Cellular response to conditional expression of the hepatitis B virus precore and core proteins in cultured hepatoma (Huh-7) cells

Journal of Clinical Virology 32 (2005) 113–121

Cellular response to conditional expression of the hepatitis B virus precore and core proteins in cultured hepatoma (Huh-7) cells S. Locarninia,∗ , T. Shawa , J. Deana , D. Colledgea , A. Thompsona , K. Lib , S.M. Lemonb , G.G.K. Lauc , M.R. Beardd a

b

Victorian Infectious Diseases Reference Laboratory, 10 Wreckyn Street, North Melbourne, Vic 3051, Australia Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1019, USA c University of Hong Kong, Faculty of Medicine, Queen Mary Hospital, Hong Kong d Infectious Diseases Laboratories and Hanson Institute, Institute of Medical and Veterinary Science; School of Molecular and Biomedical Science, The University of Adelaide, Adelaide 5000, Australia Received 1 October 2004; accepted 4 October 2004

Abstract Background: The expression of the hepatitis Be antigen (HBeAg) is one of several strategies used by hepatitis B virus (HBV) to ensure persistence. The HBeAg may function as a toleragen in utero and has been shown to regulate the host’s immune response. Aim: The aim of this study was to examine the effect of the HBV precore and core protein on cellular gene expression in the hepatoma cell line Huh-7. Study design: Huh-7 cells with tight regulated expression of the HBV core or precore protein were produced using the Tet-Off tetracycline gene expression system. Changes in cellular gene expression in response to core/precore expression compared to Huh-7 cells not expressing the proteins were determined using a commercial high-density oligonucleotide array (Affymetrix Hu95A GeneChip) containing probes for 12,626 full-length human genes. Results: Analysis of differential mRNA gene expression profiles at 7 days post precore and core expression revealed 45 and 5 genes, respectively, with mRNA changes greater than three-fold. The most striking feature was in Huh-7 cells expressing the precore protein in which 43/45 genes were downregulated 3–11-fold. These included genes that encoded products that regulate transcription/DNA binding proteins, cell surface receptors, cell-cycle/nucleic acid biosynthesis and intracellular signalling and trafficking. The only known gene, which was upregulated encoded a cytoskeletal protein. For the core cell line, 4/5 genes were downregulated 3–15-fold upon core induction and included genes that encoded products that affect intermediary metabolism, cell surface receptors and intracellular signalling. The one gene, which was upregulated was a cytokine gene. Conclusion: The results of this study show that HBV precore protein has a much greater effect on cellular gene expression in comparison to the core protein, suggesting that core and precore proteins may have diverse effects on cellular functions and equally different roles in modulating HBV pathogenesis. © 2004 Elsevier B.V. All rights reserved. Keywords: Hepatitis B e antigen; Huh-7; Gene expression system

1. Introduction Hepatitis B virus (HBV) infects more than 350 million people and is a major cause of acute and chronic liver disease in the world. HBV infection has been linked unequiv∗

Corresponding author. Tel.: +61 3 9342 2637; fax: +61 3 9342 2666. E-mail address: [email protected] (S. Locarnini).

1386-6532/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2004.10.002

ocally to the development of hepatocellular carcinoma. The HBV is a DNA virus with four open reading frames in its genome encoding its viral proteins. The virus can persistently infect the liver and several studies have suggested that the pre Core–Core (preC–C) gene plays an important role in this process (Ou, 1997). A unique feature of HBV is the production of a secreted, non-particulate form of the nucleoprotein, the hepatitis Be

