Hepatitis B Virus X Gene Expression Is Activated by X Protein but Repressed by p53 Tumor Suppressor Gene Product in the Transient Expression System

VIROLOGY

216, 80–89 (1996) 0036

ARTICLE NO.

Hepatitis B Virus X Gene Expression Is Activated by X Protein but Repressed by p53 Tumor Suppressor Gene Product in the Transient Expression System SHINAKO TAKADA,* NORIKO KANENIWA,* NOBUO TSUCHIDA,† and KATSURO KOIKE*,1 *Department of Gene Research, The Cancer Institute (JFCR), 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan; and †Department of Molecular and Cellular Oncology, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan Received September 27, 1995; accepted November 27, 1995 Hepatitis B virus (HBV) X gene is known to exhibit a transcriptional activation function and is considered to play a major role in hepatocarcinogenesis. We determined a 20-bp promoter element for the HBV X gene transcription and found a binding protein to this promoter element, designated as an X-PBP. We then examined the effects of HBV X protein and p53 tumor suppressor gene product on X gene transcription from the 20-bp promoter element using the transient expression technique. Activity of the X gene promoter was stimulated by X protein expression, but, in contrast, was repressed by transfected normal p53 gene. On the other hand, mutant p53 gene product exhibited no repression. Moreover, the p53 repression of X gene transcription was canceled by X protein coexpression. Thus, the effects of X protein and normal p53 product appear to be mutually antagonistic in the regulation of X gene expression. However, mutated promoter elements which failed to bind to X-PBP still responded to X protein or p53, indicating that the process of X transactivation or p53 respression may be independent of X-PBP binding to the promoter element. Our data suggest that X protein could disrupt function of normal p53 protein in X gene-transfected cells. q 1996 Academic Press, Inc.

INTRODUCTION

Twu et al., 1989a), but not for SV40 enhancer in CV-1 cells (Seto et al., 1990). When focused on one particular gene, the efficacy of transactivation was found to be dependent on the cell line that was used in the experiment (Seto et al., 1989). Thus, the XRE should be determined, respectively, for each cell line. Although the reported observations and the proposed mechanisms were not, in fact, always consistent, it appears that X protein exerts its transcriptional activation function via protein–protein interaction with cellular factors. Several reports have indicated that X protein activated some signal transduction factors (Kekule et al., 1993; Cross et al., 1993; Natoli et al., 1994a; Benn and Schneider, 1994) or DNA binding of some transcription factors (Natoli et al., 1994b). On the other hand, X protein directly interacted with hepatic serine proteases, tryptase TL2, and tryptase TL1 (a member of proteasome), as an inhibitor (Takada and Koike, 1990a; Takada et al., 1994a). This indicates the possibility that X protein activates the signal transduction pathway as well as transcription by inhibiting the activity of serine proteases that might be involved in proteolytic cleavage of transcription factors or signal transduction factors. However, we could not exclude the possibility that these observations were indirect outcomes of the transcriptional activation of several genes by X protein, because these serine proteases are localized in the cytoplasm (Kido et al., 1990; Tanaka et al., 1992), and X protein is detected mainly in the cytoplasm (Siddiqui et al., 1987; Benn and Schneider, 1994). As it is increasingly important to understand how X gene expression is regulated, we previously analyzed X gene expression with a particular focus on the promoter-

Hepatitis B virus (HBV) is closely related not only to acute or chronic hepatitis, but also to the development of hepatocellular carcinoma (HCC) (for review, see Tiollais et al., 1985; Ganem and Varmus, 1987). One of the HBV genes, X, encodes a basic protein of 154 amino acids and has been implicated in the carcinogenicity of this virus as a major causative factor because of its ability to induce the transformation of rodent cells (Shirakata et al., 1989; Hohne et al., 1990; Rakotomahanina et al., 1994) and to induce HCC in transgenic mice (Kim et al., 1991). The X protein is known to exhibit a transcriptional activation function for many viral and cellular genes without binding to DNA (Spandau and Lee, 1988; for review, Rossner, 1992). As to the responsive element for X protein (X-responsive element, XRE), AP-1 and AP-2 binding sequences were previously found in SV40 enhancer (Seto et al., 1990), NFkB-like sequence in the HIV LTR (Siddiqui et al., 1989; Twu et al., 1989a,b), and the 26-bp XRE in HBV enhancer I (Faktor et al., 1990). Accordingly, the X protein was expected to transactivate a certain gene through a different XRE in each cell line; for example, the AP-2 binding sequence responded to X protein in CV-1 cells (Seto et al., 1990), but not in HepG2 cells (Twu et al., 1989a), and a kB-like sequence was reported to be the XRE for the HIV LTR in HepG2 cells (Siddiqui et al., 1989;

1 To whom correspondence and reprint requests should be addressed. Fax: 81-3-5394-3902.

0042-6822/96 $12.00

80

Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

/ 6a0f$$7695

01-12-96 08:21:49

viral

AP-Virology

OPPOSITE EFFECTS BETWEEN X PROTEIN AND p53 ON HBV X GENE

containing region and found that the 58-bp region, 130 bp upstream from the first ATG codon of X ORF, has promoter activity (Nakamura and Koike, 1992). In addition, a ubiquitous protein (complex), designated as an XPBP (X promoter binding protein), was shown to bind this 58-bp DNA (Nakamura and Koike, 1992; Yaginuma et al., 1993). In this study, deletion analyses were performed on this 58-bp region and a small 20-bp region of promoter activity of the X gene was determined. We then questioned whether X protein regulates the X gene promoter. The promoter activity of the 20-bp DNA was activated by X protein expression, but we were not able to observe the direct involvement of X-PBP in the process of promoter activation by X protein. The p53 tumor suppressor gene product is mainly located in the nucleus and has pleiotropic functions, including the control of genomic instability (Harris and Hollstein, 1993). The p53 protein controls gene transcription by binding to the specific DNA sequence and to other cellular factors (such as Mdm-2, TBP, and WT1) and may be involved in DNA replication and repair processes (Seto et al., 1992; Kastan et al., 1992; Oliner et al., 1992; Momand et al., 1992; Dutta et al., 1993). Mutations on p53 were found in a wide variety of human cancers (Harris and Hollstein, 1993; Hollstein et al., 1991). p53 missense mutants not only lose normal function, but often gain oncogenic properties (Wolf et al., 1984; Dittmer et al., 1993; Gerwin et al., 1992). In virally transformed cells, p53 protein complexes with viral oncoproteins, such as the SV40 large tumor antigen(T) (Lane and Crawford, 1979; Linzer and Levine, 1979), the adenovirus E1B protein (Sarnow et al., 1982), the human papillomavirus E6 protein (Dyson et al., 1989), and the Epstein–Barr virusencoded nuclear antigen EBNA-5 (Subler et al., 1992). These protein–protein interactions can inactivate p53 functions, e.g., sequence-specific DNA binding and gene transactivation and, thus, are important to viral oncogenicity. On the basis of the general acceptance that the tumor suppressor gene p53 regulates transcriptional initiation of a variety of viral and cellular genes by direct interaction with the basal transcription factors (Zambetti et al., 1992; Kern et al., 1991; Farmer et al., 1992; Seto et al., 1992; Ragimov et al., 1993), we questioned whether the p53 gene product directly regulates the X gene promoter. As we preliminarily reported that p53 repressed X gene transcription from the 58-bp promoter-containing DNA using the CAT assay system (Takada et al., 1994b), in the present study, we examined the promoter activity of the 20-bp sequence in the presence of transfected normal p53 gene. As a result, the 20-bp promoter activity was clearly repressed by transfected normal p53 gene, but not by its mutants. In contrast, under similar conditions, the muscle creatine kinase gene promoter was activated (Takada et al., 1994b), consistent with the previous data (Weintraub et al., 1991; Zambetti et al., 1992). Interestingly, when normal p53 gene was coexpressed with X gene, the repressive effect of p53 on the promoter

