Zhen-Hua Xu, Mu-Jun Zhao, Tsai-Ping Li

Zhen-Hua Xu, Mu-Jun Zhao, Tsai-Ping Li, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
Supported by special funds for Major State Basic Research "973"of China, No. 2001CB510205.
Correspondence to: Professor Mu-Jun Zhao, P.O. Box 35, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China. mjzhao@sunm.shcnc.ac.cn
Telephone: +86-21-64374430 Ext 5295 Fax: +86-21-64338357
Received 2002-07-23 Accepted 2002-08-09

Abstract
AIM: p73, as a novel member of a family of p53-related transcription factors, shares redundant functions with p53, such as the abilities of inducing apoptosis and suppressing growth. It is well known that p53 can repress HBV expression and transcription efficiently. The aim of this paper is to investigate the transcriptional effect of p73a and p73b on hepatitis B virus (HBV) and to understand the correlation between HBV and p73.

METHODS: To construct an x-gene inactivated HBV plasmid which was cotransfected with p73a or p73b expression vectors into HepG2 cells. After transiently transfection, HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) were detected by ELISA. Viral transcripts synthesized by HBV were evaluated by Northern blotting analysis. The activities of HBV regulatory elements, including enhancer I/X promoter (ENI/Xp) and enhancer II/core promoter (ENII/Cp) were monitored by luciferase assays.

RESULTS: Both p73a and p73b could repress HBsAg and HBeAg expression by downregulating the ENI/Xp and ENII/Cp activities. But p73a exerted stronger inhibition on the activity of ENI/Xp than p73b, resulting in much lower level of viral transcripts and the antigens expression.

CONCLUSION: p73b as a novel member of p53 family can efficiently inhibit HBV transcription mainly through downregulating the activities of the HBV ENI/Xp regulatory elements.

Xu ZH, Zhao MJ, Li TP. p73b inhibits transcriptional activities of enhancer I and X promoter in hepatitis B virus more efficiently than p73a. World J Gastroenterol 2002; 8(6):1094-1097

INTRODUCTION
p73 gene maps to chromosome 1p36.1, a region which was frequently deleted in several tumors, including neuroblastoma, colorectal cancer and breast cancer^[1]. Moreover, it has been found to share significant homology with the tumor suppressor gene p53 within the transactivation domain, DNA binding domain and oligomerization domain. Both p53 and p73 have redundant functions in the regulation of gene expression, because they have amino acid sequence identity reaching to 63 % in the DNA binding domain^[2]. p73 can activate p53-regulated genes and suppress growth or induce apoptosis, and expression of p73 can be induced by DNA damage as p53 does^[3,4]. Although deletion of p73 gene is observed in neuroblastoma and a subtype of T-cell lymphoma, it is rarely mutated in human cancer^[5-9], unlike p53 which is mutated in about 50 % of human cancers^[5,7,10-12]. Other evidence suggests that p73 is important for regulation of normal development^[13]. p73 gene is expressed as p73a, a 636 amino acid polypeptide, and p73b, a 499 amino acid polypeptide that is encoded by an alternatively spliced transcript lacking 96 nucleotides corresponding to exon 13^[1]. Until now, at least six different p73 proteins (a-z) have been found^[14].
   Hepatitis B virus^[15-17] is a causative agent of chronic hepatitis and hepatocellular carcinoma. Upon infection or DNA transfection, four major viral transcripts are detected. The largest 3.5 kb mRNA is composed of precore and pregenomic mRNA, that direct the synthesis of HBV e antigen (HBeAg) and HBV core antigen (HBcAg) respectively. Pregenomic RNA also serves as a template for reverse-transcription to synthesize the viral DNA genome. The largest surface antigen is synthesized from 2.4 kb mRNA, and the middle and major surface antigen are synthesized from 2.1 kb mRNA. The smallest transcript is a 0.7 kb mRNA, which is responsible for HBx protein production^[18]. The transcription of these RNAs are governed by the core, S1, S2 and X promoters, respectively. The activities of these promoters are under the control of enhancer I and II^[19].
   In addition to a function as a tumor suppressor, p53 can defend host cell from the invading virus. p53 actively inhibits viral replication as in the case of SV40 and HBV^[20,21]. p53 binds to a sequence adjacent to the replication origin of SV40 and abrogates the helicase activity of T antigen by directly binding to it. It has been reported that p53 can bind specifically to the HBV enhancer I and repress the activity of enhancer I and X promoter, resulting in HBV gene expression downregulated^[22]. In addition, p53 can interfere with the life cycle of HBV through down-regulation of the enhancer II and pregenomic/core promoter. Although p53 can not directly bind the enhancer II, it represses the transcriptional activity of HBV through protein-protein interaction^[23].
   p73, as a novel member of a family of p53-related transcription factors, has attracted more attention during these years. However the relationship between p73 and the hepatitis B virus is not elucidated. Based on the similarity between p53 and p73, we examined whether p73, mainly p73a and p73b, can affect HBV antigens expression and the viral transcription.

