependent Manner-To determine whether the enhancement of p21 promoter activity by core protein occurred in a dose-dependent manner, WWP-luc was co-transfected with various amounts of pCXN2-core and a fixed level of pCXN2 or pCXN2-p53 into SAOS-2 cells. Core protein enhanced luciferase activity from the p21 promoter in a dose-dependent manner in the presence of exogenous p53 ( Fig. 2A). However, the enhancement was not observed in the absence of exogenous p53. To analyze the effect of core protein on endogenous p53-mediated transcriptional activation, similar experiments without pCXN2-p53 transfection were performed using HepG2 cells. Core protein also enhanced the endogenous p53-mediated transcriptional activation of the p21 promoter in a dose-dependent manner in HepG2 cells (Fig. 2B).
To elucidate the region of core protein responsible for the enhancement of p21 promoter activity, we constructed the following N-terminal deleted and C-terminal truncated mutants as HA-tagged fusion proteins expression plasmids: pCXN2-HA 1-191 aa (full-length), HA 1-173 aa (C-terminal truncated), HA 63-191 aa (N-terminal deleted), and HA 63-173 aa (both N-and C-terminal deleted). These deleted core protein expression plasmids were used for the luciferase assay with WWP-luc as a reporter plasmid. The results showed that N-terminal deleted core protein had no effect on p21 promoter activity, although the full-length or C-terminal truncated core protein enhanced p21 promoter activity in SAOS-2 cells (Fig. 2C).
Increased Induction of Endogenous p21 Protein by Core Protein-As p21 promoter activity was enhanced by core protein in the luciferase assay system, cellular p21 protein expression was examined. Endogenous levels of p21 protein were determined by Western blot analysis using SAOS-2 cell lysates. As shown in Fig. 3A, the levels of endogenous p21 expression increased in relation to the amount of core protein. To examine whether this increase in p21 expression was due to a change in the amount of p53, the expression of p53 was measured by immunoblotting. Although the dose of pCXN2-core increased, the amount of p53 protein from a fixed level of pCXN2-p53 was equivalent under this condition (Fig. 3). These results indicate that the increase of the p21 protein was not due to an increase in p53 protein level.
Without exogenous p53, a similar experiment was performed in HepG2 cells with endogenous intact p53. In HepG2 cells, the levels of p21 protein similarly increased and the amount of p53 protein was unchanged with an increasing dose of core protein (Fig. 3).
Core Protein Enhances the Transcription from p53 Responsive Elements-To determine whether the enhancement of p21 promoter activity by core protein depends on p53 responsive elements, a reporter plasmid containing only multiple copies of the p53-binding site, PG13-luc, was used for the luciferase assay. In both SAOS-2 cells and HepG2 cells, core protein enhanced the transcriptional ability of p53 in a dose-dependent manner (Fig. 4, A and B). The enhancement of luciferase activity was not observed in SAOS-2 cells without exogenous p53 (Fig. 4A). Similar results were obtained in NIH3T3, Hep3B, and weakly in HeLa cells (data not shown).
To confirm whether core protein influenced the other p53 responsive elements, MDM2-luc, a reporter plasmid containing human MDM-2 promoter linked with human luciferase gene, was used. The luciferase activity of MDM2-luc was also en-  Fig. 2. Thirty-six hours later, cells were harvested and the protein concentration was normalized and then used for immunoblotting of p53, p21, and core protein with corresponding antibodies, respectively. Representative results of the immunoblotting are indicated. hanced by core protein in a dose-dependent manner in both SAOS-2 and HepG2 cells (Fig. 4C).