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antigen [HBeAg] (Milich et al., 1998). HBeAg is an accessory protein of HBV, not required for viral replication or infection but important for natural infection in vivo (Chang et al., 1987). The HBeAg is the major product of the pre C–C gene. The HBeAg is initially translated as a 25 kDa protein (the precore protein) and during translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum (ER), its signal sequence is cleaved (Dienes et al., 1995), generating P22, which is further truncated at its C-terminal end by a cellular furin protease (Messageot et al., 2003) before secretion from the cell as HBeAg. It has been proposed that HBeAg may contribute to HBV persistence by functioning as an immune toleragen in utero as soluble HBeAg can cross the placenta (Milich et al., 1990). Furthermore, in animal models HBeAg has been shown to regulate the host’s immune response but the cellular processing of the precore protein is complex (Milich et al., 1998). Little is known about the function of HBeAg in the life-cycle of the HBV (Chen et al., 2003). It has been observed that in transfected cells an increase in the expression of the pre C–C gene leads to inhibition of HBV replication (Lamberts et al., 1993). On the other hand, mutations leading to the abolition (Carman et al., 1989) or reduction of pre C–C gene expression result in a significant increase in HBV replication (Buckwold et al., 1996; Scaglioni et al., 1997b). The negative role of the pre C–C gene in HBV replication was demonstrated to be due to cytosolic P22 molecules forming heterodimers with the HBV nucleocapsid proteins (P21), which resulted in the formation of unstable core structures (Scaglioni et al., 1997b). However, P22 in addition to its role as an HBeAg precursor, and regulator of HBV replication, may also have important effects on the host cell during HBV replication. The aim of this study, therefore, was to examine the effect of the HBV precore and core protein on cellular gene expression in Huh7 cells in which the expression of the viral proteins is tightly regulated by a tetracycline responsive promoter.

2. Materials and methods 2.1. Antibodies and immunoassays Mouse monoclonal antibodies against HBV precore and core proteins were purchased from Abcam Ltd., Cambridge, UK. Rabbit polyclonal antibody against the HBV core protein was purchased from Dako, Pvt. Ltd., UK. The HBeAg phenotype of each cell line was verified by assaying aliquots of culture medium from the Huh-7 cells for the presence of HBeAg by enzyme immunoassay (IMX Assay: Abbott Laboratories, North Chicago, IL, USA).

pBluescript II SK (Stratgene) was kindly provided by Professor H. Schaller (Delaney IV and Isom, 1998). This cDNA encoded the HBV core and precore genes, which were amplified by polymerase chain reaction (PCR), cloned into pTRE-2 (Clontech, Palo Alto, CA, USA) and verified by DNA sequencing, respectively, using standard techniques. The resulting plasmids, termed pTRE2-precore and pTRE2-core, allow expression of the HBV precore and core proteins, respectively, under the transcriptional control of the tetracycline responsive promoter. The tetracycline response expression system uses the Escherichia coli tetracycline resistance operon fused to the activating domain of VP16 (Clontech). Thus, in the presence of tetracycline, this fusion protein binds to a tet operator region on the CMV promoter and inhibits gene transcription. The Huh-7 cells were maintained in Dulbecco’s modified Eagle medium (Gibco-BRL, Grand Island, NY), supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin G and 100 U/mL streptomycin (GibcoBRL) in a humidified 37 ◦ C/5% CO2 incubator. These cells were co-transfected with a mixture of either supercoiled pTet-Off (Clontech) and pTRE2-precore, or pTRE2-core, or pTRE2 vector and Fugene 6 reagent (Roche, Indianapolis, IN, USA). Stable transfectants were selected by growth in culture medium containing 400 ␮g/mL G418 and 2 ␮g/mL tetracycline (Sigma, St. Louis, MO). G418-resistant cell lines were subsequently expanded and examined for expression of HBV proteins upon withdrawal of tetracycline for 4–7 days by indirect immunofluorescence microscopy. Cell lines expressing precore or core were confirmed by immunoblotting. 2.3. RNA isolation and analysis with high-density oligonucleotide arrays Biotinylated, single-stranded antisense RNAs were prepared from total cellular RNA isolated from cells cultured in the presence or absence of tetracycline using the RNAqueous 4PCR RNA extraction kit (Ambion, Austin, TX). Next, 25 ␮g of RNA was used as template for probe synthesis, and probes were hybridized to an Affymetrix Human Gene Chip (Hu95A) containing 12,626 probe sets for known genes, according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA). The DNA arrays were scanned using an Affymetrix confocal scanner and the data analyzed with Microarray GeneSuite software 4.0.1. The mRNA abundances were considered to be significantly different in cells grown in the presence or absence of tetracycline if the average difference between the hybridization signals from the relevant paired probe sets was greater than or equal to three-fold. 2.4. Indirect immunofluorescence microscopy

