Evidence for a bidirectional promoter complex within the X gene of woodchuck hepatitis virus

Virus Research 56 (1998) 25 – 39

Evidence for a bidirectional promoter complex within the X gene of woodchuck hepatitis virus Atsushi Shimoda 1, Fumihiko Sugata 2, Hong-shu Chen 3, Roger H. Miller 4,*, Robert H. Purcell Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA Received 19 October 1997; received in revised form 16 April 1998; accepted 16 April 1998

Abstract The genetic organization of hepadnaviruses is unusual in that all cis-acting regulatory sequences are located within genes. Thus, in the mammalian hepadnavirus genome, the presurface, surface, and X transcript promoters reside within the polymerase gene while the pregenome transcript promoter is located within the X gene. In this study we have identified two additional promoters within the woodchuck hepatitis virus (WHV) X gene that stimulate production of transcripts in vitro. First, we cloned regions of the WHV X gene into a promoterless expression vector (pGL2) to examine their ability to promote expression of firefly luciferase and mapped a previously unidentified promoter to positions 1475–1625 of the WHV8 genome. Deletion analysis revealed that the essential domain of this promoter, termed the ORF5/DX transcript promoter, mapped to nucleotides 1525 – 1625. Analysis revealed that this transcript initiated at nucleotide 1572 in both human (HuH-7) and woodchuck (WLC-3) hepatoma cell lines. Consistent with this finding, DNA footprinting analysis revealed protection of nucleotides 1567 – 1578 on the positive strand of the WHV8 genome. The function of this transcript in vivo is unclear, however, it may be used to produce a truncated form of the X protein that initiates at an AUG codon at position 1743 – 1745 on the WHV8 genome. Next, a second promoter was identified at positions 1625 – 1975 that was responsible for production of an antisense transcript. The activity of this promoter was comparable to that of the previously characterized surface transcript promoter of WHV in the absence of an enhancer. The antisense transcript promoter resides immediately upstream of open reading frame (ORF) 6, a previously identified ORF on the strand opposite of the known WHV protein-encoding sequences, that is thought to represent a vestigial gene. Analysis indicates that the antisense transcript had

* Corresponding author. Tel.: +1 301 4966430; fax: +1 301 4023211. 1 First Department of Internal Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa, 920, Japan. 2 Institute of Medical Science, St. Marianna University, Kawasaki, Japan. 3 Tzu-Chi College of Medicine, Hualien, Taiwan. 4 Targeted Interventions Branch, Basic Sciences Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. 0168-1702/98/$19.00 © 1998 Published by Elsevier Science B.V. PII S0168-1702(98)00050-1

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multiple start sites: nucleotides 1683 and 1762 on the WHV8 genome when assayed in HuH-7 cells, and nucleotide 1786 when assayed in WLC-3 cells. These data are consistent with footprinting analysis of supercoiled WHV DNA that revealed that the regions encompassing nucleotides 1696 – 1685, 1781 – 1766, and 1801 – 1787 on the negative sense DNA strand were protected from nuclease degradation. It is possible that such a transcript was once used in protein expression in an ancestral virus and may now be used for genetic control of WHV replication and/or gene expression. Overall, these data are consistent with the presence of a bidirectional promoter complex within the WHV X gene. © 1998 Published by Elsevier Science B.V. Keywords: Woodchuck hepatitis virus; Promoter; Luciferase; Gene expression; X gene

1. Introduction In the mammalian hepadnavirus genome (Blumberg et al., 1965; Summers et al., 1978; Marion et al., 1980), four long open reading frames (ORFs) have been found that encode the virus core (C), surface (S) and polymerase (P) proteins as well as a protein termed X which appears to play an important role in virus replication and/or gene expression (Chen et al., 1993; Zoulim et al., 1994; Yen, 1996). Since every region of the virus genome is a protein-encoding domain all cis-acting signal sequences reside within genes (Fig. 1). The hepatitis B virus (HBV) genome contains at least four promoters that are responsible for production of the preS1 (Courtois et al., 1988; Nakao et al., 1989), preS2/S (Shaul et al., 1986), pregenome, or C (Yaginuma and Koike, 1989; Lopez-Cabrera et al., 1990), and X transcripts (Treinin and Laub, 1987; Nakamura and Koike, 1992). All of the transcripts share an identical 3% end since they utilize a common poly (A) addition signal sequence located within the C gene. Other cis-acting regulatory sequences present are enhancers (ENH) I (Vannice and Levinson, 1988) and II (Yee, 1989) and a silencing element (Yee, 1989; Gerlach and Schloemer, 1992; Su and Yee, 1992). The HBV pregenome transcript promoter has been localized to the X gene and shown to be more active in liver cells than in non-liver cells (Honigwachs et al., 1989). Multiple mRNA start sites for the pregenome mRNA have been reported (Yaginuma and Koike, 1989). Recently, ENH II has been found to overlap the promoter. This enhancer was shown to consist of two interacting elements: the a- and b-boxes (Yuh and