Core Protein Enhances p53 DNA Binding Activity-To elucidate how core protein enhances the transcription of p53 responsive elements, the following experiments were conducted. Because p53 protein levels were not increased by core protein, the possibility of whether or not core protein modulates p53 DNA binding activity was investigated. Electrophoretic mobility shift assay was performed using the nuclear extracts of SAOS-2 cells, which had been co-transfected with pCXN2-p53 and pCXN2-core, or empty vector (as a control). As shown in Fig.  5A, p53-DNA binding activity was enhanced by core protein expressed cells compared with that observed in empty vector transfected cells (Fig. 5A, lanes 2 and 3). These bound bands observed were specific since they were ablated by an excess of unlabeled wild type competitor but not by the mutated oligonucleotide (Fig. 5A, lanes 4 and 5) and, in addition, they were supershifted with the p53 N-terminal antibody pAb1801 (Fig.  5A, lane 6). Densitometrical analysis showed that core protein enhanced p53-DNA binding activity 1.7-fold (Fig. 5A, compare  lanes 2 and 3). Because the p53 expression levels were unchanged in each nuclear extract by Western blotting (Fig. 5B), increase of p53-DNA binding was not due to a change in p53 expression level.
Similar experiments were performed using recombinant GST-p53 protein. Pretreatment of recombinant p53 with in vitro translated core protein resulted in an increase in p53-DNA binding activity (Fig. 5C, lanes 2 and 3). The bound bands were competed with an excess of wild type oligonucleotide encompassing the p53-binding site but not with excess of mutated oligonucleotide (Fig. 5C, lanes 4 and 5). Densitometrical analysis showed that core protein enhanced p53-DNA binding activity 2.1-fold (Fig. 5C, compare lanes 2 and 3). These results showed that p53 DNA binding affinity was enhanced by the core protein.
Core Protein Enhances p53 Transcriptional Ability-To elucidate whether core protein modulates p53 transcriptional ability itself irrespective of its DNA binding activity, pCXN2-Gal4BD-p53, a Gal4 DNA-binding domain-p53 fusion protein expression vector, and pFR-luc, Gal4 DNA-binding sequences linked with luciferase gene, were used. Since DNA binding of Gal4BD-p53 was mediated by Gal4 sequence in this system, the luciferase activity reflected the transcriptional ability of p53 itself. Various amounts of pCXN2-core with fixed amounts of pCXN2-Gal4BD-p53 and pFR-luc were co-transfected into SAOS-2 cells. Thirty-six hours later, cells were assayed for the luciferase activity. As shown in Fig. 6, core protein enhanced the luciferase activity by Gal4BD-p53 in a dose-dependent manner. To confirm this effect is neither general on transcriptional machinery nor due to the increasing affinity of DNA-Gal4 binding, pCXN2-Gal4BD-VP16, a Gal4BD-fused activa- FIG. 4. Core protein enhances the transcription from p53 responsive elements. In all samples, total amounts of the transfected plasmids were adjusted by adding empty vector (pCXN2) to 0.6 g. Thirty-six hours after transfection, cells were harvested and luciferase activities were measured and normalized on the basis of seapansy luciferase activities. Results are expressed as the mean (bar) ϩ S.D. (line) of at least three experiments. *, p Ͻ 0.05; **, p Ͻ 0.01. A, SAOS-2 cells were transfected with 0.39 g of PG13-luc, 0.01 g of pRL-TK, and indicated amounts of pCXN2-core with or without 0.01 g of pCXN2-p53. Luciferase activities are expressed by taking the activity of pCXN2-transfected cell lysates without exogenous p53 protein as 1. B, HepG2 cells were transfected described as in A but without pCXN2-p53. C, SAOS-2 cells were transfected with 0.39 g of MDM2-luc, 0.01 g of pRL-TK, and the indicated amounts of pCXN2-core with or without 0.01 g of pCXN2-p53. HepG2 cells were transfected similarly but without pCXN2-p53. tion domain of VP16 mammalian expression vector, was used for similar experiments. Core protein had no effect on the luciferase activity mediated by Gal4BD-VP16 (data not shown). These results showed that core protein enhanced p53-dependent transcriptional ability and the enhancement of p53 function by core protein might depend not only on augmentation of the p53-DNA binding affinity but also on enhancement of transcriptional ability of p53 itself.