/ 6a0f$$7695

01-12-96 08:21:49

viral

81

activity was counteracted by the presence of X protein. From the viewpoint of functional disruption of normal p53 in the nucleus by X protein coexpression, the transactivation mechanism in X gene-transfected cells is discussed. MATERIALS AND METHODS Plasmid DNAs pX58pCAT, pX58pM1CAT, and pX58pM2CAT were previously described by Nakamura and Koike (1992). pX58pM4 Ç pX58pM7CAT, pX58pM7mCAT (SphI site was mutated from GCATGC to TTTTTT), pXMP1CAT Ç pXMP3CAT, and pXMP2mCAT (the SphI site was mutated from GCATGC to GACTGC) were constructed by ligation of each chemically synthesized double-stranded mutant X promoter DNA into the HindIII site of pSV00CAT (Araki et al., 1988). pHBVX-1 (Kobayashi and Koike, 1984; Yaginuma et al., 1987), which was constructed by insertion of a 0.87-kb StuI/BglII (nt 872–1858) fragment of HBV DNA containing the X gene expression unit into the BamHI site of pBR322, was used as the X gene expression plasmid. Expression plasmids pEFX and pEFX-DRsaI were also used as the wild-type or mutant X gene expression, where the wild-type or DRsaI (Arii et al., 1992) mutant X gene ORF was driven under the control of the EF1a (elongation factor 1a) gene promoter (Kim et al., 1990). p53 expression plasmids pCMVp53(normal), pCMVOM1, pCMVp53d(346-393), pCMVp53d(302393), pCMVp53d(283-393), and pCMVp53DN were constructed by inserting the normal or mutant p53 ORFs into a CMV vector (CLONTECH). pCMVOM1 is a point mutant p53, in which Glu was replaced by Gly (aa 266) (Sakai and Tsuchida, 1992). pCMVp53d(346-393), pCMVp53d(302-393), and pCMVp53d(283-393) are mutant p53s which have lost the C-terminal region from aa 346, 302, and 283, respectively, to the C-terminus (Tsutsumi-Ishii et al., 1995). pCMVp53DN is an N-terminal deletion (aa 1–159) mutant. Cell line, DNA transfection, CAT activity assay, and Northern blot hybridization HepG2 is a hepatoblastoma cell line containing normal p53. Saos-2 (ATCC HTB85), which lacks endogenous p53 (Masuda et al., 1987), is a human osteosarcoma cell line. HuH-7 is a hepatocellular carcinoma cell line which has mutant p53 (Bressac et al., 1990). DNA transfection, CAT activity assay, and Northern blot hybridization were carried out as described (Takada and Koike, 1990b). To quantify the reaction products of the CAT assay, radioactive spots were cut out from the silica gel plate and their radioactivity was measured with a liquid scintillation counter. Relative activity (RA) was calculated and depicted in each respective figure. Electrophoretic mobility shift assay Nuclear extracts were prepared according to the method of Dignam et al. (1983). Binding reactions were carried out in 10 ml of the buffer containing 10 mM Tris–

AP-Virology

82

TAKADA ET AL.

HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 5% glycerol, 200 mg/ml poly(dA-dT)-poly(dA-dT) (Pharmacia), 300 mg/ml protein concentration of nuclear extract, and 2 1 104 cpm of 32P-labeled probe DNA for 20–30 min at room temperature, unless otherwise indicated. One microliter of dye solution [10 mM Tris–HCl (pH7.5), 50% glycerol, 0.4% bromophenol blue, 0.4% xylene cyanol] was added to each sample, before it was loaded onto a 4% acrylamide gel containing 6.7 mM Tris–HCl (pH7.5), 1 mM EDTA, 3.3 mM NaoAc, and 2.5% glycerol and then run in 6.7 mM Tris–HCl (pH7.5), 3.3 mM NaoAc, and 4 mM EDTA. In the supershift experiments, 300 ng of antip53 antibody PAb421, PAb1801 (Oncogene Science), or anti-SV40 T-Ag antibody (Oncogene Science) was included in the binding reaction. RESULTS Determination of a small essential region for the promoter activity of HBV X gene The promoter-containing region of the HBV X gene was previously identified by CAT activity assay as the 58-bp BalI/MboI DNA fragment (nt 1085–1142) (Nakamura and Koike, 1992). This 58-bp DNA fragment has no enhancer activity, but contains the major start site of X mRNA (Nakamura and Koike, 1992; Yaginuma et al., 1993). It was interesting to determine a small essential sequence for the promoter activity of X gene, because the promoter-containing region (58-bp DNA fragment) (nt 1085–1142) possesses none of the typical promoter sequences, such as the TATA, GC, and CAAT boxes. As shown in Fig. 1, various promoter-containing DNAs within the 58-bp DNA fragment were synthesized, introduced into the pSV00CAT plasmid, and analyzed for their promoter activity by CAT activity assay, using HepG2 cells as the host. The promoter activity was found to reside in the 20-bp MP2 DNA (nt 1102–1121) in the pXMP2CAT plasmid, whose sequence is shown at the bottom of Fig. 1. This 20-bp sequence overlaps the X-PBP protected region (nt 1096–1117), which was detected as a major shifted band by a mobility shift assay using sonicated salmon sperm DNA as a nonspecific competitor instead of poly(dI-dC) (Nakamura and Koike, 1992). Mutation at the SphI site (GCATGC to GACTGC) in the 20-bp sequence greatly reduced the promoter activity (see pXMP2mCAT), indicating that the CA sequence in the SphI site of 20-bp DNA is essential for the activity. Truncation or deletion of half of the MP2 DNA resulted in loss of the promoter activity (see pX58pM5CAT and pXMP3CAT). The exact reason why pX58pM6 CAT exhibited slightly lower activity compared to pX58pM4CAT is not known, but as a result of a deletion, a negative element at the 3* end region was close to the 20-bp sequence and may have had an effect on it. Actually, pX58pM7CAT without the 3* end region exhibited higher activity. In addition, pX58pM2CAT, containing a mutation at the SphI site (GCATGC to TTTTTT) in the 58-bp region