Ting, 1993). A silencing element, defined as a cis-acting repressing element that blocks or diminishes transcription, has been identified upstream of the promoter, and negatively regulates its activity (Yee, 1989; Gerlach and Schloemer, 1992; Su

Fig. 1. Genome structure of WHV8. The unique EcoRI recognition site is designated position 0 and 3323. The presurface (PRE-S), surface, precore (PC), core, polymerase, and X genes are shown. Cis-acting regulatory elements highlighted are enhancer I (ENH I), enhancer II (ENH II), direct repeats 1 and 2 (DR1 and DR2), the RNA packaging signal (PKG) and the poly(A) addition signal (POLY-A). ORF5 and ORF6 are shown. The black circle represents the 5% binding protein involved in initiation of first strand DNA synthesis. Modified from Chen et al. (1993).

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and Yee, 1992). It is unclear whether ENH II exerts any effect on the activity of the pregenome transcript promoter. All hepadnavirus genomes examined contain two additional ORFs, designated ORF5 and ORF6, that may have encoded proteins in an ancestral virus (Miller, 1990; Chen et al., 1993) (Fig. 1). ORF5 is located on the same strand as the four known viral genes and is 70 – 100 codons in size. ORF6 is located on the opposite strand, and is approximately 210 codons in length. ORFs 5 and 6 lack an ATG initiation codon in most hepadnaviruses and are unlikely to encode proteins (Chen et al., 1993). However, several potential poly(A) addition signal sequences are found near the end of ORF6 suggesting that a transcript may be produced. While the ancestral virus may have expressed a protein from ORF5 using any of the four known viral transcripts, it is possible that a unique transcript was utilized. On the other hand, expression of a protein from ORF6 would require production of a novel antisense mRNA. Therefore, it is possible that the ancestor of the hepadnaviruses employed a bidirectional promoter in gene expression. In this study we examined the WHV X gene for evidence of such a promoter complex.

2. Materials and methods

2.1. Construction of plasmids Regions of the infectious HBV (ayw) and WHV8 genomes (Girones et al., 1989) were amplified by the polymerase chain reaction (PCR) as previously described (Kaneko and Miller, 1990) using oligonucleotide primers with recognition sites for restriction endonuclease XhoI or BglII. The PCR-generated WHV DNA fragments were digested with restriction endonucleases XhoI and BglII and cloned into the XhoI and BglII sites respectively of promoterless vector pGL2-Basic (Promega, Madison, WI) for expression of firefly luciferase (De Wet et al., 1987). Recombinants were designated according to the map position of the insert using the numbering system of the WHV8 genome. Thus, plasmid p(1350 – 1650)GL2

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contains a WHV insert spanning nucleotides 1350–1650 of the WHV8 genome (Girones et al., 1989). In addition, 12 recombinants were constructed using vector pGL2-Basic that contained inserts spanning the complete WHV8 genome. The overlapping inserts, overlapping by 25 nts on both ends, encompassed the following nucleotides: 12–275, 250–550, 525–825, 800–1100, 1075–1375, 1350–1650, 1625–1975, 1950–2250, 2225–2535, 2510–2810, 2785–3085, and 3060– 37. All WHV inserts were sequenced (Sanger et al., 1977) with Sequenase, a T7 DNA polymerase (United States Biochemical, Cleveland, OH), to verify that no changes occurred during the amplification and cloning processes. Two clones per construct were assayed for promoter activity to avoid selecting an inactive reporter gene. Within a given pair, the construct that produced the most luciferase was selected for further analysis. It should be noted that our WHV constructs were made without an enhancer element present.

2.2. Cells Two continuous cell lines were used for assaying promoter activity. The HuH-7 cell line was derived from a well-differentiated hepatocellular carcinoma of human origin and was negative for HBV surface antigen (Nakabayashi et al., 1982). The WLC-3 cell line was derived from woodchuck hepatocytes treated with diethylnitrosamine in vivo (Lee et al., 1987). Neither cell line possesses integrated hepadnaviral DNA sequences. The cells were cultivated in Dulbecco’s modified Eagle medium (DMEM) (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Bio Whittaker, Walkersville, MD) that was inactivated for 30 min at 56°C.