MATERIALS AND METHODS
Construction of plasmid
p3.8II, kindly provided by Prof. Wang Yuan, is an HBV plasmid which contains terminally redundant HBV genome and can replicate in liver cell. The x-gene inactivated p3.8IIXm was constructed by changing the start condon of the X open reading frame on p3.8II. The pENI/XpLuc and pENII/CpLuc reporter plasmids were constructed by inserting nt1067-1403 and nt1430-1879 which contain enhancer I/X promoter and enhancer II/core promoter respectively in front of the pGL3 basic vector (Promega). The mammalian expression vectors pcDNA3-HA-73a and pcDNA3-HA-73b encode epitope tagged p73 proteins which were kind gifts from Dr. Lu Hua. p53 expression plasmid pRC/CMV hp53 was provided by Dr. Judith Roth. All the constructs were confirmed by restriction enzyme analysis and DNA sequencing.

Cell culture and transfection
HepG2 cells were cultured in DMEM supplemented with kanamycin (250 IU/ml), gentamycin (40 IU/ml) and 10 % fetal calf serum in 5 % CO₂ at 37 ℃. Transfection was carried out by the calcium phosphate method^[24]. Each transfection reaction contained a constant amount of 10 mg DNA per 6 cm dish.

Preparation of cell lysate and measurement of HBV antigens
Five days after transfection, cells were washed twice with PBS and were detached from dishes with 10 mM EDTA in PBS. After centrifugation (5 000 rpm, 1 min), cells were resuspended in 100 ml 250 mM Tris-HCl (pH7.5) and lysed by three thawing and freezing cycles. After centrifugation, the supernatant was transferred and stored at 4 ℃. The protein concentration was estimated by Bradford method (Biocolor). Culture medium was collected every day after transfection. HBsAg and HBeAg were measured with ELISA kits (Sino-America). All procedures were performed according to the descriptions of the manufacturers.

RNA analysis
Total RNA was extracted from transfected cells using TRIzol reagent (Gibco BRL). The RNA samples were treated with RNase-free DNaseI (Pharmacia). For Northern blotting analysis, 20 mg of total cellular RNA per sample was separated on 1 % formaldehyde-agarose gel and blotted to a Hybond-N nylon membrane (Amersham). The membrane was prehybridized in Quick-Hyb buffer (Amersham) for at least 1 hour, followed by 2 hr hybridization at 65 ℃ with the probes of a-³²p-dCTP labeled 3.2 kb HBV DNA and G3PDH fragments. After hybridization, the membrane was washed in 2 %SSC, 0.1 %SDS buffer (RT, 20 min), 0.2 % SSC, 0.1 %SDS (65 ℃,30 min), then exposed to an X-ray film at -70 ℃.

Luciferase assay
Cells were lysed and analyzed with a luciferase assay system (Promega). Corrections of the luciferase activity were made based on the protein concentrations of the lysates. The luciferase activity was measured with a Lumat LB9507 luminometer (Berthold) in 10 ml of the lysate after addition of 100 ml assay reagent.

RESULTS
p73b represses HBsAg and HBeAg expression more efficiently than p73a
Before investigating whether p73 could affect HBV transcription like p53, we established the X protein-minus HBV mutant p3.8IIXm to avoid the interaction between p73 and the HBV X protein. This HBV mutant had no ATG start codon of the X-ORF (Figure1). ELISA results revealed that the HBV antigens expression in mutant type of p3.8IIXm was just a half of that in wild type p3.8II.

Figure 1 (PDF)Partial DNA sequences of p3.8II and p3.8IIXm is indicated in the left panel (A). The point mutation A→C results in the amino acid changes in the ORF of HBV polymerase (P) and X protein(X) (B).