/ 6a0f$$7695

01-12-96 08:21:49

viral

FIG. 1. Determination of a small essential region for the HBV X gene promoter. The upstream region of the X gene is shown schematically at the top. Enh I means the enhancer I of HBV. St, Ba, Sp, and Mb indicate the cleavage sites of StuI, BalI, SphI, and MboI, respectively. The BalI/MboI fragment is the 58-bp DNA fragment which contains the X gene promoter (Nakamura and Koike, 1992). Various sections of this 58-bp DNA or their mutants indicated in the figure were chemically synthesized and connected to pSV00CAT, and their promoter activities were measured by CAT activity assay using 2 or 10 mg of each CAT construct per 10-cm dish. Relative promoter activity of each inserted DNA is shown to the right. Location of the 20-bp promoter is presented by the hatched box and its nucleotide sequence is shown below. In the MP2m mutant, the sequence GCATGC at the SphI site in MP2 DNA was changed to GACTGC.

of pX58pCAT, was tested for its CAT activity. pX58pM2CAT exhibited about one-eighth (162 versus 1250) the CAT activity of pX58pCAT, which corresponded to 12.5-fold that of pX58pM4CAT (data not shown). The essential region for the promoter activity could thus be restricted to the 20-bp MP2 DNA sequence (nt 1102– 1121), where the major start site of X mRNA was present. The 20-bp sequence is separated from the NF-1 binding site (nt 1088–1100) (Kobayashi and Koike, 1984). We previously demonstrated the interaction of X-PBP with the promoter region of the X gene by a mobility shift assay using the 58-bp BalI/MboI DNA fragment as the probe (Nakamura and Koike, 1992). DNaseI footprinting experiments localized the binding site at the sequence between nt 1096 and 1117, which contains an 8-bp palindromic sequence with an SphI cleavage site (nt 1110) at its center. The mutations introduced into this target sequence for X-PBP binding remarkably reduce transcription from the major start site of CAT mRNA using the BalI/MboI DNA–CAT construct (pX58pCAT) (Nakamura and Koike, 1992). Therefore, the binding of X-PBP was shown to be essential for the initiation of X gene transcription. These data indicate an important role of X-PBP in the regulation of X gene expression. To further examine the binding region for X-PBP, a mobility shift assay was performed using various X gene promoter-containing DNAs. Results indicated that M7 DNA, the insert of the pX58pM7CAT plasmid (Fig. 1), had the capacity to bind X-PBP. Competition experiments against M7 DNA were then carried out with various deletion mutant DNAs (Fig. 2A). A mobility shift assay was also

AP-Virology

OPPOSITE EFFECTS BETWEEN X PROTEIN AND p53 ON HBV X GENE

performed using wild-type or mutant DNAs as the probe (Fig. 2B). The 20-bp MP2 DNA (nt 1102–1121) was found to be sufficient for the binding of X-PBP, although X-PBP protected a larger area in the DNaseI footprinting experiments (Nakamura and Koike, 1992; Yaginuma et al., 1993). The point mutant MP2m (the insert of pXMP2mCAT plasmid) showed much reduced X-PBP binding activity (Fig. 2B), suggesting that the sequence ‘‘CA’’ in the SphI site is in the recognition site of X-PBP. MP2m DNA also exhibited much reduced promoter activity (Fig. 1). Although the physiological function of X-PBP has not yet been fully investigated, the binding activity of X-PBP to normal or mutant promoter DNA is well correlated with the promoter activity, consistent with previous observations (Nakamura and Koike, 1992; Yaginuma et al., 1993). Activation of the X gene promoter activity by X protein Since X protein is known to activate transcription of many genes through various DNA elements, it was of interest to determine whether X protein is able to activate the X gene promoter. CAT activity assay was performed using various X gene promoter CAT constructs and the X gene expression plasmid (pHBVX-1) or the control plasmid (pBR322). As the basal CAT activity of the reporter plasmid varied (see Fig. 1), the amount of each transfected reporter plasmid DNA was adjusted (2 mg for pXMP2CAT, pX58pM4CAT, and pX58pM7CAT and 10 mg for pXMP2mCAT and pXMP3CAT), in order to clearly distinguish the difference in activity between basal and activated promoters. Interestingly, transcription from the small 20-bp DNA (MP2 or MP2m) was clearly activated by X protein, but MP3 was not (Fig. 3). All reporter plasmids, except pXMP3CAT, were similarly activated by X protein coexpression in their extent. The data indicated that an

FIG. 2. Determination of the essential region for X-PBP binding. (A) Competition experiment. A mobility shift assay was carried out using 32 P-M7 DNA (see Fig. 1) as the probe, HepG2 nuclear extract, and 100 ng of the indicated DNAs as the competitors. (B) X-PBP binding to the promoter DNA. A mobility shift assay was carried out using various 32 P-labeled promoter DNAs as the probes. Specific activities (cpm/ pmol) of the probes were M7m, 1.3 1 106; M7, 1.9 1 106; MP2, 1.1 1 106; MP2m, 9.6 1 105; and MP3, 9.0 1 105, respectively. Black triangles indicate the positions of the X-PBP band. The M7m DNA was used as a negative control.

/ 6a0f$$7695

01-12-96 08:21:49

viral

83

FIG. 3. Transcriptional activation of the X gene promoter by HBV X protein. The CAT plasmid containing wild-type or mutant X promoter was cotransfected with the X gene expression plasmid (pHBVX-1 indicated as /) or the control plasmid (pBR322 indicated as 0) of 10 mg per 10-cm dish into HepG2 cells, and then CAT activity was assayed. As the basal activity of the reporter plasmid varied (see Fig. 1), the amount of the transfected reporter plasmid DNAs was adjusted; 2 mg for MP2 (pXMP2CAT), M4 (pX58pM4CAT), and M7 (pX58pM7CAT), and 10 mg for MP2m (pXMP2mCAT) and MP3 (pXMP3CAT), in order to clarify the difference between the basal and activated promoter activities. Similar results were obtained when 10 mg of pEFX was used as the X expression plasmid. Relative activity (RA) for the basal activity of each reporter plasmid was calculated and is shown at the top.

X-responsive element was present in X gene promoter and that X protein was able to activate X gene expression as long as the promoter activity was detected. When Northern blot analysis of the CAT mRNA that was expressed in X gene-transfected or nontransfected cells was carried out, CAT mRNA was found to have increased in the X-transfected cells as a function of the amount of X gene transfection (data not shown). These data support our indication that activation of the X gene promoter activity by transfected X gene is not merely caused by overexpression of X protein in the transient expression system. Next, we investigated whether the X-PBP is directly involved in the transactivation function of X protein. A mutated X gene promoter (MP2m), to which X-PBP was weakly bound (Fig. 2B), was analyzed for its response to X protein. As shown in Fig. 3, the mutant pXMP2mCAT was still responsive to transactivation by X protein, although the mutant had much weaker promoter activity (see Fig. 1) than that of the wild-type construct (pXMP2CAT), indicating that X-PBP was not a direct mediator of the X protein function. In addition, pX58pM2CAT was tested for its response to X protein, since pX58pM2CAT does not bind X-PBP at all (Nakamura and Koike, 1992). pX58pM2CAT was transactivated by transfected pHBVX-1 DNA (data not shown). These observations suggest that X promoter activation by X protein is not directly mediated by X-PBP. Repression of the X gene promoter activity by transfected p53 gene It has been reported that the p53 tumor suppressor gene product was directly involved in transcription initiation through the TATA box binding protein (Seto et al., 1992) and TBP-associated factors (TAFII40 and TAFII60) (Thut et al., 1995). However, the 58-bp BalI/MboI DNA