2.3. Transfection Plasmid DNA for transfection of cells was purified using an anion-exchange resin (Qiagen, Germany). Cells were seeded at 1.5×105 cells per well in 12-well tissue culture plates (Costar, Cambridge, MA) and were incubated at 37°C in 5% CO2 for 15–18 h. Transfections were performed in triplicate by the method of Chen and Okayama

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(1987) using BES [N,N-bis(2-hydroxyethyl)-2aminoethane-sulfonic acid] (Aldrich, Milwaukee, WI) when the cell monolayer was 60 – 70% confluent. A mixture of plasmid DNAs containing 3.5 mg of a specific WHV-pGL2 construct and 0.1 mg of plasmid pSVTKGH, a plasmid that expresses human growth hormone (HGH) (Selden et al., 1986), which was used as a control for transfection efficiency, was mixed with 0.15 ml of 0.25 M CaCl2. Then 0.15 ml of 2X BES-buffered saline [50 mM BES (pH 6.96 with NaOH), 280 mM NaCl, and 1.5 mM Na2HPO4] was added and the mixture was incubated for 10 – 20 min at room temperature (RT). The BES-DNA mixture (0.3 ml) was added dropwise to the cells, which were then overlaid with 3 ml of fresh DMEM. The cells were incubated for 15 – 18 h at 37°C in 2% CO2. Next, the cells were washed twice with fresh media, 3 ml of complete DMEM was added, and the cells were incubated for 45 – 48 h at 37°C in 5% CO2. Plasmid pGL2-Basic served as a negative control and plasmids pGL2-Control or pGL2Promoter served as positive controls (Promega, Madison, WI). pGL2-Control contains the SV40 early promoter and enhancer while plasmid pGL2-Promoter contains only the SV40 early promoter.

2.4. Luciferase assay Transfected cells were rinsed twice with 3 ml of PBS and lysed directly with 0.1 ml of lysis buffer [25 mM Tris–phosphate, pH 7.8, 2 mM dithiothreitol (DTT), 2 mM 1,2-diaminocyclohexaneN,N,N%,N%-tetraacetic acid, 10% glycerol, 1% Triton X-100] for 10 – 15 min at RT. A total of 20 ml of cell extract at RT was mixed with 100 ml of luciferase assay reagent at RT [20 mM Tricine, pH 7.8, 1.07 mM (MgCO3)4Mg(OH)2 · 5H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT, 270 mM coenzyme A, 470 mM luciferin, 530 mM ATP] (Promega). The production of light by luciferase was immediately monitored for 10 s (Wood et al., 1984; Wood and DeLuca, 1987) in a luminometer (Model 20e, Turner Designs, Sunnyvale, CA, Jago et al., 1989). Extracts from untransfected cells were used as blanks in the assay. Light units were normalized to the level of HGH, an

internal control protein secreted into the media (described below). The mean and standard deviation (S.D.) of corrected activities were calculated for the results of the assay from three wells. Experiments were repeated at least twice, unless otherwise noted, with positive and negative controls present. All luciferase assays were performed within the linear response range of the assay.

2.5. HGH assay The growth media from the cells was tested for the presence of HGH by immunoassay (AllUgro HGH, Nichols Institute, Los Angeles, CA). The level of HGH, always measured within the linear response range of the assay, was used as a determination of transfection efficiency.

2.6. 5 % rapid amplification of cDNA ends (RACE) The 5% ends of the mRNAs were determined by the method of Frohman et al. (1988). Total RNA was extracted using guanidium thiocyanate–phenol–chloroform (Chomczyuski and Sacchi, 1987) 3 days after transfection of HuH-7 or WLC-3 cells with 10 mg WHV-pGL2 plasmid DNA in a 6 cm dish. Next, poly(A) containing RNA was purified using Oligotex (Qiagen, Germany). Firststrand cDNA synthesis was performed by incubating 0.5 mg of each RNA sample in a total reaction volume of 30 ml containing 10 mM Tris– HCl pH 8.3, 50 mM KCl, 8 mM MgCl2, 1 mM DTT, 260 mM of each dNTP, 10 pmol of an arbitrary hexamer primer (Gibco BRL), 50 U RNasin, and 10 U AMV reverse transcriptase (Promega) at 52°C for 30 min. RNA hydrolysis was achieved by adding 2 ml of 6 N NaOH and incubating at 65°C for 30 min. The solution was neutralized by addition of 2 ml of 6 N acetic acid. cDNA was purified using Geno-Bind (Clontech, Palo Alto, CA), alcohol precipitated, pelleted, and resuspended in 6 ml of DEPC-treated water. Anchor-cDNA ligation reactions (Tessier et al., 1986) were performed at 22°C with 3 ml of cDNA and 4 pmol of the phosphorylated, blocked anchor which was 5%P-CACGAATTCACCGATTCTGGAACCTTCAGAGG-NH3 3% (Clontech). The reaction volume was 10 ml containing 50 mM

