[CANCERRESEARCH57.5137-5142, November15, 19971 The Transactivation and p53-interacting Functions of Hepatitis B Virus X Protein Are Mutually Interfering but Distinct1 Yong Lin, Takahiro Nomura, Tatsuya Yamashita, Dorjbal Dorjsuren, Hong Tang, and Seishi Murakami2 Department of Molecular Biology, Cancer Research Institute, Kanazawa University, Takara-machi /3-1, Kanazawa 920, Japan ABSTRACT teins that interact with p53. These include the SV4O large T antigen, adenovirus E1B, EBV EBNA-5, human cytomegalovirus 1E84, and Transactivation of viral and host genes expression by hepatitis B virus the human papillomavirus E6 proteins (28—30).Disruption of the p53 x protein(HBx)isbelievedtobeinvolvedinhepatocarcinogenesis.The functions, such as transcriptional activation and p53-dependent apop interaction of HBx with the tumor suppressor p53 and Its inhibitory effect on p53 functIons have been reported recently. However, the question of tosis by viral oncoproteins, is not only beneficial to virus infection but whetherp53 is directlyinvolvedin HBxtransactivationhasnot yet been also important in virally induced carcinogenesis. addressed. l.a this study, we delineated the interaction sites of HBx and The direct interaction of HBx and p53 both in vitro and in vivo has p53 using far-Western blotting and glutathione S-transferase-resm pull been reported recently (31—35).HBx was shown to inhibit p53 tans down assays The results indicate that the HBx-binding sites are located activation and p53-dependent apoptosis (32, 33, 36). However, there within the oligomerization and specific DNA-binding domains of p53 and have been no reports showing that HBx tansactivation is affected by that the p53-binding site was confined to a small region in the HBx p53. The relationship between transactivation and the p53-binding transactivatlon domain. Mutual interference of the transactivations by functions of HBx has not yet been addressed. In this report, we HBx and p53 was detected by CAT assays in a transient transfection mapped the interaction sites of both p53 and HBx and investigated system. Strikingly, transactivation by HBx was observed in the p53- negativecells,Saos-2and Hep3B,indicatingthatthe transactivationand whether p53 binding is involved in HBx transactivation. The results the p53-inhibitingfunctionsof HBxare mutuallyinterferingbutdistinct. show that the p53-binding site is within the transactivation domain of HBx, and p53 has two independent HBx-binding sites located in the specific DNA-binding domain and oligomerization domain, respec INTRODUCTION tively. p53 and HBx interfere with each other's transactivation func There is a great deal of epidemiological evidence to suggest that tion. However, because HBx tansactivation was clearly detected in chronic HBV3 infection is a major risk factor associated with hepa p53-negative cells (Saos-2 and Hep3B), HBx tansactivation is ‘us tinct from its p53-binding function. tocarcinogenesis (1). However, the mechanisms by which HBV con tributes to liver cell transformation remain elusive. No HBV gene acts as a dominant oncogene or acutely transforms liver cells. The wood MATERIALS AND METHODS chuck and ground squirrel hepatitis viruses, the genomes of which harbor the X gene, have been associated with the development of Plasmid Constructions. The full-length and truncatedHBx-5Dl, -5D2, HCC, whereas the oncogenic potential of avian hepadnaviruses, -3D5, -3D39, -Dl6, and -D26 expression plasmids have been described pre which are devoid of the X gene, has not been established (2, 3). viously (37—39). Construction of the truncated HBx mutant HBx-Dl9 (5 1—99 Importantly, the HBV X gene has been shown to have the ability to an) was carried out by using PCR cloning. p53 and TBP cDNA derived from induce transformation in rodent cells (4—6)and HCC in some tans pSGHS3 and HlO (40), which were kindly provided by Drs. M. Fujii (Tokyo Medical and Dental University, Tokyo, Japan) and M. Hirokoshi (Tokyo genic mice lines or to potentiate oncogenesis by oncogene or chemical University, Tokyo, Japan), respectively, were inserted into the the EcoRI and carcinogens (7—10).The X gene is frequently integrated into the host BamHI sites of pSG5UTPL and pGENKI (37). The resultant plasmid, genome and expressed in HCC (1 1—13).HBx, a 154-na protein pSG5UTPL-p53, was used to construct p53-dl (1—100 as), pS3-d5 (101—314 encoded by the HBV X gene, has been shown to deregulate cell cycle an), and p53-d3 (315—393 an) using PCR cloning. The p53 mutant R273H checkpoint controls (14). On the other hand, HBx is a viral transac coding sequence was constructed by PCR-directed mutagenesis. The histidine tivator that can activate the expression of many viral and cellular tagged protein