AP-Virology

84

TAKADA ET AL. TABLE 1 Repression of X Gene Transcription by Transfected Tumor Suppressor Gene p53 and Its Mutant Relative CAT activity Reporter plasmid Expression plasmid

pX58pCAT

pX58pM1CAT

pEtCAT

None (vector) p53 p53 mutant DN p53 mutant OM1

1 0.1 1.7 2.6

1 0.1 1.8 2.9

1 16.9 0.8 1.7

Note. Saos-2 cells were cotransfected with the indicated plasmids and CAT activity was determined relative to that of the controls. The vector for the expression plasmid contained the CMV promoter. For most experiments, the quantity of reporter plasmid containing 58-bp DNA (pX58pCAT) or its mutant (pX58pM1CAT) was 1 mg per dish, that of the expression plasmid was 1 mg per dish, and the corresponding vector was used to adjust the amount of total DNA in each experiment. pEtCAT, a CAT construction of the creatine kinase gene promoter as the positive control. DN, amino-terminal deletion (aa 1–159). OM1, point mutation Gly (aa 226) r Glu.

has neither a p53 binding site nor a TATA box, and Saos2 cells are known to be p53-negative cells (Masuda et al., 1987). Therefore, we carried out DNA transfection experiments to Saos-2 cells using the BalI/MboI DNA– CAT construct (pX58pCAT) and its mutant pX58pM1CAT, together with normal or mutant p53 expression plasmids (Table 1). When normal or mutant p53 gene and pX58pCAT or pX58pM1CAT were cotransfected, normal p53 gene repressed CAT expression, while mutant p53 (p53 mutant DN or OM1) did not and, in contrast, slightly stimulated CAT expression for some unknown reason. In other words, transfected normal p53 is able to repress X gene promoter activity. Under the same conditions, the creatine kinase gene promoter (pEtCAT) was activated by transfected p53 gene (Table 1), consistent with the previous data (Weintraub et al., 1991; Zambetti et al., 1992). Although there is no detailed information available yet on the kind of basal transcription factor involved in X gene transcription, it was of interest to clarify whether normal p53 protein regulates the 20-bp sequence for promoter activity of the X gene. To examine the effect of p53 protein on this minimal promoter sequence, different amounts of the p53 expression plasmid and the constant amount of reporter plasmid pXMP2CAT containing the 20-bp sequence were cotransfected to HepG2 cells. As shown in Fig. 4A, activity of the X gene promoter was clearly repressed by the transfected p53 gene in a dosedependent manner. On the other hand, transcriptional activation of the creatine kinase gene promoter was observed. More specifically, the same amount of p53 expression plasmid brought about more than 15-fold activation in HepG2 cells (data not shown) or p53-negative Saos-2 cells (Table 1 and Takada et al., 1994b). When

/ 6a0f$$7695

01-12-96 08:21:49

viral

Northern blot analysis of X mRNA transcribed from the expression plasmid pHBVX-1 or pHBV dimer in the p53transfected or nontransfected cells was further carried out, X mRNA was found to have been reduced in the p53 gene-transfected cells (Fig. 4B). These data support our proposal that repression of X gene promoter activity by transfected normal p53 gene is not caused merely by overexpression of p53 protein in the transient expression system, but may reflect regulation of X gene expression in HBV DNA-infected cells (Takada and Koike, 1990b). Next, we investigated whether X-PBP is directly involved in the transcriptional repression function of normal p53 protein. A mutated X gene promoter, MP2m (twonucleotide substitution at the SphI site), to which X-PBP bound weakly (Fig. 2B), was analyzed in terms of its response to the p53 protein. As shown in Fig. 4C, the wild-type X promoter (pXMP2CAT) and the mutant X promoter (pXMP2mCAT) were similarly responsive to p53 repression in their extent, although X-PBP binding activity was much weaker in the mutant than in the wild-type (pXMP2CAT), indicating that X-PBP was unlikely to be a direct mediator of the p53 function. pX58pM2CAT (Nakamura and Koike, 1992) was also tested for its response to p53 repression, since pX58pM2CAT does not bind XPBP at all because of a five-nucleotide substitution at the SphI site. pX58pM2CAT was also responsive to normal p53 protein (data not shown). Thus, repression of the X gene promoter activity by normal p53 protein appears to be closely correlated with the basal transcription factor. To clarify the region of normal p53 protein responsible for the repression of X gene promoter activity, several p53 mutants were examined. Consistent with the recent observations by others (Shiio et al., 1992; Subler et al., 1992; Sang et al., 1994), any amino-terminal deletion (DN) or carboxy-terminal deletion (345, 301, or 282) or a point mutation at the central region (OM1) (Sakai and Tsuchida, 1992) abolished the transcriptional repression function of the p53 protein (Fig. 4D). We then prepared a nuclear extract from p53-negative Saos-2 cells to examine whether X-PBP migrates at a different position in the mobility shift assay without p53 protein. The Saos-2 cell extract showed the same X-PBP band as that from HepG2 cells (Fig. 5A). In addition, antip53 antibodies (PAb421 or PAb1801) did not supershift or eliminate the X-PBP band (Fig. 5A). Data so far obtained indicate that the p53 protein is not a major constituent of the X-PBP complex. Therefore, p53 binding to X-PBP was tested using immunopurified normal p53 from the baculovirus expression system and the HepG2 nuclear extract (NE) with or without anti-p53 antibody (PAb421) or anti-SV40 T-Ag antibody (as a control). Any significant interaction between X-PBP and normal p53 protein was not detected under the conditions used (Fig. 5B). To confirm that our p53 protein retains the sequence-specific DNA binding activity, normal and mutant HOS(156Pro) p53 proteins were used with the consensus p53 binding

AP-Virology

OPPOSITE EFFECTS BETWEEN X PROTEIN AND p53 ON HBV X GENE

85

FIG. 4. Repression of transcription from the X gene promoter by normal p53. (A) Dose-dependent repression of the X gene promoter activity by normal p53. Various amounts of p53 expression plasmid, pCMVp53 (normal), were cotransfected with 10 mg of the X gene promoter – CAT plasmid, pXMP2CAT, per 10-cm dish into HepG2 cells and CAT activity was assayed. The amounts of transfected p53 expression plasmid are indicated at the bottom. Relative activity (RA) was calculated and is shown at the top. (B) Northern blot analysis of X mRNA from p53transfected or control cells. The X gene expression plasmid (pHBVX-1) or the HBV genomic plasmid (pHBV-dimer) in the quantity of 10 mg per 10-cm dish was transfected into HuH-7 cells with or without 2 mg per dish of p53 expression plasmid pCMVp53. Total RNAs were analyzed using a 32P-labeled X gene probe. Later, the same filter was rehybridized with a 32P-labeled b-actin probe (WAKO Pure Chemical Industry Ltd.) to confirm the amount of blotted RNAs. (C) Mutant X gene promoter at X-PBP binding site preserves response to p53. p53 expression plasmid, pCMVp53 (normal), indicated as / or the control plasmid, pCMV, indicated as 0 (each 2 mg per 10-cm dish), was cotransfected with 10 mg per 10-cm dish of the X gene promoter CAT plasmid (pXMP2CAT) or 10 mg of the point mutant at the X-PBP binding site (pXMP2mCAT). For structures of the reporter plasmids, see Fig. 1. Relative activity (RA) was calculated and is shown at the top. (D) Inability of mutant p53 to repress the transcription from the X gene promoter. Mutant p53 constructs were tested for their ability to repress transcription of the X gene. Two micrograms per 10-cm dish of mutant p53 expression plasmid was transfected with the reporter plasmid pXMP2CAT (10 mg). Transfected p53 expression plasmids were Vecter, pCMV; Wild, pCMVp53 (normal); 345, pCMVp53d(346-393); 301, pCMVp53d(302-393); 282, pCMVp53d(283-393); OM1, pCMVOM1; and DN, pCMVp53DN. Data for the plasmid 301 were introduced from the separate experiment where 301 plasmid exhibited the same level of the activity as the control vector only.