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Tris –HCl, pH 8.0, 10 mM MgCl2, 10 mg/ml bovine serum albumin, 25% w/v polyethylene glycol 8000, 1 mM hexamine cobalt chloride, 20 mm ATP and 10 U T4 RNA ligase (Clontech). Ligations were terminated after 20 h and diluted 1:10 for PCR amplification. PCR was performed using 2 ml of the diluted ligation reactions in a 50 ml volume containing 10 mM Tris – HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl2, 200 mM of each dNTP, 10 pmol of each specific primer, 0.4 ml TaqStart antibody (Clontech), and 2 U Taq polymerase (Perkin Elmer Cetus, Norwalk, CT). The oligonucleotide primer specific for the anchor was 5ANCH1 (5%-CCTCTGAAGGTTCCAGAATCGATAG-3%) and the cDNA-specific primer used in the WHV mutant-pGL2 construct was GL2196R (5%-AATTGTTCCAGGAACCAGGGCGTAT-3%). The reactions were performed for 35 cycles using a programmable DNA Thermal Cycler (Perkin Elmer Cetus) as follows: 45 s at 94°C, 45 s at 67°C, and 2 min at 72°C. The nested PCR was done using another set of primers: 5ANCH2 (5%-TGAAGGTTCCAGAAGATAGTGAATT-3%) and GL2-166R (5%-TCATAGCCTTATGCAGTTGCTCTCC-3%). The PCR product to be ligated was purified using a QIAquick PCR-purification kit (Qiagen, Germany) as previously described. 4 ng of insert and 50 ng of pGEM-T (Promega) were ligated with T4 DNA Ligase for 3 h at 16°C. After transformation of JM109 high efficiency competent cells (Promega), colonies were selected on isopropyl-b-D-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-Gal) plates. White colonies were placed into 7 ml of GeneAmp DNA amplification reagent (Perkin Elmer Cetus) using T7 and SP6 promoter sequencing primers for PCR screening (1 min at 94°C, 1 min at 50°C, and 2 min at 72°C for 25 cycles) (Gu¨ssow and Clackson, 1989). At least three clones containing appropriate inserts were sequenced and determined to have identical 5% ends.

2.7. Nuclear extracts Both HuH-7 and WLC-3 cells were harvested and 1×108 cells were used to prepare nuclear extracts according to the method of Dignam et al.

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(1983) with the following modifications. Cells were washed twice with cold washing buffer (10 mM Tris–HCl, pH 7.5, 130 mM NaCl, 5 mM KCl, 8 mM MgCl2), and homogenized using a Dounce homogenizer with a loose fitting (type B) pestle in hypotonic buffer [20 mM N-2hydroxyethylpiperazine-N%-2-ethanesulfonic acid (HEPES), pH 7.9, 5 mM KCl, 0.5 mM MgCl2, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1% NP-40] on ice (Pitluk and Ward, 1991). Nuclei were collected by centrifugation at 1000× g for 5 min at 4°C. All pelleted nuclei were suspended with extraction buffer (20 mM HEPES, pH 7.9, 0.5 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 25% glycerol, 0.5 mM PMSF, 10 mg/ml pepstatin A, 1.28 (g/ml spermidine), and stirred on ice for 1 h. The suspension was centrifuged at 280000×g for 90 min at 4°C.

2.8. DNase I footprinting A total of 20 fmol of supercoiled WHV-pGL2 plasmid DNA was incubated with 40 mg nuclear proteins extracted from HuH-7 or WLC-3 cells in a 40 ml reaction mixture containing 20 ml of 2X FP buffer (10 mM HEPES, pH 7.6, 50 mM KCl, 0.2 mM EDTA, 2 mM DTT, 12 mM MgCl2, 20% glycerol), and 5 mg of poly(dI-dC)(poly (dI-dC) (Pharmacia Biotech, Piscataway, NJ) as described (Gralla, 1985). After 30 min of incubation at 22°C, 2 ml of 50 mM CaCl2 was added and the mixture was treated with freshly prepared DNase I (Promega, Madison, WI) for 1 min at 22°C. The amounts of DNase I required for our study were 0.02 U in the negative control and 0.1 U in the reaction containing nuclear proteins. Digestion was stopped with 40 ml of stop solution [10 mM HEPES pH 7.6, 20 mM EDTA, 1% sodium dodecyl sulfate (SDS)], and DNA was purified through QIAquick as previously described. Samples were resuspended in 50 ml of PCR mixture [10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 (M each dNTP, 2.5 U of Taq DNA polymerase (Perkin Elmer Cetus), and 0.5 pmol of a 32 P-labeled primer]. Primers were labeled with [g32 P]ATP (6000 Ci/mmol) (Amersham, Arlington Heights, IL) to a specific activity of 1× 106 cpm/ pmol by T4 polynucleotide kinase (Gibco BRL) in

