INTRODUCTION

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Hepatitis B viruses (HBVs; hepadnaviruses) are small, enveloped DNA viruses that produce persistent hepatic infections and are strongly associated with the development of hepatocellular carcinoma (5). The viral genome is a partially duplex, relaxed circular species of approximately 3 kb that replicates within cells via reverse transcription. Upon entry into cells, the viral genome is converted into covalently closed circular DNA that serves as the template for the synthesis of subgenomic RNA and pregenomic RNA (pgRNA) by host RNA polymerase (19). Studies on transcription of human HBV have shown that this step is controlled by regulatory elements that include four promoters and two enhancers (1, 11, 13, 15, 16, 24, 26, 27). The preS, S, and X promoters drive the transcription of subgenomic RNAs encoding the envelope and X proteins. The C promoter controls the production of the pgRNA that encodes the core protein and polymerase and that also serves as the template for viral reverse transcription.

Two enhancers, enhancer I (EnI) and EnII, located upstream of the HBV X and C promoters, respectively, have been shown to be capable of upregulating homologous and heterologous promoters in transient transfection (15, 21, 29). EnI has been proposed to be widely involved in the regulation of HBV transcription (18). However, despite the shared genomic organization and replication cycle of mammalian hepadnaviruses, EnI activity has not been found in other viruses of the family (2, 22). EnII is known to enhance the C promoter in transient assays, suggesting that it might regulate the transcription of pgRNA (18, 28). The expression of pgRNA is highly liver specific, a fact that correlates well with the known liver specificity of EnII activity (28). Thus, it is attractive to speculate that EnII, together with the C promoter, may account for the liver-specific expression of pgRNA that determines (at least in part) the hepatotropism of hepadnaviruses (6, 14, 28).

Woodchuck hepatitis virus (WHV) is a mammalian hepadnavirus that is closely related to HBV and is strikingly oncogenic in its natural host (12, 20). Analyses of WHV-induced woodchuck hepatocellular carcinomas have demonstrated that activation of the proto-oncogene N-myc2 by viral DNA insertion is commonly involved in carcinogenesis (4, 7, 25). Our studies of viral DNA sequences in N-myc2 activation suggested that the region corresponding to HBV EnII played a major role in the activation (22). Accordingly, we and others have mapped and characterized EnII in WHV (3, 23). The activity of WHV EnII maps to an 88-nucleotide DNA fragment (nucleotides 1772 to 1859) upstream from the transcription initiation site of pgRNA. Biochemical and genetic studies have identified three host transcription factors that recognize elements within this region: the liver-enriched factors HNF-1 and HNF-4 and the ubiquitous factor Oct-1 (23). Deletion analyses suggested that HNF-1 and HNF-4 are the main contributors to the EnII activity in transient assays and together account for its strong liver specificity.

Most analyses of HBV and WHV enhancer activity have been carried out by using transient assays with cell lines transfected with artificial reporter constructs. The role(s) of the host transcription factors that regulate viral En elements in viral replication in vivo have not been fully defined in the mammalian viruses, although efforts in this direction have been made for duck HBV (9). In this study, we undertook in vitro and in vivo analyses of the role of WHV EnII in the viral life cycle. First, we introduced mutations in WHV EnII that abolish the binding of HNF-1, HNF-4, or Oct-1 to the element and assessed the effects of these mutations, singly and in combination, on EnII activity, as assayed on a chloramphenicol acetyltransferase (CAT) reporter driven by a minimal promoter linked to EnII. We then constructed 1.5-mer WHV genomes bearing these mutations and tested their abilities to transcribe and replicate viral DNA in cultured cells. Finally, selected mutant genomes were injected intrahepatically into woodchucks to investigate their replication competence in vivo. We show here that EnII primarily governs the production of pgRNA in vivo and that HNF-1 is required for this activity.