Figure 2 (PDF)Time course of HBV antigen expression. HepG2 cells were transiently transfected with X-minus p3.8IIXm, and cotransfected with pcDNA3, p53, p73a, or p73b individually. Culture medium were harvested at certain days, and detected by ELISA kit.

As shown in Figure2A, P3.8IIXm could secret HBsAg into the medium continuously starting from day 2. Cotransfection of p3.8IIXm with p73b expression plasmid resulted in a reduced level of HBsAg (87 % reduction on day 5 post transfection), which was similar to p53 (89 % reduction on day 5 post transfection). Cotransfection of p3.8IIXm with p73a expression plasmid exhibited weak repression on HBsAg synthesis (only 63 % reduction on day 5 post transfection). The time-dependent alteration in the level of HBeAg (Figure2B) displayed a similar pattern to that of HBsAg. On day 5 post transfection, the expression of HBeAg was changed at the level of 36 % reduction for p73a and 64 % reduction for p73b. Also as shown in Figure2B, p53 could inhibit HBeAg more efficiently (78 % reduction on day 5 post transfection) than p73a and p73b. It is concluded that both p73a and p73b can downregulate HBV expression including HBsAg and HBeAg, but p73b can repress the HBV antigens expression more efficiently than p73a.

p73b can repress the synthesis of viral transcripts including pregenomic/precore RNA and preS/S RNA
The roles of p73a and p73b in viral transcription were assessed at the HBV RNA levels after cotransfection of p3.8IIXm and p73 expression plasmids. Using Northern blot hybridization with ³²P-labeled 3.2 kb HBV fragment as a probe, we detected the pregenomic RNA and precore RNA as well as preS/S mRNA. As Figure 3 shown, cotransfection of p73b resulted in the reduction of the viral transcript level like p53, but p73a seems to exhibit very weak repression on HBV transcription. These results are in accordance with the above ELISA data.

Figure 3 (PDF)Northern blotting analysis of HBV viral transcripts, including pregenomic/precore RNA and preS/S RNA. HepG2 cells were transiently transfected with p3.8IIXm, and also with the expression plasmids. Lane 1 represents pcDNA3, lane 2, p53, lane 3, p73b, lane 4, pHBX and lane 5, p73a respecitively. Total cellular RNA were extacted and a-³²p-dCTP labeled 3.2 kb HBV DNA or G3PDH used as probe.

p73b inhibits HBV transcription mainly through downregulating the activity of HBV ENI/Xp regulatory elements
To explain the molecular mechanism of the p73b mediated repression of HBV transcription, we investigated the possibility of p73 regulating the enhancer and promoter activity. pENI/Xp reporter plasmid containing HBV enhancer I and X promoter was cotransfected into HepG2 cells with p73a and p73b expression plasmids, and the levels of luciferase activity were determined. As shown in Figure 4A, the luciferase activity of the pENI/Xp was decreased with transfection of p73b and p73a, down to 65 % and 35 % of the control value respectively. Therefore, p73b can repress the activity of enhancer I and X promoter more efficiently than p73a. The activity of HBV enhancer II and core promoter were repressed by p73a and p73b at a similar level, but the effects of inhibition were weaker than that by p53 (Figure 4B). Therefore, it is concluded that p73b represses HBV gene expression mainly through the enhancer I and X promoter.

Figure 4(PDF) Effects of p73a and p73b on the regulatory sequences of hepatitis B virus. HepG2 cells were transfected with expression plasmids, pcDNA3(1), p53(2), p73a (3), and p73b (4). In each case, 1 mg of reporter plasmids, pENI/XpLuc, containing the enhancer I/X promoter (A) and pENII/CpLuc, containing enhancer II/core promoter (B), were individually cotransfected. 42 hours after transfection, the cells were harvested and luciferase activity was determined. The value obtained with reporters alone was arbitrarily set to 100 %, and the other values were normalized accordingly. The column heights reflect the average of at least three independent experiments.