HCV Core Protein Binds p53 in Vitro-To clarify the molecular mechanisms underlying the enhancement of p53-DNA binding affinity and transcriptional ability of p53 by core protein, direct interaction between core protein and p53 was examined by GST binding assay. Either in vitro translated core protein or the cell lysate of COS-7 cells, which had been transfected with pCXN2-core, were incubated with purified GST-p53 protein, and bound proteins were checked by Western blotting. As shown in Fig. 7, core protein bound to GST-p53 but not to GST.
To determine which region of p53 was required for interaction with core protein, N-terminal deletion (p53C; aa 72-393) and C-terminal truncation (p53N; aa 1-71) mutants of p53 were fused to GST protein and used for the GST binding assay. As shown in Fig. 7, core protein bound strongly to GST-p53C but not to GST-p53N, suggesting that aa 72-393 of p53 was important for core protein binding. GST binding assays using these GST-p53 fusion proteins with biotinylated in vitro translated core protein confirmed not only an interaction with fulllength p53, but revealed that the C-terminal domain of p53 was required for the interaction.
Interaction between HCV Core Protein and hTAF II 28 -As the interaction between p53 and core protein was demonstrated, we examined whether core protein itself had transcriptional activity. To elucidate the transcriptional activity of core protein, pBIND-core, a GAL4BD-core hybrid protein expression vector, constructed by subcloning core gene into the pBIND vector (Promega, Madison, WI) was used. pBIND-core and pFR-luc were co-transfected into SAOS-2 cells and the cell lysates were assayed for luciferase activity. However, GAL4BD-core fusion protein did not activate transcription from pFR-luc, showing that core protein itself had not transcriptional activity (data not shown).
Although core protein itself had no transcriptional ability, it was possible that core protein might act as a transcriptional co-activator. Prior studies have established that p53 interacts with hTAF II 32/31, hTAF II 70 (53), and TBP (54) in a transcriptional initiation complex. Thus, to investigate the possible contribution of TFIID comprised of TBP and hTAFs to the enhancement of p53 transcriptional ability by core protein, the ability of core protein to interact with TFIID was examined by co-immunoprecipitation assay. COS-7 cells were transfected with pCXN2-core and vectors expressing HA-hTAF II 18, hTAF II 20, hTAF II 28, hTAF II 70, and TBP. Transfected cell extracts were immunoprecipitated with anti-HA antibody, and the precipitated proteins were analyzed by immunoblotting with anti-core protein. As shown in Fig. 8A, core protein interacted with hTAF II 28. The same result was observed using p53-deficient SAOS-2 cells. This interaction was confirmed by using in vitro translated core and recombinant hTAF II 28 protein. In vitro translated core protein was incubated with either FIG. 5. Core protein enhances the p53 DNA binding affinity. A, 2.5 ϫ 10 6 SAOS-2 cells were transfected with 1.5 g of pCXN2-p53 and 1.5 g of pCXN2-core. Thrity-six hours later, nuclear extracts were prepared and assayed for p53 DNA binding affinity by gel shift assay. The following were added to the reaction: lane 1, nuclear extract from cells without transfection as negative control; lane 2, nuclear extract from cells transfected with pCXN2-p53 and pCXN2; lane 3, nuclear extract from cells transfected with pCXN2-p53 and pCXN2-core; lanes 4 -6, the same nuclear extracts as lane 2, except that either excess p53 consensus binding oligonucleotide probe or excess unlabeled mutant oligonucleotide probe was added for competition, respectively; lane 6, anti-p53 N-terminal antibody (pAb1801) was added for supershift analysis. Densitometrical analysis demonstrated that the relative intensities of the specific bands of lanes 2-6 in A are 1, 1.71, 0.06, 0.98, 0.97, respectively. B, nuclear extracts from the cells that were transfected with the indicated expression plasmids were immunoblotted with anti-p53 antibody. C, 1 g of recombinant GST-p53 protein was added to the reaction (except lane 1, GST protein as a control), in the absence (lanes GST-hTAF II 28 fusion protein or GST control protein and then precipitated with the glutathione-Sepharose beads. As shown in Fig. 8B, the core protein could be pulled down specifically by GST-hTAF II 28 protein. This result indicated that core protein can directly bind to hTAF II 28, a component of the transcriptional factor complex. DISCUSSION In this study, we showed that, among investigated HCV proteins, only core protein augmented the promoter activity and the expression of p21 waf1 . We showed that this augmentation occurs in a p53-dependent and p53-specific manner from several results. First, in