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a forward labeling reaction. Ten cycles of PCR amplification (1 min at 95°C, 1 min at 55°C, and 2 min at 72°C) were performed and unincorporated nucleotide was removed by QIAquick purification of DNA. The samples were electrophoresed in 6% acrylamide denaturing sequencing gels. A sequence marker was obtained by sequencing the original template with the same oligonucleotide used for direct incorporation of [a-35S]dATP (1000 Ci/mmol, Amersham). Sequencing was performed with Exo-Pfu polymerase as recommended by the manufacturer (Stratagene, Menasha, WI).

deletion of 50 nts from the 5% end of the region resulted in a slight decrease in promoter activity in both cell lines studied, deletion of an additional 50 nts from the fragment spanning nts 1525–1625 resulted in a seven-fold decrease in promoter activity in WLC-3 cells and nearly a two-fold decrease in HuH-7 cells. While regions spanning nts 1475–1525 and 1525–1575 exhibited weak promoter activity in HuH-7 cells their activity in WLC-3 cells was limited. Overall, deletion mapping of the promoter suggests that this element resided within the region spanning nts 1475–1625, with the crucial domain localized between nts 1525–1625.

3. Results

3.2. Analysis of antisense transcript promoter acti6ity in the WHV X gene

3.1. Identification of a pre6iously undescribed promoter within the WHV X gene The purpose of our investigation was to determine whether the X gene region of the WHV genome possessed promoters upstream of the pregenome transcript promoter. In preliminary experiments using the chloramphenicol acetyltransferase (CAT) assay (Sugata et al., 1994) we mapped a previously undescribed promoter, termed the ORF5/DX promoter, to a region spanning nucleotides (nts) 1508 – 1651 of the WHV genome as assayed in both WLC-3 (woodchuck) and HuH-7 (human) hepatoma cell lines (unpublished data). However, in order to study the activity of this relatively weak promoter in the absence of homologous or heterologous enhancers we used the more sensitive luciferase reporter system. Thus, we cloned the region spanning nts 1475 – 1625 into vector pGL2-Basic in both polarities as described in Section 2. One advantage of using this vector is that a poly(A) addition site has been placed upstream of the cloning site, which serves to limit background transcription from spurious prokaryotic promoters in the vector sequence (Langner et al., 1986; Heard et al., 1987). Analysis of the production of luciferase by sense transcription from this promoter revealed comparable values in both WLC-3 and HuH-7 cell lines (Fig. 2). Deletion of 50 nts from the 3% end of this region reduced luciferase activity by three-fold. While

A second aspect to this study was the search for the presence of a promoter capable of producing a transcript of opposite polarity from the previously characterized transcripts of WHV. In the previous experiment the antisense insert spanning nts 1475–1625 exhibited promoter activity in HuH-7 and WLC-3 cells (Fig. 2). Therefore, we constructed a number of recombinants with inserts from this general region of the WHV genome. Analysis indicated that the region spanning nts 1625–1825 exhibited antisense transcript promoter activity in both cell lines and the activity was stronger in HuH-7 than in WLC-3 cells (unpublished data). However, to place this finding in a broader context we examined the entire WHV plus genomic DNA strand for promoter activity. We cloned 12 slightly overlapping fragments that spanned the virus genome into the luciferase reporter vector in both orientations (see Section 2). In order to compare accurately the potential promoter activity from the various regions we measured luciferase in a single, comprehensive experiment. We found that two regions of the WHV8 genome possessed significant promoter activity in both WLC-3 and HuH-7 cells. The region spanning nts 1625–1975 had the greatest activity: 962942 light units in WLC-3 cells and 1542 9 54 light units in HuH-7 cells. The other region of the WHV8 genome that yielded an antisense transcript spanned nts 3060–37 of the circular genome

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Fig. 2. Mapping the WHV ORF5/DX transcript promoter. Progressive deletions were made at both ends of the region of the WHV8 genome spanning map positions 1475–1625, which were known to contain the promoter. Luciferase assays were performed in HuH-7 and WLC-3 cells as described in Section 2. Results are expressed in light units (mean 9S.D.). pGL2-Basic served as the negative control and pGL2-Control as the positive control in all experiments. The activity of the positive control was very strong due to the presence of the SV40 early promoter and enhancer. It should be noted that for this and subsequent experiments, the values represent data collected in triplicate for each recombinant, and all constructs in the figure were assayed in the same experiment to permit comparison. The experiment was repeated and yielded comparable results.

and possessed three-fold less activity in both cell lines: 288941 light units in WLC-3 cells and 4519 144 light units in HuH-7 cells. It was noteworthy that the strength of the ORF6 transcript promoter compared favorably with that of the S transcript promoter (i.e. 21109 248 light units in WLC-3 cells and 14389169 light units in HuH-7 cells) in the absence of homologous or heterologous enhancers.