	MATERIALS AND METHODS

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Plasmids. All of the WHV EnII mutants we used are based on pBluescript II KS+ (Stratagene). The fragment from nucleotide 1697 to nucleotide 1919 (the nucleotide sequence and numbering are according to Kodama et al. [8]) encompassing EnII was prepared by PCR on the template WHV monomer (pWHVmono), in which 5'-end primer WBam1697-1711 (5'-GACTGGATCCCTCCGGTCCGTGTTG-3') and 3'-end primer WBam1919-1905 (5'-GACTGGATCCTCGCATGCATTTATG-3') were used. The PCR product was gel purified and cloned into the BamHI site in pBSKS+. The resulting plasmid, pW1697-1919WT, served as a template for PCR to introduce mutations into the EnII region.

EMSA. The fragments used as probes for electrophoretic mobility shift assay (EMSA) were generated by PCRs in which pW1697-1919WT, -IIA(), -IIB(), -IIC(), -IIAB(), IIAC(), -IIBC(), and -IIABC() were amplified with Taq polymerase and primers with the sequences 5'-CTTTGTCTCCTGGC-3' (nucleotides 1760 to 1773) and 5'-GCCCTCCTCCCATT-3' (nucleotides 1859 to 1846). The fragments (spanning nucleotides 1760 to 1859) were then labeled with T4 polynucleotide kinase and [³²P]ATP and incubated with nuclear extracts from HepG2 cells as previously described (23).

Transfection of HepG2 cells and CAT assay. HepG2 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% bovine serum and penicillin-streptomycin. Transfection was performed by the calcium phosphate coprecipitation method. At 16 h after transfection, cells were washed twice with phosphate-buffered saline without calcium and magnesium and incubated for another 24 h. The CAT assay was carried out as previously described (23). All assays were performed in duplicate, and each experiment was replicated at least three times.

Nucleic acid analysis. Total RNA was isolated from transfected HepG2 cells with RNAzol (TEL-TEST), electrophoresed through a standard 1% agarose-2.2 M formaldehyde gel, and transferred to a Hybond N membrane (Amersham). Nucleic acids in cytoplasmic nucleocapsids were prepared as previously described (10, 17) and analyzed by electrophoresis, transfer into Hybond N+ (Amersham) in 0.4 N NaOH, and hybridization with WHV genomic DNA. To prepare DNA from woodchucks, liver biopsy samples were incubated in 10 mM Tris-HCl (pH 8.0)-10 mM NaCl-10 mM EDTA-1% sodium dodecyl sulfate-120-µg/ml proteinase K at 4°C overnight, phenol extracted three times, chloroform extracted once, and ethanol precipitated. DNA was digested with PvuII and subjected to Southern blotting.

In situ transfection of woodchucks. The experimental woodchucks were born and raised in laboratory animal facilities and, prior to inoculation, were negative for serologic markers of infection (woodchuck hepatitis surface antigen [WHsAg], anti-woodchuck hepatitis core antigen antibody [anti-WHc], and anti-WHsAg antibody). DNA of the 1.5-mer WHV genome was prepared by using the Qiagen plasmid kit. For transfection in vivo, laparotomies were performed under general anesthesia and 50 µg of DNA (suspended in 0.5 ml of phosphate-buffered saline was injected at multiple sites into the parenchyma of the left lateral lobe of the liver. Groups of three animals were transfected with the mutant EnIIA(), EnIIB(), EnIIC(), or EnIIABC() or the WT 1.5-mer WHV genome.

Serological assay. Serum samples were collected serially, beginning 4 weeks postransfection, at ca. 2-week intervals for a total of 7 months. The sera were tested for the presence of WHsAg and anti-WHc by using enzyme-linked immunosorbent assays (1a). At 13 weeks after transfection, the animals were anesthetized and percutaneous needle biopsies of the liver were performed under ultrasound guidance.

	RESULTS AND DISCUSSION

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Effect of EnII mutations in transient transfection assays. In previous studies (23), we mapped WHV EnII to an 88-bp fragment (nucleotides 1772 to 1859) and demonstrated the binding of transcription factors Oct-1, HNF-1, and HNF-4 to subregions designated IIC, IIB, and IIA, respectively (Fig. 1A). In preliminary deletion analyses using CAT reporter genes driven by EnII and a minimal TATA box, we showed that the absence of the binding site for Oct-1 reduced EnII activity only twofold, while more extensive deletions involving the A and B sites resulted in more dramatic losses of activity (23).