the absence of exogenous p53 in p53-null SAOS-2 cells, the enhancement of p21 promoter activity by the core protein was not observed (Fig. 2A). This suggests p53-dependent enhancement of p21 promoter activity by core protein. Second, the enhancement by core protein was also observed using endogenous p53. However, the effect was weak in HeLa cells. This phenomenon could be explained by the fact that p53 is bound by human papillomavirus E6 protein in HeLa cells, resulting in polyubiquitination and subsequent degradation of p53 (17). Similarly, the ability of enhancement by core protein was slightly weak in NIH3T3 cells. Recently, it was reported that p19 ARF stabilizes p53 by antagonizing MDM2 (55). Since p19 ARF is not detected in NIH3T3 cells resulting in degradation of p53, the enhancement in NIH3T3 cells might be weaker than that in other cell lines (Fig. 1C). These also suggest p53-dependent enhancement of p21 promoter activity by core protein. Third, when using artificial reporter bearing Gal4-binding sites, core protein enhanced the luciferase activity mediated by Gal4-p53 but had no effect on the activity by Gal4-VP16. This result showed that core protein trans-activates transcription mediated by p53 specifically. These results strongly suggest that core protein enhances p21 promoter activity specifically in a p53-dependent manner.
Several viral proteins have been reported to enhance p53 function (26 -32). These proteins are non-structural proteins, which are known to be related to viral replication and act as a promiscuous trans-activator of a variety of viral and cellular genes. Therefore, the mechanism of this enhancement is explained partly by the increase in p53 protein level during the transcription process. In this study, however, we showed that core protein, a structural protein, enhanced p53 function without increasing the amount of p53 protein. Alternatively, two possible mechanisms were demonstrated. First, core protein enhanced the amount of p53 DNA binding as shown in electrophoretic mobility shift assay (Fig. 5). We also showed the direct binding between core and the C terminus of the p53 protein in vitro (Fig. 7). There are a couple of reports indicating that the C terminus of p53 protein negatively regulates its DNA binding affinity (56 -58), and that 14-3-3 protein, a member of a family of proteins that regulate cellular activity by binding and sequestering several proteins, could associate with the C terminus of p53 to suppress negative regulator and increase its DNA binding affinity (59). Thus, it is possible that binding of the core protein to p53 protein masks the C terminus of p53 and act like 14-3-3 protein and, hence, increases the p53 DNA binding affinity. Second, core protein also enhanced the transcriptional ability of p53 irrespective of p53-DNA binding affinity (Fig. 6). One possibility extrapolated from the results is the interaction between core protein and hTAF II 28 (Fig. 8). This interaction was detected even using either p53-deficient SAOS-2 cell lysates or recombinant proteins, suggesting that core protein binds hTAF II 28 directly without p53. p53 interacts with TBP when exerting its transcriptional activity (54), and hTAF II 28 was reported to interact with TBP (60). Although core protein itself did not possess transcriptional ability, these findings lead us to speculate that core protein works as a transcriptional co-activator, possibly resulting in a complex formation of p53, core protein, hTAF II 28, and TBP that influences the transcriptional ability of p53.
p53 and transcriptional factors are known to exist mainly in the nucleus. Therefore, it could be questioned whether core protein might move into the nucleus and interact with the transcriptional factors. Three independent nuclear localization signals are present in the N-terminal one-third of the core protein (61,62). It was previously reported that core protein resided predominantly in the cytoplasm but was also found in the nucleus (63, 64). Thus, we examined whether N-terminal FIG. 7. Interaction of core protein with p53 in the GST fusion protein binding assay. COS-7 cells were transfected with 0.6 g of pCXN2-core. Thirty-six hours after transfection, the lysates were prepared by 200 l of immunoprecipitation buffer and were mixed with 10 g of GST, GST-p53, GST-p53N, or GST-p53C fusion proteins bound to glutathione-Sepharose 4B beads, respectively, as indicated. Bound proteins were then analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with anti-core antibody. The similar experiments were conducted using in vitro translated biotinylated core protein instead of the cell lysates. Bound proteins were blotted with streptavidin conjugated with horseradish peroxidase kit. The core protein bands are shown by arrows.