DISCUSSION
HBV, an important risk factor of hepatitis and hepatocellular carcinoma, employs a reverse transcription step which is controlled by cellular transcription factors and some cellular signal transduction pathways, including the tumor suppressor gene p53^[25,26]. Physiologically activated p53 can repress HBV transcription, and this repression can be abrogated by physiological levels of HBx protein. It is generally believed that p53 can interact with HBx, and the latter destroys p53 function, including the repression of virus transcription. To exclude the interaction between p73 and the X protein, we constructed the X-minus HBV strain p3.8IIXm which could replicate in hepatoma cells without producing X protein.
   HBsAg and HBeAg are translated from preS/S mRNA and precore mRNA individually. HBV core promoter is in charge of the transcription of pregenomic RNA as well as precore mRNA. ENI and ENII are the two enhancers in the viral genome. ENI is usually able to upregulate all the HBV promoters, and ENII has a particularly significant stimulatory effect upon the core promoter. In this report, we found that p73b repressed HBsAg and HBeAg expression more efficiently, and it inhibited the viral transcripts level mainly through downregulating the enhancer I and X promoter. In the ENI/Xp construct, there were two sites with homology to the 10 bp half-consence (RRRCWWGYYY) for p53 binding, which had been reported to bind specifically to the HBV enhancer I and repress its activity^[22]. Liver-specific enhancer II had also been found to be the target for the p53-mediated inhibition of hepatitis B viral gene expression. p53 could not directly bind to the enhancer II, and it repressed the ENII activity through protein-protein interaction^[23]. p73 protein has significant homology with p53, and p73 can interact with the consensus p53-responsive sequences. But that whether the mechanism of repression on enhancer of HBV by p73 and p53 is the same awaits further investigation. In our results, p73a exerted a very weak repression on HBV transcription and antigen expression, while Doitsh et al reported that p73b did not repress but activated HBV transcription^[27], probably due to the high dose of the protein we used in this study.
   It was noted that p73 was less active than p53, whereas p73b displayed stronger transcriptional activity than p73a. The difference between p73a and p73b in the potential to regulate transcription may be due to the ability of these proteins to form oligomers. p73b is the truncated form of full-length p73a. While p73b is known to oligomerize with itself efficiently, p73a forms oligomers with a poor efficiency, at least when assayed in the yeast two-hybrid system^[2]. Therefore it can be used to explain why p73b exerts more efficient inhibition on HBV transcription than p73a.
   Although p73b has been found to interfere with the HBV transcription in this report, whether HBV can inactivate the function of p73 to affect the viral-infected cell fate, and the relationship between the X protein and p73b remain to be investigated.

ACKNOWLEDGEMENTS
The authors would like to thank Prof. Wang Yuan for providing p3.8II plasmid, Dr. Lu Hua for pcDNA3-HA-p73a, pcDNA3-HA-p73b, and Dr. Vogelstein for the pRC/CMV-p53.