2.2. Plasmids, cloning and establishment of cell lines with conditional expression of HBV proteins A replication competent plasmid containing a 1.3 times genome-length HBV construct (genotype D, subtype ayw) in

Cells were grown on chamber slides in tetracyclinecontaining medium, which was replaced by fresh tetracycline-free medium at selected times. Cells were washed 3 times in phosphate-buffered saline (PBS) before

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fixing in 4% paraformaldehyde for 20 min, followed by membrane permeabilization with 0.2% Triton X-100 for 5 min at room temperature. After washing in PBS, fixed cells were stained with antibodies to HBV precore or core proteins diluted in PBS containing 3% bovine serum albumin (BSA) for 2 h at room temperature. Cells were incubated with a goat anti-mouse or goat anti-rabbit IgG fluorescein isothiocyanate-conjugated secondary antibody (Sigma), counterstained with 4 ,6-diamidino-2-phenylindole (DAPI), and examined with a Zeiss Axioplan2 microscope. 2.5. Immunoblot detection of HBV proteins Cells were lysed in a buffer containing 150 mmol/L NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 50 mmol/L Tris, 1 mmol/L phenylmethylsulfonyl fluoride, 2 ␮g/mL aprotinin, and 1 ␮g/mL leupeptin. Protein concentrations were determined using a bicinconinic acid kit (Sigma). Samples were subjected to SDS 12–15% polyacrylamide gel electrophoresis, followed by transfer onto Hybond-ECL nitrocellulose membranes (Amersham Pharmacia Biotech, Mississauga, Ont., Canada). Membranes were blocked with 5% skimmed milk in PBS and incubated with primary antibodies diluted in PBS containing 1% BSA. Following three washes in PBS, membranes were incubated for 1 h at room temperature with a secondary horseradish peroxidase-conjugated antibody (Amersham Pharmacia Biotech) diluted in PBS containing 1% BSA. Following three additional washes in PBS, proteins bound to antibody were visualized by enhanced chemiluminesence (ECL; Amersham Pharmacia Biotech). 2.6. Analysis of cell proliferation and cell viability To assess proliferation, 4 × 104 cells were seeded into each well of a six-well tissue-culture plate in tetracyclinecontaining medium and incubated overnight under standard conditions. The cells were refed with medium containing various concentrations of tetracycline, and cells from triplicate wells were harvested and counted visually every 48 h over a period of 10 days. For bromodeoxyuridine (BrdU) incorporation experiments, 3 × 105 cells were seeded in 25 cm2 flasks in tetracycline-containing medium. After 16 h, the cells were refed with either tetracycline-free or tetracycline-containing medium, incubated for an additional 72 h, and then pulselabeled with 10 ␮mol/L BrdU (BD Pharmingen, San Diego, CA) for 45 min. Cells were then harvested, stained with anti-BrdU fluorescein isothiocyanate conjugate and 7-aminoactinomycin D (7-AAD) (BD Pharmingen), and analyzed by flow cytometry. A colorimetric assay for cell viability was used to detect possible cytotoxic or cytostatic effects due to expression of the HBV proteins. Triplicate sets of Huh-7 cells were seeded into multiwell tissue culture plates at semi confluency and allowed to adhere overnight in the presence or absence of tetracycline. After 7–10 days, cell viability in each culture

Fig. 1. (A) Time course immunoblot of core expression in the HBV core cell line cultured with (T) and without tetracycline from day 0 (D0) to day 10 (D10). The 21 kDa protein band of the hepatitis B core antigen (HBcAg) is marked. Below the immunoblot is an immune electron micrograph following the incubation of anti-HBc antibody with cell extracts of the cell line at D7 after tetracycline removal. It shows 27 nm nucleocapsid-like structures composed of the HBcAg. Magnification ×250,000. (B) Immunoblot showing the time course of precore expression in the HBV precore cell line cultured with (T) and without tetracycline from day 0 (D0) to day 10 (D10). The 14–22 kDa protein bands representing precore protein processing by the cell are shown.