3.3. 5 % end mapping of mRNA The determination of specific transcriptional initiation sites is important for verification of an authentic transcript. One technique that is in standard use for 5% end mapping is primer extension.

However, this technique is unable to identify the presence of a capped G residue at the 5% end of mRNA molecules. Furthermore, the amount of mRNA produced by weak promoters is not always sufficient for analysis by primer extension. To circumvent both problems we used the anchored 5% RACE method (Tessier et al., 1986; Edwards et al., 1991) to map the 5% ends of the major transcripts produced by the ORF5/DX and ORF6 promoters. After transfection of cells with the p(1475–1625)GL2 chimera we ascertained the 5% end of the mRNA (Fig. 3(A)). Analysis revealed that the anchor sequence used in the 5% RACE amplification adjoined the WHV plus strand at nt 1572 in RNA obtained from both HuH-7 and WLC-3 cells. This result is in excellent

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Fig. 3. Determination of the 5% ends of transcripts using a modified 5% RACE procedure. The autoradiograph shows an anchor sequence connected to the 5% end of the sense and antisense transcripts. (A) The sense RNA transcript starts at nt 1572 of the WHV plus strand. (B) The antisense transcript starts at nt 1683 of the WHV minus strand as assayed in HuH-7 cells. (C) Summary of results showing the span of promoters (closed boxes) and 5% ends of transcripts (arrows).

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agreement with the results of the luciferase assay defining the ORF5/DX promoter to nts 1525 – 1625 of the WHV genome (Fig. 3(C)). Similar analysis was used to map the 5% end of the ORF6 transcript. Several initiation sites were found: nt 1683 in HuH-7 (Fig. 3(B)), nt 1762 in HuH-7, and nt 1786 in WLC-3 cells. The presence of multiple initiation sites could be due to the fact that this promoter is no longer under selective pressure to produce a defined transcript and the specificity of the promoter is decreasing. However, it is clear that all of the initiation sites were located within the region spanning nts 1625 – 1975, which corresponds to the domain identified in the luciferase reporter assay as possessing antisense promoter activity (Fig. 3(C)).

3.4. Identification of the protein binding sites of the promoter elements DNase I footprinting has been widely used to identify DNA sequences that can be recognized by regulatory factors. In our study, protein binding to DNA was examined on supercoiled templates that were then digested with DNase I as described in Materials and Methods. This approach was taken due to the fact that analysis using end-labeled template DNA was unsatisfactory, most likely due to the high G +C content (64%) of the region under study. Specific cleavage was found by primer extension on DNase I-digested DNA using a 32P-labeled specific primer. The DNA-protein binding region was found to reside between nts 1567 – 1578 of the plus (sense) strand of WHV genome by using supercoiled DNA from construct p(1350 – 1650)GL2 (Fig. 4(A)). This demonstrates that the target site for binding was located upstream of the initiation site of mRNA. This result is consistent with the previous finding of the initiation site of nt 1572 (Fig. 3(A)). Upstream of this site, other regions protected against DNase I were identified (i.e. nts 1545–1558). We also found protected regions downstream of nt 1572 (i.e. nts 1582 – 1596, nts 1596–1606, and nts 1606 – 1615). It is possible that these regions are involved in production of minor RNA transcripts. Protein binding sites on the WHV8 genome were also mapped on the opposite DNA strand. The

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DNA-protein binding regions were localized to nts 1696–1685, 1781–1766, and 1801–1787, consistent with our previous finding of the 5% ends of the antisense RNAs at nts 1683, 1762, and 1786, respectively (Fig. 4(B)). Taken together, these data are consistent with the presence of a bi-directional promoter complex within the X gene of WHV.