REFERENCES
1    Ikawa S, Nakagawara A, Ikawa Y. p53 family genes: structural comparison, expression and mutation. Cell Death
      Differ 1999; 6:1154-1161
2    Kaghad M, Bonnet H, Yang A, Creancier L, Biscan JC, Valent A, Minty A, Chalon P, Ferrara P, Mckeon F, Caput
      D. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other
      human cancers. Cell 1997; 90: 809-819
3    Steegenga WT, Shvarts A, Riteo N, Bos JL, Jochemsen AG. Distinct regulation of p53 and p73 activity by adenovirus E1A,
      E1B, and E4orf6 proteins. Mol Cell Biol 1999; 19: 3885-3894
4    Jost CA, Marin MC, Kaelin WJ. p73 is a human p53-related protein that can induce apoptosis. Nature 1997; 389: 191-194
5    Ichimiya S, Nimura Y, Kageyama H, Takada N, Sunahara M, Shishikura T, Nakamura Y, Sakiyama S, Seki N, Ohira M,
      Kaneko Y, McKeon F, Caput D, Nakagawara A. p73 at chromosome 1p36.3 is lost in advanced stage neuroblastoma but
      its mutation is infrequent. Oncogene 1999; 18: 1061-1066
6    Nimura Y, Mihara M, Ichimiya S, Sakiyama S, Seki N, Ohira M, Nomura N, Fujimori M, Adachi W, Amano J, He M, Ping
      YM, Nakagawara A. p73, a gene related to p53, is not mutated in esophageal carcinomas. Int J Cancer 1998; 78: 437-440
7    Nomoto S, Haruki N, Kondo M, Konishi H, Takahashi T, Takahashi T, Takahashi T. Search for mutations and examination
      of allelic expression imbalance of the p73 gene at 1p36.33 in human lung cancers. Cancer Res 1998; 58: 1380-1383
8    Mihara M, Nimura Y, Ichimiya S, Sakiyama S, Kajikawa S, Adachi W, Amano J, Nakagawara A. Absence of mutation of the
      p73 gene localized at chromosome 1p36.3 in hepatocellular carcinoma. Br J Cancer 1999; 79: 164-167
9    Alonso ME, Bello MJ, Lomas J, Gonzalez-Gomez P, Arjona D, De Campos JM, Gutierrez M, Isla A, Vaquero J, Rey JA.
      Absence of mutation of the p73 gene in astrocytic neoplasms. Int J Oncol 2001; 19: 609-612
10  Yang SM, Zhou H, Chen RC, Wang YF, Chen F, Zhang CG, Zhen Y, Yan JH, Su JH. Sequencing of p53 mutation in
      established human hepatocellular carcinoma cell line of HHC4 and HHC15 in nude mice. World J
      Gastroenterol 1998; 4: 506-510
11  Peng XM, Peng WW, Yao JL. Codon 249 mutations of p53 gene in development of hepatocellular carcinoma.
      World J Gastroenterol 1998; 4: 125-127
12  Wang D, SHI JQ. Overexpression and mutations of tumor suppressor gene p53 in hepatocellular carcinoma. China Natl
      J New Gastroenterol 1996; 2: 161-164
13  Yang A, Walker N, Bronson R, Kaghad M, Oosterwegel M, Bonin J, Vagner C, Bonnet H, Dikkes P, Sharpe A, Mckeon F,
      Caput D. p73-deficient mice have neurological pheromonal and inflammatory defects but lack spontaneous tumors.
      Nature 2000; 404:99-103
14  Ueda Y, Hijikata M, Takagi S, Chiba T, Shimotohno K. New p73 variants with altered C-terminal structures have
      varied transcriptional activities. Oncogene 1999; 18: 4993-4998
15  Wands JR, Blum HE. Primary hepatocellular carcinoma. N Engl J Med 1991; 325: 729-731
16  Tang ZY. Hepatocellular carcinoma-cause, treatment and metastasis. World J Gastroenterol 2001; 7: 445-454
17  Feitelson MA, Sun B, Satiroglu Tufan NL, Liu J, Pan J, Lian Z. Genetic mechanisms of hepatocarcinogenesis.
      Oncogene 2002; 21:2593-2604
18  Nassal M, Schaller H. Hepatitis B virus replication. Trends Microbiol 1993; 1: 221-226
19  Seeger C, Mason WS. Hepatitis B virus biology. Micro MolBioRev 2000; 64: 51-68
20  Bargonetti J, Friedman PN, Kern SE,Volgestin B, Prives C. Wild-type But Not Mutant p53 Immunopurified Proteins
      Bind to Sequences Adjacent to the SV40 Origin of Replication. Cell 1991:1083-1091
21  Wang EH, Friedman PN, Prives C. The Murine p53 Protein Blocks Replication of SV40 DNA In Vitro by Inhibiting the
      Initiation Functions of SV40 Large Antigen. Cell 1989; 57: 379-392
22  Ori A, Zauberman A,Doitsh G, Paran N, Oren M, Shaul Y. p53 binds and represses the HBV enhancer: an adjacent
      enhancer element can reverse the transcription effect of p53. EMBO J 1998;17: 544-553
23  Lee H, Kim HT, Yun YD. Liver-specific enhancer II is the target for the p53-mediated inhibition of hepatitis B viral
      gene expression. J Biol Chem 1998; 273: 19786-19791
24  Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning, a Laboratory Manual, 2nd edn. New York: Cold Spring
      Harbor Laboratory Press 1989: 788-791
25  Arbuthnot P, Kew M. Hepatitis B virus and hepatocellular carcinoma. Int J Exp Pathol 2001; 82: 77-100
26  Brechot C, Gozuacik D, Murakami Y, Paterlini-Brechot P. Molecular bases for the development of hepatitis B virus
      (HBV)-related hepatocellular carcinoma (HCC). Semin Cancer Biol 2000;10: 211-231
27  Doitsh G, Shaul Y. HBV transcription repression in response to genotoxic stress is p53-dependent and abrogated by
      pX. Oncogene 1999; 18: 7506-7513

Edited by Zhang JZ