set conditions was assayed by MTT reduction as described previously (Chin et al., 2001; Mosmann, 1983). 2.7. Experimental design for cellular gene expression Each of the three cell lines was grown in both the presence and absence of (2 ␮g/mL) tetracycline for up to 10 days. Viral protein expression was typically maximal by day 7 (see Fig. 1). Total mRNA was extracted and its yield and quality estimated from the 260 nm:280 nm absorbance ratio and by electrophoresis. The effects of viral proteins on cellular gene expression in each cell line was determined by analysing matched pairs (Tet-On and Tet-Off) of RNA samples using Affymetrix Hu95A high-density oligonucleotide arrays.

3. Results 3.1. Establishment and characterization of the cell lines expressing hbv core and precore proteins The plasmids, pTRE2-precore, pTRE2-core and pTRE2were co-transfected with pTet-Off into Huh-7 cells and G418resistant clones were expanded and subsequently screened by immunofluorescence microscopy for protein expression after

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tetracycline removal. At least 50 G418-resistant clones were screened, resulting in the identification of five or six clones, which expressed HBV precore or core proteins, respectively. Single clones that demonstrated tightly regulated expression of the HBV precore and core proteins, respectively, were selected for subsequent characterisation (see Fig. 1A and B). A G418-resistant cell-line, which was co-transfected with pTetOff and pTRE2, had no demonstrable expression of HBV proteins, was also expanded as a control cell line. The presence of both plasmid DNAs in the cell genome was confirmed by PCR (results not shown). Replicate samples of each of the three cell lines were individually processed in either Tet-On or Tet-Off conditions from the day of splitting (day 0) and observed for 10 days. Cells and cell culture supernatants were collected and processed for detection of HBV core and precore antigens by enzyme immunoassay, immunoblot and immune electron microscopy. The core-producing cell line produced a 21 kDa protein, which reacted with anti-HBc antibody in immunoblots (Fig. 1A). Immune electron microscopy of cell extracts revealed aggregates of 27 nm particles that had the typical appearance of HBV cores (Fig. 1A). The precore producing cell line was found to produce 14–22 kDa protein bands that reacted with anti-HBe antibody in immunoblots (Fig. 1B). Immune electron microscopy of extracts from these cells did not reveal any virus-like or core-like particles (data not shown). The control cell line did not produce any protein products that reacted with antibodies to HBV precore or core proteins. Expression of HBV precore or core protein in Huh-7 cells had no measurable adverse effect on cell proliferation or viability (results not shown). Each cell line has been continuously passaged for more than 12 months, during which time they have maintained both the morphological characteristics of the parent Huh-7 cells and the capacity to respond appropriately to the presence and absence of 2 ␮g/mL tetracycline. Core protein or precore protein expression became detectable by immunoblotting within 2–4 days, respectively, after tetracycline withdrawal (Fig. 1A and B). Dose-dependent expression of the HBV precore protein and subsequent secretion into the cell culture supernatant as HBeAg, was observed when the precore producing line was exposed to concentrations of tetracycline less than 2.0 ␮g/mL and was greatest in the absence of tetracycline (Fig. 2).

Fig. 2. The level of HBeAg production in the cell culture supernatant, as measured by enzyme immunoassay (EIA), of the pTRE2-precore producing cell line in the presence of decreasing doses of tetracycline at 24 h post antibiotic removal.