expression plasmid pLHis was derived from pETlld by replac genes through a wide variety of cis-elements (15—19).Although the ing the NdeI—BamHI fragment with the annealed complementary oligonucleo mechanism has not been definitely elucidated, HBx transactivation tides, TATGAATI'CCATGAAGCTTGGATC, so as to create the EcoRI, seems to be important for its oncogenic role, because HBx tansacti HindIII, and BamHI digestion sites. Plasmids pLHis-HBx and pLHis-p53 were yates not only HBV genes but also various host genes that engage in constructed by inserting the HBx and p53 coding sequence into the EcoRI and cell proliferation and acute inflammatory responses (15, 20—23). BamHI sites of pLHis. All of the constructs were sequenced using Taq sequencing kits and a DNA sequencer (370A; Applied Biosystems). The tumor suppressor p53 is involved in maintenance of the Preparation of Recombinant Proteins. GST-fused proteins were cx genomic integrity of cells (24). p53 has multiple functions, including pressed in Escherichia coli by induction with 0.4 mM isopropyl @3-D-thioga transcriptional activation and repression, modulation of factors en lactopyranoside for 3 h at 30°C. Cells were harvested and sonicated in PBST gaged in DNA repair, cell cycle control, and apoptosis regulation buffer (PBS containing 1% Triton X-lOO). After centrifugation, the extract (25—27).Various DNA tumor viruses encode transforming oncopro (supematant) was collected and stored at —80°C.Forpurification, the extracts were incubated with glutathione-Sepharose 4B resin (Pharmacia) for 1 h at Received 6/12/97; accepted 9/19/97. room temperature. The beads were collected and washed four times with PBST Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage buffer and then eluted with 10mMreduced glutathione in 50 nmiTris-HCI (pH charges. This article must therefore be hereby marked advertisement in accordance with 8.0). The eluted proteins were divided into aliquots and stored at —80°C. 18U.S.C.Section1734solelyto indicatethisfact. Histidine-tagged HBx and p53 were expressed in the BL2I (DE3 pLys) I This study was partly supported by a Science Grant from the Ministry of Education andCultureof Japan. transformants of pLHis-HBx and pLHis-p53 by 0.4 mM isopropyl @-D-thioga 2 To whom requests for reprints should be addressed. Phone: 8 1-76-265-273 1 ; Fax: lactopyranoside induction at 30°C.Cells were harvested 3 h postinduction and 81-76-234-4501; E-mail: [email protected]. sonicated in denaturing binding buffer (6 M guanidine chloride, 20 m@i sodium 3 The abbreviations used are: HBV, hepatitis B virus; HBx, HBV X protein; HCC, phosphate, and 500 nmiNaC1,pH 7.8). Histidine-tagged proteins were purified hepatocellular carcinoma; as, amino acid(s); NF-.cB, nuclear factor KB; GST. glutathione S-transferase; TBP, TATA box binding protein; RPB5, RNA polymerase II subunit 5; by incubating the sonication supernatant with nickel resin and following TFIIB, transcription factor IIB. extensive washing with denature binding buffer and then denaturing washing S 137 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1997 American Association for Cancer Research. ItBx-p53 INTERACTION buffer (6 Mguanidine chloride, 20 mMsodium phosphate, and 500 mr@iNaCl, have been described previously. (42). Total cell lysates were prepared from pH 6.0) and were eluted with denaturing elution buffer (20 msi sodium cells that were harvested 48 h after transfection. The CAT assay reactions phosphate and 500 mM NaCl, pH 4). The eluted proteins were renatured by proceeded for 60 mm at 37°Cusing20 @gofprotein from the transfected cell dialysis against a buffer with a sequentially reducing concentration of urea (20 lysates (42). The fractionated TLC plates were exposed to imaging plates, and mM sodium phosphate and 500 mr@iNaCl, pH 7.8). After the final dialysis in the CAT activities were measured as the percentage of the conversion to PBS, the proteins were divided into aliquots and stored at —80°C. acetylated forms of [‘4C]chlorumphethcol (% acetylation; Amersham) using a Far-WesternBlotting.Thefar-Westernblottingwasperformedasprevi Bioimage analyzer (BAS1000; Fuji). Transfection and CAT assays were ously described (39). The target proteins (200 ng each) were fractionated on performed at least three times with each combination of transactivator and SDS-PAGE gels and then electrotransferred onto nitrocellulose membranes, CAT reporter constructs. Representative data are shown. which were denatured, renatured, and blocked with5% skim milk in modified GBT buffer (10% glycerol, 50 nmiHepes-NaOH, pH 7.5, 170mi@KC1,7.5 mai RESULTS MgCl2,0.1 IflMEDTA, 0.1 mMDU, and 1% Triton X-lOO)before subjected to far-Western blot (38). The binding reaction proceeded