DNA in the presence or absence of PAb421, which was reported to activate the sequence-specific DNA binding of p53 (Hupp et al., 1992). As also shown in Fig. 5B, the small arrowhead to the right indicates the complex of normal p53 protein and p53 binding DNA. The large arrowhead to the right indicates the triple complex of PAb421, normal p53 protein, and p53 binding DNA. These observations strongly suggest that repression of the X gene promoter activity by normal p53 protein is not mediated through X-PBP. X protein cancels the transcriptional repression by transfected p53 gene As described above, when the CAT plasmid containing wild-type or mutant X promoter was cotransfected with the X gene expression plasmid (pHBVX-1) or the control plasmid (pBR322) into HepG2 cells, transcription from

/ 6a0f$$7695

01-12-96 08:21:49

viral

the X gene promoter was found to be clearly activated by X protein expression (Fig. 3), indicating that the Xresponsive element is present in the X gene promoter region. Because the X gene promoter activity was regulated negatively by normal p53 protein or positively by X protein, CAT activity assay was then carried out to determine the effect of cotransfected X expression plasmid on the transcriptional repression function of p53 protein. As shown in Fig. 6, p53 repression of the X gene promoter activity (pXMP2CAT) was significantly recovered by increasing the amount of cotransfected X gene. This recovery depended on the Kunitz domain-like structure of the X protein, which is essential for the transactivation function of X protein (Takada et al., unpublished data). The data clearly indicated that the p53 transcriptional repression of X gene promoter activity can be counteracted with X protein coexpression in the transient expression system using HepG2 cells. Normal endogenous

AP-Virology

86

TAKADA ET AL.

FIG. 5. Normal p53 protein was not detected in the X-PBP complex. (A) Absence of normal p53 protein in the X-PBP complex. Left: A mobility shift assay of X-PBP using 5 mg of protein of the nuclear extract from Saos-2 (lane 1) or HepG2 (lane 2) cells. Right: A mobility shift assay of X-PBP using HepG2 nuclear extract in the absence (lane 3) or presence [100 ng (lanes 4 and 6) or 300 ng (lanes 5 and 7)] of anti-p53 antibodies. 32P-labeled M7 DNA was used as the probe. Arrowheads indicate the position of the X-PBP bands. (B) No direct interaction of X-PBP with normal p53 protein. A mobility shift assay was performed using 32P-labeled MP2 DNA (left, lanes 1–7) or p53 binding DNA (Hupp et al., 1992) (right, lanes 8–11) as the probe. Poly(dA-dT)/poly(dA-dT) was used as the nonspecific carrier. To see whether p53 protein binds to X-PBP, immunopurified normal p53 (50 ng) from the baculovirus expression system was added to the HepG2 nuclear extract (NE), with or without anti-p53 antibody (PAb421) or anti-SV40 T-Ag antibody (as a control). Arrowhead to the left indicates the simple X-PBP band from NE. Left: lane 1, NE alone; lane 2, NE and p53 protein; lane 3, NE, p53 protein and PAb421; lane 4, NE, p53 protein and anti-SV40 T-Ag; lane 5, NE and mutant p53, HOS(156Pro); lane 6, p53 alone; lane 7, normal p53 and pAB421 together. To indicate that our p53 protein retains the sequence-specific DNA binding activity, normal (lanes 8 and 9) and mutant HOS(156Pro) (lanes 10 and 11) p53 proteins were mixed with the consensus p53 binding DNA in the presence (lane 9 and 11) or absence (lanes 8 and 10) of PAb421.

p53 protein is probably one dominant determinant of the occurrence of transcriptional activation by the X protein. It is not certain whether there is a direct interaction between p53 and X protein under the conditions used; however, X protein can up-regulate X gene transcription in the presence of normal p53. This indication was supported by the fact that transactivation of the X gene as well as cellular genes by the X protein can be observed in several different cell lines expressing normal p53, but not in its mutant cell lines (Takada et al., unpublished data; see Discussion).

as the chromatin structure or by modification of the DNA (Weintraub, 1985; Ptashne, 1988; Johson and McKnight, 1989). The amount of transcription factors in cells may also affect the transcription activity (Rechesteiner, 1991). A difference in responsiveness to X protein seems not to be species-specific or simply tissue-specific (Seto et al., 1989), so one can speculate that X protein has a repertory of interaction partners, selects an appropriate one among them according to the condition of the cell or the concentration of factors in the cell, and transactivates a specific set of genes in the cell in consequence. Alternatively, X protein may catalytically modify a specific set of transcription factors. Considering the previous observations, it is important to examine interaction between X protein and cellular factors and/or the interaction partner with X protein. Maguire et al. (1991) demonstrated that X protein could bind to CREB or ATF-2, making each bind tightly to a CRE-related sequence in the HBV enhancer I. However, a direct interaction of X protein with other XRE-binding proteins, such as NFkB or AP-1, has not yet been published. In this context, Kekule et al. (1993) and Natoli et al. (1994b) detected increased AP-1 binding to its specific binding DNA site in X proteinexpressing cells, but X protein was not detected in the DNA–protein (AP-1) complex. Therefore, further investigations are necessary to understand the exact role of X protein in the increased binding of cellular factors to the X-responsive elements. Previous observations indicated that X protein is able to transcriptionally activate a number of upstream sequences of viral and cellular genes. This is the first report to demonstrate that X protein can activate the promoter activity without enhancer sequences. It is not clear why investigators previously did not point out the promoter activation function of X protein. Perhaps little attention has been paid to the difference in promoter activity in the presence or absence of X protein. In fact, we also found that X protein activated the core promoter of the SV40 early gene (data not shown). Among the elements for transcription initiation, the TATA box is the most common sequence with which the transcription factor, TFIID,

DISCUSSION Promoter activation by X protein and the state of p53 protein The activity of transcription is determined for each gene by transcriptional factors and their responsive elements in the regulatory DNA region of each gene as well

/ 6a0f$$7695

01-12-96 08:21:49

viral

FIG. 6. Disruption of transcriptional repression function of normal p53 by X protein. Indicated amount (mg per 10-cm dish) of normal p53 and X expression plasmids (pCMVp53 and pHBVX-1, respectively) were cotransfected to HepG2 cells and CAT activity assay was carried out using 2 mg of pXMP2CAT as the reporter plasmid. Relative activity (RA) was calculated and is shown at the top.