3.2. mRNA profiling by Affymetrix GeneChip analysis Microarrays, which display sets of oligonucleotide probes for 12,626 different human genes were purchased from Affymetrix. Seven days after the induction of HBV protein expression by tetracycline withdrawal, cellular RNA was extracted from each cell line and purified as described above. RNA was also extracted and purified from replicate control cultures grown for 7 days in the presence of tetracycline. Microarray analysis of RNA samples derived from each pair of Tet-On and Tet-Off cultures identified two cellular genes that were upregulated and 43 that were repressed by the presence of the HBV precore protein. In cells that expressed the core protein, one cellular gene was upregulated and four repressed. In the control cell line, six and eight genes were up- and downregulated, respectively, by tet withdrawal. Up- and downregulated genes were classified into six different broad functional groups: (1) regulators of nucleotide metabolism; (2) signal transducers and cell trafficking proteins; (3) regulators of cell cycle and apoptosis; (4) cytokines and other secreted proteins; and (6) unknown (Table 1). Specific genes in each category are listed with additional details in Tables 2–6. Expression of the precore protein in Huh-7 cells produced the greatest change in cellular gene expression. Expression of mRNAs for 45 different genes was altered by more than three-fold. Gene repression, affecting all function groups was the predominant effect: 43/45 mRNAs were downregulated 3–11-fold, representing 0.3% of all mRNAs detectable by this assay (see Fig. 3). In particular, regulators of nucleotide metabolism and transcription (Table 2) as well as signal transducers and cell trafficking proteins (Table 3) were most affected. Only two genes were upregulated: one encoded the haemoglobin beta chain, the other a product of unknown function. Details are provided in Tables 2–6. In contrast, expression of only five cellular genes (representing 0.03% of mRNAs detectable) was changed by expression of the precore protein. A single cytokine gene was upregulated; the remaining four that were downregulated by up to 15-fold included two enzymes of intermediary metabolism (DHF-reductase and phospholipase C), and two structural proteins associated with membranes and signalling (see Tables 2–6). Interestingly, neither genes that control the cell cycle nor those that regulate apoptosis were affected by expression of the core protein and assembly into viral nucleocapsid-like structures. Importantly, in the control cell line the presence and absence of tetracycline altered expression of only a few cellular genes, changing the abundance of individual mRNAs by less than four-fold (see Tables 2–6). The small number of mRNAs differentially regulated in the control and core-producing lines suggests that neither tetracycline at the concentration used nor core expression has much effect on the regulation of cellular gene transcription. Collectively these results strongly suggest that the precore protein, but not the core protein has a significant repressive effect on transcription.

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Table 1 Affymetrix analysis: number of mRNAs that increase or decrease >three-fold after precore or core gene expression in Huh-7 cells Functional grouping

Cell line Precore (45) Down

1. 2. 3. 4. 5. 6.

Core (5)

Control (8)

Up

Down

Up

Down

Up

Regulators of nucleotide metabolism and transcription Signal transducers, membrane-associated and cell trafficking proteins Regulators of cell cycle and apoptosis Structural and metabolic proteins Cytokines and other secreted proteins Unknown

12 11 6 3 4 7

– – – 1 – 1

1 3 – – – –

– – – – 1 –

1 2 – 1 – 2

– – 1 1 –

Total

43

2

4

1

6

2

Table 2 Functional grouping; regulators of nucleotide metabolism and transcription Fold change

Accession number

Gene

Chromosomal location

Product description

Cellular location

Precore −10.7 −7.7 −7.5 −6.7 −3.9 −3.7 −3.7 −3.7 −3.4 −3.2 −3.0 −3.0

X90858 X14850 M29039 Z97029 L04731 AF059252 X54199 AB016816 W27050 M64228 X6228 AA143321

UP H2AFX JUNB RNASEH1 MLL DOM3Z GART MASL1 SFPQ ECGF1 NUP214 DFKZP586

7 11 19 19 11 6 21 8 1 22 9 18

Uridine phosphorylase Histone 2A Jun-B transcription factor (oncogenic) Ribonuclease H1 Transcription factor Exoribonuclease binding protein De novo purine synthesis Probable transcription regulator Pre-mRNA splicing factor Thymidine phosphorylase Nuclear pore protein Probable RNA polymerase I subunit

Cytosol Nucleus Nucleus Nucleus Nucleus Nucleus Cytosol Nucleus Nucleus Cytosol Nucleus Nucleus