4. Discussion The goal of this investigation was to examine the WHV X gene for evidence of additional promoter elements. Although our preliminary studies were performed using the CAT gene as a reporter gene, we changed to the firefly luciferase gene because of the increased sensitivity and linearity of the luciferase assay. This was important since we performed the promoter analysis in the absence of homologous or heterologous enhancers that could have served to complicate the study. The distinction between enhancers and promoters appears to be blurred, with enhancers viewed in some cases as elements containing promoters that are grouped closely together with the ability to function at increased distances from the transcription initiation point. In our experiments all constructs were sequenced to insure that mutations did not occur during the subcloning process, and were tested in triplicate with values normalized to the levels of secreted HGH, which served as an internal control. Both the luciferase and HGH assays were performed within the linear response range of the given assay. WHV promoter levels, although low due to the absence of enhancers, compared favorably to the well-studied HBsAg promoter examined in parallel experiments (data not shown). A new promoter, termed the ORF5/DX promoter, was found to reside between nts 1475–1625, with the main element spanning nts 1525–1625 of the WHV8 genome. The 5% end of the transcript mapped to nt 1572 near the center of the predicted promoter domain. Furthermore, an experiment using DNA footprinting to reveal the binding profile of nuclear proteins to the promoter region revealed that nts 1567–1578 were protected from nuclease digestion. This finding is in excellent agreement with the mapping of the 5% end of the

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Fig. 4. DNA footprinting analysis. The binding of proteins to DNA was analyzed on supercoiled templates that were then digested with DNase I. Specific cleavage was found by primer extension on DNase I-digested DNA using a 32P-labeled specific primer. (A) A DNA-protein binding region was found between nts 1567 – 1578 of the plus (sense) strand of WHV genome using supercoiled p(1350 – 1650)GL2. This demonstrates that the target site for binding was located upstream of the initiation site of mRNA corresponding to the initiation site at nt 1572. (B) DNA-protein binding sites at nts 1696 – 1685, 1781 – 1766, and 1801 – 1787 corresponded to the 5% RNA ends (nts 1683, 1762, and 1786, respectively) on the antisense strand of the WHV genome. The binding sites on the WHV genome were shown to be virtually the same in both HuH-7 (H lane) and WLC-3 (W lane) nuclear extracts. Both nuclear protein extracts exhibited strong binding to the basal core promoter region of WHV (data not shown).

transcript to nt 1572. Taken together, the data from these three experiments are consistent with the hypothesis that an authentic promoter resides between nts 1525 – 1625 within the WHV X gene.

The function of the transcript produced by the ORF5/DX promoter is open to speculation. One possibility is that the transcript is utilized to produce a truncated form of the X protein (DX) initiating at a second AUG codon known to be

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present in all mammalian hepadnavirus X sequences. In support of this hypothesis, a truncated form of the HBV X protein has been identified in patient sera and corresponds to an X protein lacking N-terminal sequences (Feitelson and Clayton, 1990). Another possibility is that the transcript is involved in production of a protein from ORF5 that initiates at a codon other than AUG (Miller, 1990). If so, the protein would probably be present in very low amounts in vivo since our earlier studies did not detect antibody directed against such a protein in the sera of acutely or chronically infected woodchucks (Chen et al., 1993). It is also possible that the ORF5/DX promoter, once responsible for production of a transcript in the ancestral virus, is under negative control and is no longer utilized in vivo in the virus replication cycle. Important future experiments are to determine whether a truncated version of the X protein can be produced from this transcript in vitro and to determine whether a transcript is produced from the ORF5/DX promoter in the liver cells of woodchucks infected with WHV. Identification of an ORF, termed ORF6, on the DNA strand opposite the one that encodes the known hepadnavirus proteins previously led us to search for an antisense transcript (Kaneko and Miller, 1988). We were unsuccessful in detecting an antisense transcript with a strand-specific probe in total RNA, poly(A)-selected RNA, or nuclear RNA from 21 woodchucks with either an acute or chronic WHV infection (Kaneko and Miller, 1988). It was not possible to express a protein from ORF6 using in vitro translation of a synthetic transcript. Also, there were no antibodies in the serum of acutely or chronically infected animals specifically directed against the synthetic peptide CHTGSNSMIQRH representing the carboxyl terminus of the predicted ORF6 protein (Chen et al., 1993). However, analogous HBV transcripts have been reported (Standring et al., 1983; Zelent et al., 1987). Thus, this study was to determine whether a promoter existed on the WHV genome that could produce an antisense transcript in vitro. Our analysis, using the highly sensitive luciferase reporter gene system, revealed the presence of an antisense transcript