AP-Virology

OPPOSITE EFFECTS BETWEEN X PROTEIN AND p53 ON HBV X GENE

interacts. Another initiation sequence, for instance, the GC box, is also known in a variety of genes. Since no homology has been found between the X-PBP binding sequence and the known initiation sequences, the sequence stretch including the X-PBP binding site may be another type of initiator sequence. Although the physiological function of X-PBP has not yet been fully characterized, the present data indicate that activation of the X gene promoter activity by X protein is unlikely to be mediated through X-PBP. It is known that activation by X protein is observed in different degrees according to the cell type (Seto et al., 1989; Rossner, 1992). In this respect, it should be noted that some of the p53-defective cell lines, such as HuH7 cells (Bressac et al., 1990), Cos cells (because of the expression of SV40 T-Ag), and CV-1 cells, when transfected with SV40 T-Ag expression plasmid, exhibited no transcriptional activation by X protein (Koike et al., 1989; Spandau and Lee, 1988). The state of p53 protein is probably one of the dominant determinants of cell type specificity for occurrence of efficient transcriptional activation by X protein. Transcriptional repression of X gene promoter by p53 protein p53 was previously demonstrated to interact with the TBP-associated factors TAFII40 and TAFII60 when activating transcription through p53 binding DNA (Thut et al., 1995) and the basal transcription factor TFIIH (Xiao et al., 1994). Viral and cellular genes have been reported to be negatively regulated by normal p53 (Deffie et al., 1993; Shiio et al., 1992; Ginsberg et al., 1991; Santhanam et al., 1991; Subler et al., 1992). It was also reported that normal p53 binds to TBP and inhibits transcriptional initiation by interfering with binding of the basal transcription factors to the TATA motif (Ragimov et al., 1993). In this study, we first determined the 20-bp region for X gene promoter activity, which possesses none of the typical promoter sequences and then examined whether normal p53 protein positively or negatively regulated X gene transcription. We demonstrated that the promoter activity was repressed by normal p53 under the conditions used. Although the requirement of TBP for transcription from the X gene promoter is unknown because of the absence of the TATA motif, the mechanism of repression of the X gene promoter may involve direct interference of TBP function or TFIIH function with normal p53 protein. Normal p53 protein (synthesized and purified from the baculovirus expression system) exhibited no sequence-specific binding activity to this 20-bp promoter DNA under the conditions used (Figs. 5A and B). We also observed that X-PBP binding correlated with the X gene promoter activity (Nakamura and Koike, 1992; this paper); however, further analyses on the basal transcription factors for the X gene promoter activity and their relationship with the component(s) of X-PBP are necessary for under-

/ 6a0f$$7695

01-12-96 08:21:49

viral

87

standing the mechanism of X gene transcription. These analyses are currently under investigation. Disruption of normal p53 function by X protein and transactivation As we found that the X gene promoter activity was negatively or positively regulated by p53 or X protein, respectively, the transcriptional activation function of X protein may be explained by the interference of normal p53 function with coexpressed X protein. Recently, several studies have suggested that X protein influenced signal transduction pathways; for example, X protein stimulated Ras–GTP complex formation and promoted downstream signaling through Raf and MAP kinases (Benn and Schneider, 1994; Cross et al., 1993). It would be possible to speculate, therefore, that X protein influences one of the signal pathways, which may affect any of the cytosolic factors involved in the disruption of p53 function. Binding of p53 to its DNA consensus sequence is necessary for the transactivation of genes adjacent to these binding sites (Kern et al., 1991; Weintraub et al., 1991). It has also been postulated that normal p53 modulates gene transcription by interacting with the basic transcriptional machinery, TFIID, a part of the transcription initiation complex that has been shown to be a p53 target, resulting in down-regulation of TFIID-dependent genes (Seto et al., 1992; Liu et al., 1993). HBV X protein seems to have such an effect on the p53 suppressor function, similar to that of oncogene products of other DNA tumor viruses, including SV40 T-antigen, human papilloma virus E6, and adenovirus E1B. Viral replication may be triggered through a disruption of the p53 suppressor function by X gene expression without any mutation of p53. Recently, the in vitro interaction between p53 and X proteins was studied by fusing the entire X or normal and mutant p53 ORFs to the GST (glutathione S-transferase) gene (Wang et al., 1994). Only the GST normal p53, but not GST mutant p53 or GST alone, bound the in vitro translated X protein. These results are consistent with those of a previously reported study (Feitelson et al., 1993). On the other hand, when the X gene expression plasmid was cotransfected with the p53 expression plasmid into HepG2 cells, we observed the major localization of X protein in the cytoplasm (Takada et al., unpublished data). Thereby, the repression function of p53 may be canceled by this cytoplasmic X protein by some unknown mechanism in the cotransfected HepG2 cells. Further elucidation of the mechanism by which X protein, particularly its serine protease inhibitor-like domain (Takada and Koike, 1990a), disrupts the repressor function of p53 protein may prove to be fruitful in understanding the pleiotropic effects of X protein. ACKNOWLEDGMENTS We thank F. Hanaoka of The Institute of Physical and Chemical Research (RIKEN) for kindly providing purified normal and mutant p53

AP-Virology

88

TAKADA ET AL.

proteins of the baculovirus expression system. We also thank M. Kobayashi for expert help and advice. This work was partly supported by a grant-in-aid from the Ministry of Education, Science and Culture of Japan to S.T., a grant from the Uehara Memorial Foundation to S.T., a grant-in-aid from the Ministry of Health and Welfare of Japan to K.K., and by a grant-in-aid from the Ministry of Education, Science and Culture of Japan to K.K.

