INTRODUCTION

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Human infection with the hepatitis B virus (HBV) will lead either to an acute, self-limiting disease, to fulminant hepatic failure, or to chronic HBV infection. Despite the fact that only 3 to 10% of all individuals who acquire HBV will become chronic carriers, more than 400 million people worldwide are chronically infected with the virus (2). The risk of developing liver cirrhosis or hepatocellular carcinoma for these persons is 100 to 200 times higher than the risk for the rest of the population (16, 44).

After infection with HBV, a specific T- and B-cell response against viral proteins leads to clearance of the virus from the organism and development of lifelong immunity. However, in the chronic carrier state, the immune system fails to eliminate the virus, leading to a persistent infection with HBV (13, 32). There is growing evidence that escape from elimination by the immune system may be caused by the selection of mutant viruses during the course of HBV infection (7). The high frequency of mutations found in the HBV genome compared to those of other DNA viruses is caused by the inaccuracy of the viral polymerase, which lacks proofreading activity (30, 31, 35).

An example of the selection of a mutant virus has been provided by the replication of HBV in a newborn whose mother was chronically infected. Even though the baby received passive immunization immediately after birth to prevent HBV infection, HBV replication still occurred. Sequence analysis of the HBV S gene revealed a mutation in the "a" determinant, which disrupts antibody binding and thus prevents elimination of the virus by the immune system (8-10).

In addition to mutations which affect the immunogenicity of the virus, changes which influence its replication competence may be of similar importance. During chronic HBV infection, two different stages can be differentiated. The time immediately after the first contact with the virus is characterized by high levels of HBV DNA and a strong inflammatory response in the liver (44). At a later stage, there is a strong decrease in the level of replication, which is frequently associated with seroconversion from hepatitis B virus e antigen (HBeAg) to anti-HBe (28).

The amount of viral gene products, including the pregenomic RNA, is controlled at the level of transcription. Four promoter and two enhancer elements have been characterized in the HBV genome. In these control regions, DNA binding motifs for liver-specific transcription factors have been identified which regulate transcription in a tissue-specific manner and thus directly contribute to the hepatotropism of HBV (1, 18, 23, 30, 34). Transcription of the pregenomic RNA is controlled through the core promoter and the enhancer I region. Besides the pregenomic RNA, the enhancer I region regulates transcription of the core and X genes (1, 19, 21, 26, 46). The activity of enhancer I is regulated by the complex interaction of hepatocyte-specific and ubiquitous transcription factors which can bind in this region (Fig. 1A) (24, 33, 45). The enhancer I region has been described as consisting of three domains (Fig. 1A). The modulator element contains ubiquitous and liver-specific transcription factor binding sites and spans the 5' end of the enhancer I region. A central region, called the enhancer core domain, is responsible for the strong influence of the enhancer I region on the different promoters in the HBV genome, and the 3' end overlaps with the X promoter (11, 15, 18, 20, 22, 24, 30, 41). At least four nuclear proteins have been identified which are able to bind to the core domain (3, 18, 22, 30). The external motifs 5' and 3' of the inner core domain bind nuclear factor 1 and rheumatoid factor 1, respectively. However, the tissue-specific regulation of enhancer I is mediated by two motifs located in the central core domain which bind proteins from the nuclear receptor family. One motif binds hepatocyte nuclear factor 4 (HNF4) (18, 22); the other motif represents a binding site for HNF3, a transcription factor that is expressed in several isoforms in the liver (24, 30).

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Enhancer I mutants derived from 10 patients chronically infected with the hepatitis B virus. (A) DNA binding sites in the HBV enhancer I-X promoter between nt 2364 and 2768 of the HBV genome (pre-C ATG = 1) are depicted. The binding motifs of the liver-specific transcription factors HNF3 and HNF4 are marked in boldface letters. Binding sites for other ubiquitous or liver-specific factors are also shown. (B) Sequence analysis of patient-derived enhancer I regions showing deletions and mutations in comparison to the wt HBV enhancer I region. Between nt 2526 and 2558, the enhancer I sequences are highlighted (boldface italics) to show the mutations found in patients 1 and 3.