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promoter, termed the ORF6 promoter, within the X gene of the WHV genome between nts 1625– 1975, immediately adjacent to the newly identified ORF5/DX transcript promoter located between nts 1525–1625. A second region of the WHV8 genome, spanning nts 3060–37, possessed antisense promoter activity in both cell lines examined, but the activity was three-fold less than that of the ORF6 transcript promoter. Analysis of the 5% end of the ORF6 transcript revealed the presence of three such ends at nts 1683 and 1762 in HuH-7 cells and at nt 1786 in WLC-3 cells. This finding is in contrast to the 5% end mapping experiment for the ORF5/DX transcript where the 5% terminus was localized to a single nt (i.e. nt 1572) on the WHV8 genome as assayed in both WLC-3 and HuH-7 cells. The finding of multiple 5% ends for the antisense transcript could be due to a lack of selective pressure to maintain a discrete start site for the transcript. Analysis of the binding of nuclear proteins using DNA footprinting methodology revealed that nts 1696–1685, 1781–1766, and 1801–1787 were protected from DNase degradation, which is in complete agreement with the genomic location of the 5% ends of the antisense transcripts. Since the 5% ends of these transcripts are close to the region that encodes the N-terminus of the predicted ORF6 protein, which begins at nt 1719, it is possible that this antisense promoter was once used in expression of an authentic gene product in the virus that was the ancestor of the hepadnaviruses (Chen et al., 1993). In support of our findings, Velhagen et al. (1995) have recently identified an antisense promoter within the X gene of HBV. Using a comparable luciferase assay, these investigators have mapped the promoter to a 241 bp Sty I fragment corresponding to a domain at the 3% end of the X gene, a location coinciding to that of the antisense promoter we have identified within the WHV genome. Furthermore, we also have independently mapped an antisense promoter to this location within the HBV genome (AS, RHM, RHP, unpublished data). Thus, both the WHV and HBV genomes appear to possess a promoter within the X gene that is capable of producing an antisense transcript in vitro.

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While it is unlikely that ORF6 currently encodes a protein, it is possible that the antisense transcript we characterized in vitro plays a role in virus replication or gene regulation. The role of antisense RNA in the regulation of gene expression in prokaryotes and eukaryotes has been well studied (Inonye, 1988; Simons, 1988; Simons and Kleckner, 1988; Vannice and Levinson, 1988; Eguchi et al., 1991). Furthermore, intriguing findings are emerging on the role of antisense transcripts in virus replication and gene expression. One example is the latency-associated transcript of herpes simplex virus known to be complementary to the immediate-early gene encoding infected cell protein 0 (Farrell et al., 1991). This protein is a potent transactivator of gene expression in transient assays, suggesting an important role in the virus replication cycle. Thus, the latency-associated transcript may be involved in the regulation of expression of this virus protein. In addition, antisense transcripts have been identified in cells or tissues infected by papillomaviruses (Vormwald-Dogan et al., 1992), Epstein Barr virus (Smith et al., 1993), and the human immunodeficiency virus (HIV) (Michael et al., 1994). Regarding the latter, HIV has been shown to possess an ORF on the genomic plus strand that exactly correlates with the position of the antisense transcript (Miller, 1988). Furthermore, there is evidence that a protein is expressed from this ORF during virus replication (Vanhee´Brossollet et al., 1995). Thus, antisense transcripts appear to play a role in some aspects of cellular and viral gene expression and replication. Due to the unique organization of the hepadnavirus genome, an antisense transcript produced from the ORF6 promoter would be complementary to all of the previously characterized viral transcripts since the latter share a common poly(A) addition site located within the C gene. Therefore, an antisense transcript may be used to regulate protein translation from particular transcripts at discrete time points in the hepadnavirus replication cycle. In this regard, the X transcript and the transcript produced from the ORF5/DX promoter would have the least overlap with the antisense transcript, which could allow more X, or more of the putative truncated form of the X protein, to be synthesized as needed.

In summary, we have provided evidence in vitro for a bidirectional promoter complex in the WHV genome. Bidirectional promoter complexes, while not common, have been found in a wide range of cellular genomes. For example, bidirectional promoters have been found associated with the c-Haras gene of humans (Lowndes et al., 1989), the a-actin gene of chickens (Grichnik et al., 1988), the Xenopus laevis mitrochondrial genome (Bogenhagen and Romanelli, 1988), and several genes in the genomes of rats (Huh et al., 1991) and mice (Efrat and Hanahan, 1987; Burbelo et al., 1988; Doyen et al., 1989; Linton et al., 1989; Lennard and Fried, 1991). Furthermore, such promoters have been found in retroposons (Friesen et al., 1986) and some viruses (Laux et al., 1989; Chen and Velicer, 1991). One unusual feature of the bidirectional promoter of WHV is that it is in the middle of a gene (i.e. the X gene) whose product is crucial for virus replication in vivo (Yen, 1996). The mechanism by which this genetic arrangement evolved is unclear. Future work should help to unravel the complexities of the organization of the hepadnavirus genome and may provide insight into the organization of the ancestral virus. This, in turn, could provide a clearer picture of the evolution of genetic systems in general.

Acknowledgements We thank T. Kitagawa for providing WLC-3 cells, T. Tsareva for assistance in oligonucleotide synthesis and purification, S. Murakami and M.-S. Yu for helpful suggestions, and T. Heishman and C. Hutton for editorial assistance.

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