REFERENCES Araki, E., Shimada, F., Shichiri, M., Mori, M., and Ebina, Y. (1988). pSV00CAT: Low background CAT plasmid. Nucleic Acids Res. 16, 1627. Arii, M., Takada, S., and Koike, K. (1992). Identification of three essential regions of hepatitis B virus X protein for transactivation function. Oncogene 7, 397–403. Benn, J., and Schneider, R. T. (1994). Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. Proc. Natl. Acad. Sci. USA 91, 10350– 10354. Bressac, B., Galvin, K. M., Liang, T. J., Isselbacher, K. J., Wands, J. R., and Ozturk, M. (1990). Abnormal structure and expression of p53 gene in human hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 87, 1973–1977. Cross, J. C., Wen, P., and Rutter, W. J. (1993). Transactivation by hepatitis B virus X protein is promiscuous and dependent on mitogen-activated cellular serine/threonine kinases. Proc. Natl. Acad. Sci. USA 90, 8078–8082. Deffie, A., Wu, H., Reinke, V., and Lozano, G. (1993). The tumor suppressor p53 regulates its own transcription. Mol. Cell. Biol. 13, 3415– 3423. Dignam, J. D., Lebovitz, R. M., and Reoder, R. G. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489. Dittmer, D., Pati, S., Zambetti, G., Chu, S., Teresky, A. K., Moore, M., Finlay, C., and Levine, A. J. (1993). Gain of function mutations in p53. Nature Genet. 4, 42–46. Dutta, A., Ruppert, J. M., Aster, C., and Winchester, E. (1993). Inhibition of DNA replication factor RPA by p53. Nature 365, 79–82. Dyson, N., Howley, P. M., Munger, K., and Harlow, E. (1989). The human papilloma virus-16E7 oncoprotein is able to bind the retinoblastoma gene product. Science 243, 934–937. Faktor, O., Budlovsky, S., Ben-Levy, R., and Shaul, Y. (1990). A single element within the hepatitis B virus enhancer binds multiple proteins and responds to multiple stimuli. J. Virol. 64, 1861–1863. Farmer, G., Bargonetti, J., Zhu, H., Friedman, P., Prywes, R., and Prives, C. (1992). Wild-type p53 activates transcription in vitro. Nature 358, 83–86. Feitelson, M. A., Zhu, M., Duan, L. X., and London, W. T. (1993). Hepatitis B X antigen and p53 are associated in vitro and in liver tissues from patients with primary hepatocellular carcinoma. Oncogene 8, 1109– 1117. Ganem, D., and Varmus, H. E. (1987). The molecular biology of hepatitis B viruses. Annu. Rev. Biochem. 56, 651–695. Gerwin, B. I., Spillare, E., Forrester, K., Lehman, T. A., Kispert, L., Welsh, J. A., Pfeifer, A. M. A., Lechner, J. F., Baker, S. J., Vogelstein, B., and Harris, C. C. (1992). Mutant p53 can induce tumorigenic conversion of human bronchial epithelial cells and reduce their responsiveness to a negative growth factor, transforming growth factor b. Proc. Natl. Acad. Sci. USA 89, 2759–2763. Ginsberg, D., Mechta, F., Yaniv, M., and Oren, M. (1991). Wild-type p53 can down-regulate the activity of various promoters. Proc. Natl. Acad. Sci. USA 88, 9979–9983. Harris, C. C., and Hollstein, M. C. (1993). Clinical implications of the p53 tumor suppressor gene. N. Engl. J. Med. 329, 1318–1327. Hohne, M., Schaefer, S., Scifer, M., Feitelson, M. A., Paul, D., and Gerlich, W. H. (1990). Malignant transformation of immortalized transgenic hepatocyte after transfection with hepatitis B virus DNA. EMBO J. 9, 1137–1145.

/ 6a0f$$7695

01-12-96 08:21:49

viral

Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. C. (1991). p53 mutations in human cancers. Science 253, 49–53. Hupp, T. R., Meek, D. W., Midgley, C. A., and Lane, D. P. (1992). Regulation of the specific DNA binding function of p53. Cell 71, 875–886. Johnson, P. F., and McKnight, S. L. (1989). Eukaryotic transcriptional regulatory proteins. Annu. Rev. Biochem. 58, 799–839. Kastan, M. B., Zhan, Q., El-Deiry, W. S., Carrier, F., Jacks, T., Walsh, W. V., Plunkett, B. S., Vogelstein, B., and Fornace, A. J., Jr. (1992). A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71, 587–597. Kekule, A. S., Lauer, U., Weiss, L., Luber, B., and Hofschneider, P. H. (1993). Hepatitis B virus transactivator HBx uses a tumour promoter signalling pathway. Nature 361, 742–745. Kern, S. E., Kinzler, K. W., Bruskin, A., Jarosz, D., Friedman, P., Prives, C., and Vogelstein, B. (1991). Identification of p53 as a sequencespecific DNA-binding protein. Science 252, 1708–1711. Kido, H., Fukutomi, A., and Katsunuma, N. (1990). A novel membranebound serine esterase in human T4/ lymphocytes immunologically reactive with antibody inhibiting syncytia induced by HIV-I. J. Biol. Chem. 265, 21979–21985. Kim, C.-M., Koike, K., Saito, I., Miyamura, T., and Jay, G. (1991). HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature 351, 317–320. Kim, D. W., Uetsuki, T., Kaziro, K., Yamaguchi, N., and Sugano, S. (1990). Use of the human elongation factor 1a promoter as a versatile and efficient expression system. Gene 91, 217–223. Kobayashi, M., and Koike, K. (1984). Complete nucleotide sequence of hepatitis B virus DNA of subtype adr and its conserved gene organization. Gene 30, 227–232. Koike, K., Shirakata, Y., Yaginuma, K., Arii, M., Takada, S., Nakamura, I., Hayashi, Y., Kawada, M., and Kobayashi, M. (1989). Oncogenic potential of hepatitis B virus. Mol. Biol. Med. 6, 151–160. Lane, D. P., and Crawford, L. V. (1979). T antigen is bound to a host protein in SV40-transformed cells. Nature 278, 261–263. Linzer, D. I., and Levine, A. J. (1979). Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell 17, 43–52. Liu, X., Miller, C. W., Koeffler, P. H., and Berk, A. J. (1993). The p53 activation domain binds the TATA box-binding polypeptide in HoloTFIID, and a neighboring p53 domain inhibits transcription. Mol. Cell. Biol. 13, 3291–3300. Maguire, H. F., Hoeffler, J. P., and Siddiqui, A. (1991). HBV X protein alters the DNA binding specificity of CREB and ATF-2 by proteinprotein interaction. Science 252, 842–844. Masuda, H., Miller, C., Koeffler, H. P., Battifora, H., and Cline, M. J. (1987). Rearrangement of the p53 gene in human osteogenic sarcomas. Proc. Natl. Acad. Sci. USA 84, 7716–7719. Momand, J., Zambetti, G. P., Olson, D. C., George, D., and Levine, A. J. (1992). The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-medicated transactivation. Cell 69, 1327– 1245. Nakamura, I., and Koike, K. (1992). Identification of a binding protein to the X gene promoter region of hepatitis B virus. Virology 191, 533– 540. Natoli, G., Avantaggiati, M. L., Chirillo, P., Puri, P., Ianni, A., Balsano, C., and Levrero, M. (1994a). Ras- and Raf-dependent activation of cJun transcriptional activity by the hepatitis B virus transactivator pX. Oncogene 9, 2837–2843. Natoli, G., Avantaggiati, M. L., Chirillo, P., Costanzo, A., Artini, M., Balsano, C., and Levrero, M. (1994b). Induction of the DNA-binding activity of c-Jun/c-Fos heterodimers by the hepatitis B virus transactivator pX. Mol. Cell. Biol. 14, 989–998. Oliner, J. D., Kinzler, K. W., Meltzer, P. S., George, D. L., and Vogelstein, B. (1992). Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature 358, 80–83. Ptashne, M. (1988). How eukaryoric transcriptional activators work. Nature 335, 683–689. Ragimov, N., Krauskopf, A., Navot, N., Rotter, V., Oren, M., and Aloni,