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Mutations abolishing binding of transcription factors to WHV EnII. (A) schematic representation of the EnII region. The mutated nucleotides in the binding sites of Oct-1, HNF-4, and HNF-1 are shown below the diagram. (B) Mutations block binding of the factors. The indicated WT and mutant labeled EnII fragments were incubated with HepG2 nuclear extract and then examined by EMSA as described in Materials and Methods. IIA(), HNF-4 binding site mutated; IIB(), HNF-1 binding site mutated; IIC(), Oct-1 binding site mutated.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Effects of EnII mutations on CAT reporter gene activity in transient transfection assays. EnII mutant and WT DNA segments were cloned into an E1b TATA-CAT vector. The results of CAT assay were normalized to the level of CAT activity of E1b TATA-CAT (arbitrarily set at 1.0).

HNF-1 is required for the production of pgRNA in HepG2 cells. To investigate the effects of these mutations on viral replication in cultured cells, we recombined the mutations into overlength (1.5-mer) viral genomes and transfected them into HepG2 cells, which are known to be permissive for WHV replication (14); WT WHV DNA transfected in parallel served as a positive control. Three days after transfection, total cellular RNA was examined by Northern blot hybridization with WHV DNA. The results of three independent experiments showed that viral genomes bearing lesions in any of the three individual sites had only modest effects (twofold or less) on the production of the S mRNA transcripts (Fig. 3A). In sharp contrast, some of the lesions had pronounced effects on the production of pgRNA. Most dramatic was the impact of mutations in the HNF-1 site: these lesions nearly totally ablated detectable pgRNA synthesis [Fig. 3A, lanes EnIIB(), EnIIAB(), EnIIBC(), and EnIIABC()]. Mutations in the Oct-1 site had no impact on pgRNA [or on the subgenomic transcripts; lane EnIIC()]. HNF-4 site mutations had a subtler phenotype: they reduced the levels of pgRNA to levels that were roughly equimolar with the S mRNA [lanes ENIIA() and EnIIAC()]. This phenotype, while modest, was clearly different from that of WT WHV, in which pgRNA was clearly present in a two- to fourfold excess over S mRNA [Fig. 3A, lanes WT and EnIIC()].

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Effects of the mutations on virus expression and replication in cultured cells. (A) Northern blot analysis of total RNA from HepG2 cells transfected with WT or mutant 1.5-mer viral genomes, as indicated above the gels; following transfer, the filters were probed with 32P-labeled WHV DNA. (B) Analysis of DNA in viral nucleocapsids from transfected cells by Southern blot hybridization with a WHV DNA probe. Each lane corresponds to the same transfection as in the RNA samples in panel A. ssDNA, single-stranded DNA. Degraded plasmid DNA is shown as a loading control. (C) Control for RNA loading, 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe hybridized to the Northern blot of panel A.

Mutations in the HNF-1 and HNF-4 binding sites affect viral replication in woodchucks. HepG2 is a human hepatoma cell line and therefore is derived from a heterologous host for WHV replication. In addition, hepatoma cell lines (and hepatocyte cultures generally) are known to lack many phenotypic features of fully differentiated hepatocytes in vivo. Both of these considerations raise the possibility that the regulation of viral replication in HepG2 cells is different from that observed in the livers of intact host animals. We therefore investigated the impact of some of the preceding EnII mutations on viral replication in woodchucks. Given the limited availability of susceptible woodchucks, we selected the single mutations in the HNF-1, HNF-4, and Oct-1 sites and the triple mutation, which inactivates all three sites. Each of the mutant DNAs was transfected into three individual woodchucks by intrahepatic inoculation. Starting at 4 weeks posttransfection, serum samples were collected at 2-week intervals for 7 months and assayed for WHsAg antigenemia and anti-WHc by enzyme immunoassay (Table 1).

View larger version (58K): [in this window] [in a new window]   FIG. 4.   Replication of mutant viral genomes in woodchuck liver. DNA from liver biopsies was digested with PvuII and analyzed by Southern blot hybridization with a WHV DNA probe. Animal numbers and corresponding mutants injected are presented above and below the gel, respectively (upper panel). The lower panel shows ethidium bromide-stained DNA as a loading control.