Core −15.2

J00140

DHFR

5

Dihydrofolate reductase

Cytosol

Control −3.3

AI610467

SMG1

16

Nonsense mRNA iirveillance protein

Nucleus

Table 3 Functional grouping: signal transducers, membrane-associated and cell trafficking proteins Fold change

Accession number

Gene

Chromosomal location

Product description

Cellular location

Precore −6.7 −5.5 −4.5 −4.1 −4.0 −3.9 −3.7 −3.6 −3.3 −3.1 −3.0

AB009462 U75362 D16815 U73704 L36240 Z50194 X14968 AJ001014 Z26317 U58970 AC002400

LRP3 USP13 NR1D2 FAP48 ENIGMA PHLDA1 PRKAR2A RAMP1 DSG2 TOM34 UBPH

19 3 3 1 5 12 3 2 18 20 16

Lipoprotein receptor Ubiquitin specific protease 13 Nuclear hormone receptor Membrane anchoring rrotein Actin-binding signal transducer Plekstrin homologue Regulatory subunit of a protein kinase Transporter for calcitonin receptor Desmoglein 2 (mediates cell adhesion) Mitochondrial translocase Ubiquitin binding protein

Membrane Cytosol Nucleus Membrane Cytosol Membrane Membrane Membrane Membrane Membrane Cytosol

Core −6.1 −3.9 −3.4

AF038897 J00277 Z16411

STX16 HRAS1 PLCB3

20 11 11

Syntaxinl6 Oncogenic GTPase c-H-Ras Phospholipase C beta 3

Membrane Membrane Membrane

Control −4.0 −4.0

AF067724 X95632

NMS5 ABI2

5 2

NDP kinase homologue Modulates Abl signalling pathway

Cytosol Cytosol

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Table 4 Functional grouping: regulators of cell cycle and apoptosis Fold change

Accession number

Gene

Chromosomal location

Product description

Cellular location

Precore −6.5 −5.7 −5.4 −5.1 −4.5 −3.1

AB024401 Z82244 Z11584 X84213 AB017430 AF015451

ING1 MCM5 NuMA1 BAK1 KNSL4 CFLAR

6 22 11 6 16 2

Tumour suppressor DNA replication licensing factor Nuclear mitotic apparatus component Bcl-2 binding protein (pro-apoptotic) Kinesin-like Caspase homologue

Nucleus Nucleus Nucleus Nucleus Nucleus Nucleus

Table 5 Functional grouping: structural and metabolic proteins Fold change

Accession number

Gene

Chromosomal location

Product description

Cellular location

Precore −3.6 −3.1 −3.0 +4.0

M121215 U08021 AF040965 L48215

TPM2 NNMT RES4-25 HBB

9 11 4 11

␤-tropomyosin Nicotinamide-N-methyltransferase Endoplasmic reticulum component Haemoglobin ␤ chain

Cytoskeleton Cytosol Cytosol Cytosol

Control −3.3 +3.2

M24895 D78367

AMY2B KRT12

1 17

␣ amylase 2B Cytokeratin 12

Cytosol Cytoskeleton

Table 6 Functional grouping: cytokines and other secreted proteins Fold change

Accession number

Gene

Chromosomal location

Product description

Cellular location

Precore −5.5 −4.5 −4.0 −3.4

M77140 M30704 M36821 L08044

GAL AREG GR03 TFF3

11 4 4 21

Galarin-neuropeptide 28 kDa Glycoprotein growth factor Autocrine growth factor Trefoil factor

Secreted Secreted Secreted Secreted

Core +3.8

M36821

GRO3

4

Autocrine growth factor

Secreted

Control +3.0

U81234

SCYB6

4

12 kDa Cytokine

Secreted

Fig. 3. Graphic summary of the effects of precore protein expression on the expression of cellular genes in Huh-7 cells. Expression of a large majority of genes belonging to all functional groups is repressed.