AP-Virology

OPPOSITE EFFECTS BETWEEN X PROTEIN AND p53 ON HBV X GENE Y. (1993). Wild-type but not mutant p53 can repress transcription initiation in vitro by interfering with the binding of basal transcription factors to the TATA motif. Oncogene 8, 1183–1193. Rakotomahanina, C. K., Hilger, C., Fink, T., Zentgraf, H., and Schroder, C. H. (1994). Biological activities of a putative truncated hepatitis B virus X gene product fused to a polylysin stretch. Oncogene 9, 2613– 2621. Rechstreiner, M. (1991). Natural substrates of the ubiquitin proteolytic pathway. Cell 66, 615–618. Rossner, M. T. (1992). Hepatitis B Virus X-gene product: A promiscuous transcriptional activator. J. Med. Virol. 36, 101–117. Sakai, E., and Tsuchida, N. (1992). Most human squamous cell carcinomas in the oral cavity contain mutated p53 tumor-suppressor genes. Oncogene 7, 927–933. Sang, B.-C., Chen, J.-Y., Minna, J., and Barbosa, M. S. (1994). Distinct regions of p53 have a differential role in transcriptional activation and repression functions. Oncogene 9, 853–859. Santhanam, U., Ray, A., and Sehgal, P. B. (1991). Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product. Proc. Natl. Acad. Sci. USA 88, 7605–7609. Sarnow, P., Ho, Y. S., Williams, J., and Levine, A. J. (1982). Adenovirus Elb-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells. Cell 28, 387–394. Seto, E., Zhou, D. X., Peterlin, B. M., and Yen, T. S. B. (1989). Transactivation by the hepatitis B virus X protein shows cell-type specificity. Virology 173, 764–766. Seto, E., Mitchell, P. J., and Yen, T. S. B. (1990). Transactivation by the hepatitis B virus X protein depends on AP-2 and other transcription factors. Nature 344, 72–74. Seto, E., Usheva, A., Zambetti, G. P., Momand, J., Horikoshi, N., Weinmann, R., Levine, A. J., and Shenk, T. (1992). Wild-type p53 binds to the TATA-binding protein and represses transcription. Proc. Natl. Acad. Sci. USA 89, 12028–12032. Shiio, Y., Yamamoto, T., and Yamaguchi, N. (1992). Negative regulation of Rb expression by the p53 gene product. Proc. Natl. Acad. Sci. USA 89, 5206–5210. Shirakata, Y., Kawada, M., Fujiki, Y., Sano, H., Oda, M., Yaginuma, K., Kobayashi, M., and Koike, K. (1989). The X gene of hepatitis B virus induced growth stimulation and tumorigenic transformation of mouse NIH3T3 cells. Jpn. J. Cancer Res. 80, 617–621. Siddiqui, A., Jameel, S., and Mapoles, J. (1987). Expression of hepatitis B virus X gene in mammalian cells. Proc. Natl. Acad. Sci. USA 84, 2513–2517. Siddiqui, A., Gayner, R., Srinivasan, A., Mapoles, J., and Farr, R. W. (1989). Trans-activation of viral enhancers including long terminal repeat of the human immunodeficiency virus by the hepatitis B virus X protein. Virology 169, 479–484. Spandau, D. F., and Lee, C-H. (1988). Trans-activation of viral enhancers by the hepatitis B virus X protein. J. Virol. 62, 427–434. Subler, M. A., Martin, D. W., and Deb, S. (1992). Inhibition of viral and cellular promoters by human wild-type p53. J. Virol. 66, 4757–4762. Subler, M. A., Martin, D. W., and Deb, S. (1994). Overlapping domains on the p53 protein regulates its transcriptional activation and repression functions. Oncogene 9, 1351–1359. Takada, S., and Koike, K. (1990a). X protein of hepatitis B virus resembles a serine protease inhibitor. Jpn. J. Cancer Res. 81, 1191–1194.

/ 6a0f$$7695

01-12-96 08:21:49

viral

89

Takada, S., and Koike, K. (1990b). Trans-activation function of 3* truncated X gene–cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues. Proc. Natl. Acad. Sci. USA 87, 5628– 5632. Takada, S., Kido, H., Fukutomi, A., Mori, T., and Koike, K. (1994a). Interaction of hepatitis B virus X protein with a serine protease, tryptase TL2, as an inhibitor. Oncogene 9, 341–348. Takada, S., Mori, T., Kido, H., Nakamura, I., Yaginuma, K., Tsuchida, N., and Koike, K. (1994b). Contribution of HBV X gene expression to hepatic carcinogenesis. In ‘‘Viral Hepatitis and Liver Diseases’’ (K. Nishioka, H. Suzuki, S. Mishiro, and T. Oda, Eds.), pp. 753–756. Springer-Verlag, Tokyo. Tanaka, K., Tamura, T., Yoshimura, T., and Ichihara, A. (1992). Proteasomes: Protein and gene structures. New Biol. 4, 173–187. Thut, C. J., Chen, J.-L., Kiemm, R., and Tjian, R. (1995). p53 transcriptional activation mediated by co-activators TAFII40 and TAFII60. Science 267, 100–104. Tiollais, P., Pourcel, C., and Dejean, A. (1985). The hepatitis B virus. Nature 317, 489–495. Tsutsumi-Ishii, Y., Tadokoro, K., Hanaoka, F., and Tsuchida, N. (1995). Response of heat shock element within the human HSP70 promoter to mutated p53 genes. Cell Growth Differentiation 6, 1–8. Twu, J.-S., Chu, K., and Robinson, W. S. (1989a). Hepatitis B virus X gene can transactivate heterologous viral sequences. Proc. Natl. Acad. Sci. USA 86, 5168–5172. Twu, J.-S., Rosen, C. A., Haseltine, W. A., and Robinson, W. S. (1989b). Identification of a region within the human immunodeficiency virus type I long terminal repeat that is essential for transactivation by the hepatitis B virus gene X. J. Virol. 63, 2857–2860. Wang, X. W., Forrester, K., Yeh, H., Feitelson, M. A., and Gu, J-R. (1994). Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proc. Natl. Acad. Sci. USA 91, 2230–2234. Weintraub, H. (1985). Assembly and propagation of repressed and derepressed chromosomal states. Cell 42, 705–711. Weintraub, H., Hauschka, S., and Tapscott, S. J. (1991). The MCK enhancer contains a p53 responsive element. Proc. Natl. Acad. Sci. USA 88, 4570–4571. Wolf, D., Harris, N., and Rotter, V. (1984). Reconstitution of p53 expression in a nonproducer Ab-MuLV-transformed cell line by transfection of a functional p53 gene. Cell 38, 119–126. Xiao, H., Pearson, A., Coulombe, B., Truant, R., Zhang, S., Regier, J. L., Triezenberg, S. J., Reinberg, D., Flores, O., Ingles, C. J., and Greenblatt, J. (1994). Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol. Cell. Biol. 14, 7013–7024. Yaginuma, K., Shirakata, Y., Kobayashi, M., and Koike, K. (1987). Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. Proc. Natl. Acad. Sci. USA 84, 2678–2682. Yaginuma, K., Nakamura, I., Takada, S., and Koikra, K. (1993). A transcription initiation site for the hepatitis B virus X gene is directed by the promoter-binding protein. J. Virol. 67, 2559–2565. Zambetti, G. P., Bargonetti, J., Walker, K., Prives, C., and Levine, A. J. (1992). Wild-type p53 mediate positive regulation of gene expression through a specific DNA sequence element. Genes Dev. 6, 1143– 1152.

AP-Virology