FIG. 8. Analysis of the interaction of core protein with TFIID.
A, COS-7 cells were transfected with 0.4 g of pCXN2-core together with 0.4 g of a vector HA-tagged expressing TFIIDs as indicated above the lanes. Cell extracts were precipitated with 0.2 g of anti-HA antibody each, and the precipitated proteins were revealed by immunoblotting using anti-core antibody. The coprecipitated core protein is shown by an arrow. As for input, 1/20 of the cell extracts was used. B, the GST binding assay was conducted using in vitro translated biotinylated core protein and 10 g of GST or GST-hTAF II 28. Bound proteins were blotted with streptavidin conjugated with horseradish peroxidase kit. The core protein band is shown by an arrow. deleted core protein might retain the ability of enhancement of p53 function, and showed that N-terminal deleted core protein aa  had no effect on the p21 promoter (Fig. 2C). From this result, N-terminal deleted core protein might lose the ability to approach the nucleus and, hence, it might lose the p53-dependent activation of p21 promoter. However, it should be cautioned that only a small fraction of the full-length core protein moves into the nucleus; thus the mechanisms we mentioned here which are supposed to happen in the nucleus may not fully explain the mechanisms of the enhancement of p53 function by core protein. It has been demonstrated that p53 protein is extensively modified by phosphorylation and acetylation (65), thus whether such a modification occurs indirectly by core protein should be further examined.
Our results are in contrast to previous reports (66,67) in which core protein was shown to suppress the activities of p53 and p21 promoters by reporter assays. In their study, however, only core protein expression vector was used and no appropriate control was included. Therefore the possibilities cannot be denied that the core protein expression vector they used nonspecifically suppressed promoter activities due to a phenomenon called squelching effect. In contrast, we constructed seven HCV protein expression vectors under the same promoter and showed that only core protein specifically enhanced the p53 function significantly in a p53-dependent and p53-specific manner. Moreover, we clearly showed not only the increasing luciferase activities from p21 promoter but also the increasing expression of the endogenous p21 protein by core protein. We believe it is critical to confirm the protein expression levels of the endogenous target gene.
During preparation of this manuscript, Lu et al. (68) reported that core protein activates p53 function. However, the mechanism of activation of p53 function by core protein remains unclear. We showed here, in addition to the activation of p53 function by core protein, that this effect is derived from two possible mechanisms; the augmentation of p53-DNA binding affinity and transcriptional ability of p53 by core protein. In addition, we showed that core protein potentially interacts with hTAF II 28, a member of a general transcriptional complex. Although core protein has been reported to regulate other cellular promoters (33)(34)(35)69), the mechanisms of regulation are not understood. This interaction between core protein and hTAF II 28 might be a clue to the understanding the various effects of core protein on other promoters.
HCV is thought to be a causative agent of HCC. The core protein was immunohistochemically identified both in nonneoplastic and neoplastic tissue (70). It was reported that the majority of hepatocellular carcinoma without p53 mutation express p21 protein, while only a small fraction of hepatocellular carcinoma with p53 mutation demonstrate significant p21 expression (71). Previous sequence analysis of the p53 gene in HCC obtained from patients indicated that mutation of p53 is rare in "early" tumors, but frequent in advanced cases (72,73). p53 mutation was reported to be a poor prognostic indicator in patients with HCC (74). Therefore, it is highly likely that once p53 is mutated, the loss of the enhancement of p53 ability by core protein becomes apparent and the HCC may become progressive.
In conclusion, the present study showed that, among HCV proteins investigated, only core protein augmented the promoter activity and the expression of p21 waf1 , and bound to p53 in vitro. These results suggest that HCV core protein influences the various functions of p53 and thus may contribute to the pathogenesis of HCV.