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4. Discussion Data presented here provides a new perspective to previous studies that have implicated the HBV precore protein in the pathogenesis of HBV infection (Milich and Liang, 2003). We have generated hepatocyte-derived Huh-7 cells that conditionally express the precore protein in a tightly regulated manner (Fig. 1B and Fig. 2) in order to study the effects of precore protein expression on the expression of cellular genes. Even high levels of HBV precore protein expression failed to affect cell viability or proliferation, suggesting that its actions are not cytotoxic or cytostatic in vivo. However, the effect of the precore protein expression on Huh-7 gene expression cell was dramatic, altering the intracellular concentrations of mRNA transcripts of 45 genes by more than three-fold. A large majority (43/45, ∼96%) of the genes affected were downregulated (see Table 1 as well as Tables 2–6). Those repressed most belonged to one of two broad functional groups, comprising genes associated with the regulation of transcription or nucleoside metabolism and genes associated with cell trafficking or signal transduction, respectively. The third largest functional group included genes involved in regulation of the cell cycle and apoptosis (Fig. 3, Table 1). Most affected (∼11-fold decrease in messenger) was a gene for uridine phosphorylase, an enzyme that catalyses the reversible phosphorolysis of uridine to uracil. Uridine phosphorylase is associated with cell membranes and its activity is high in the mammalian liver, where it plays a vital role in uridine homeostasis by catabolising incoming uridine (Pizzorno et al., 2002). It may also have a regulatory role in fatty acid metabolism, since the major end-product of uridine catabolism in liver is ␤-alanine, an essential precursor for fatty acid synthesis (Zhang et al., 2004) (Fig. 4). It has been proposed that HBeAg, an alternative gene product of the core gene, promotes chronicity in vivo by acting as an immune toleragen (Milich and Liang, 2003). Acute infection with viruses that are defective in HBeAg production is frequently associated with a more severe or even fulminant disease. Similarly, heterogeneous clinical presentation, which is typical of chronic hepatitis B, often involves fluctuations between HBeAg positivity and negativity, the latter being due to HBeAg-negative mutants that predispose to more aggressive disease (Hadziyannis and Vassilopoulos, 2001). The precore G1896A stop codon mutant HBV, which is both replication competent and infectious, often emerges late in the natural course of chronic HBV and is a common cause of HBeAg negativity (Tong et al., 1991, 1992). Previous in vitro studies suggested that the G1896A mutation may be selected because of its ability to enhance HBV replication (Scaglioni et al., 1997a, 1997b) because the precore protein can mediate inhibition of HBV DNA synthesis (Lamberts et al., 1993). Point mutations that abolish expression of the precore gene, enhance the yield of progeny viral DNA (Scaglioni et al., 1997a, 1997b). Non-secreted HBeAg precursor protein (P22), rather than secreted HBeAg, has been shown to be responsible for the inhibition of viral DNA synthesis. It

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appears to act by associating with P21, the capsid protein to form empty hybrid nucleocapsid structures that are devoid of pregenomic RNA (Scaglioni et al., 1997a, 1997b) (see Fig. 5). In transgenic mice, overexpression of the precore protein eliminated nucleocapsid particles from the cytoplasm of the hepatocytes and abolished HBV replication without affecting the hepatic steady-state content of pregenomic RNA (Guidotti et al., 1996). Besides modulating the host’s immune responses to HBV-infected hepatocytes and regulating HBV replication, HBeAg is believed to serve other important functions in the viral replication cycle (see Fig. 5). A recent study from our group (Chen et al., 2003) found that the presence of the precore G1896A stop codon mutation, which prevents HBeAg synthesis, compensated for the in vitro replication deficiency characteristic of lamivudine-resistant HBV mutants, without altering their in vitro sensitivity to lamivudine or other antiviral nucleoside analogues. Following translation, the viral polymerase binds to epsilon (␧), a stem-bulge stem-loop structure at the 5 end of the viral pregenomic RNA (Junker-Niepmann et al., 1990), which triggers the assembly of core protein subunits around the pregenomic RNA to form a nucleocapsid (Bartenschlager and Schaller, 1992). Nucleocapsid formation, which is essential for subsequent reverse transcription (Nassal and Schaller, 1996; Tavis, 1996), depends on the secondary base-paired structure of the lower stem of ␧ (Pollack and Ganem, 1993). In HBV of all genotypes except A and F, the G1896A mutation increases the stability of the lower stem of ␧ by replacing T–G pairs with more stable T–A pairs (Junker-Niepmann et al., 1990). This may increase the efficiency of replication secondary to its effect on packaging. Alternatively, precore mutation may act more directly to increase the catalytic efficiency of HBV polymerase and with it, replication yield. Unfortunately, controlled studies of the processivity and other enzymatic properties of the HBV polymerase have been hampered by technical difficulties (Davis et al., 1996). The demonstration of specific associations with cellular proteins may provide additional clues to the function of HBV preC–C gene products. Recently, Lain´e et al. (2003) found that gC1qR, a mitochondrial matrix protein, interacts both in vitro and in vivo with the precore protein. They used confocal microscopy to show that gC1qR and HBV P22 co-localised in both the cytoplasm and the nucleus, but never in mitochondria (see Fig. 5), indicating that P22 might interact with the mitochondrial targeting sequence of immature gC1qR, blocking its mitochondrial import. Since gC1qR is a multi-ligand protein that also appears to be involved in mRNA splicing, the nuclear location of the P22–gC1qR complex could be interpreted as suggesting that it plays a role in the production of spliced HBV transcripts (Soussan et al., 2000). In this context, it is interesting that a gene for a pre-mRNA splicing factor is one of those repressed by precore protein expression (Table 2). Furthermore, gC1qR can bind calcium and interact with the mitochondrial permeability transition pore (Jiang et al., 1999). This observation, together with the repression of genes listed in Tables 3 and 4, which includes genes for

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Fig. 4. Summary of the precore and core protein processing pathways and their possible inter-relationship inside the hepatocyte. Note cleavage and transport of P25 sub-unit as outlined in Dienes et al. (1995) and the P22 protein (Lain´e et al., 2003).

a ubiquitin-specific protease, a ubiquitin binding protein, a mitochondrial translocase, a transporter for the calcitonin receptor, a pro-apoptiticBc1-2 binding protein and a caspase homologue all point to a role for the P22–gC1qR interaction in regulation of mitochondrially-mediated apoptosis (Lain´e et al., 2003). In contrast to the findings with the precore expression, only five genes (0.03% of mRNAs present on the gene array chip) were altered as a consequence of HBV core protein expression. The most striking effect of HBV core expression

was the down-regulation by 15-fold of DHF-reductase, a key enzyme in folate metabolism and thymidylate synthesis. The other affected genes were at a similar level to that observed in the pTRE2 control cell line. In conclusion, the expression of the HBV precore protein had a much greater effect on gene expression in Huh-7 cells than did expression of the HBV core protein. Precore protein induction resulted in the repression of many genes, the products of which are normally involved in cell-signalling, RNA transport and processing and cytosol-nuclear traffick-

Fig. 5. Precore/Core Protein Cellular Processing.

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ing. In Huh-7 cells, the precore protein seems to be behaving like a tumour suppressor protein with anti-apoptotic activity. By contrast, core protein expression affected few cellular genes. Unravelling the reasons and mechanisms for contrasting effects of HBV core and precore proteins on cellular gene expression and investigation of their consequences will be an interesting and fruitful field for future investigation. Besides increasing our understanding of the repertoire of functions that HBV pre-C–C gene products serve during infection, the results might also lay the groundwork for development of new approaches to treatment of HBV infections.

Acknowledgements The authors wish to thank Dr. Purnima Bhat at The Burnet Institute for Public Health, Prahran, Victoria, Australia for technical assistance and Dr. John Marshall, VIDRL for assistance with the electron microscopy. We also wish to thank the staff of the UTMB Genomics Core Facility for Affymetrix GeneChip analysis. K. Li is the John Mitchell Hemophilia of Georgia Liver Scholar of the American Liver Foundation.

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