In this study we tested whether enhancer I mutations have an impact on viral replication. The mutations were derived from patients suffering from chronic HBV infection prior to undergoing liver transplantation for end-stage liver cirrhosis. In addition, we examined the impact of these mutations on the control of viral replication during the early and late stages of HBV infection. Collectively, our results demonstrate that the patient-derived mutations in the binding sites for HNF3 and HNF4 which downregulate enhancer I activity contribute to decreased replication fitness of these mutant viruses. Lower virus replication may result in an advantage for the chronic persistence of HBV.

(Part of this work was contained in the M.D. thesis of Katharinna Köhler.)

	MATERIALS AND METHODS

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Patients. The 10 patients included in this study were transplanted for HBV-related end-stage liver disease between September 1985 and July 1992. The patients experienced HBV reinfection, which was defined as the reappearance of hepatitis B virus S antigen (HBsAg). Routinely, 20,000 IU of anti-HBs (hepatitis B virus immune globulin [HBIg]) was administered in the anhepatic phase, and 10,000 IU of HBIg was administered each day for up to 1 week after surgery (7). At later time points, anti-HBs titers were tested, and HBIg was given when the titers fell below 100 IU/liter. Nine out of 10 patients had the subtype adw2, and 1 patient had subtype ayw.

Serological assays. Serum samples were aliquoted and stored at 20°C until they were defrosted for testing. HBsAg, anti-HBs, HBeAg, and anti-HBe were tested by commercial enzyme immunoassays (EIAs) (Abbott Laboratories, Chicago, Ill.).

Extraction of DNA from sera and amplification of the HBV genome. DNA was extracted from sera (39) collected before orthotopic liver transplantation (OLT) and at time points after HBV reinfection was evident. Ninety microliters of serum was treated with 10 µl of proteinase K and 300 µl of DNA lysis buffer (0.67% sodium dodecyl sulfate (SDS), 13.33 mM Tris-HCl [pH 8.0], 6.6 mM EDTA [pH 8.0], and 13.3 mg of tRNA/ml). The mixture was incubated at 56°C for 2 h. Four hundred microliters of phenol-chloroform-isoamyl alcohol (25:24:1 [vol/vol]) was added and vortexed. The mixture was centrifuged for 30 min at 13,000 × g and 4°C. The DNA in 300 µl of the upper phase was precipitated by adding 660 µl of 100% ethanol and 30 µl of 3 M sodium acetate (pH 5.2) and centrifuged for 30 min at 13,000 × g and 4°C. After the pellet was briefly washed with 70% ethanol, it was dried and resuspended in 25 µl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

Plasmid construction. To determine the transcriptional activity of the different enhancer I and enhancer I-X promoter mutants in luciferase assays, the PCR fragments from the serum-derived HBV DNAs of the 10 patients were introduced into the restriction sites KpnI and BglII of the multiple cloning sites of the pGL2 luciferase basic (without any promoter) or promoter reporter (simian virus 40 [SV40] promoter; Stratagene) vector using the following primers: Enh1-sense, 5'-GGG GTA CCC TTC CTG TTA ACA GGC CTA T-3' (starting at nt 2364), and Enh1-antisense, 5'-GAA GAT CTG CTC CAG ACC GGC TGC GAG C-3' (terminating at nt 2725). For the enhancer I-X promoter constructs, the following primers were used: Enh1-sense (described above) and Enh1-long: 5'-GAA GAT CTT TTC CGC GAG AGG ACG ACA GA-3' (terminating at nt 2768). These primers had 5' extensions with either the KpnI or the BglII restriction enzyme site (underlined sequences) spanning the HBV region from nt 2364 to 2725 or from nt 2364 to 2768, respectively. The enhancer I PCR fragments (nt 2364 to 2725) in all the plasmids were sequenced and checked for the presence of mutations compared to wild-type (wt) HBV (Fig. 1B).

Cell culture, transfection experiments, and luciferase assays. HuH-7 cells (human hepatoma cells [27]), which are negative for HBV markers, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum under 5% CO₂ at 37°C. DNA transfection by the calcium phosphate coprecipitation method was performed as described previously (6). For analysis, the cells were harvested between 24 h and 6 days after transfection. Transfection efficiency was routinely checked by cotransfecting 0.2 µg of the -galactosidase pCMVGal vector as an internal standard (29). To verify the results, all transfection experiments were routinely performed in triplicate at a minimum.

Intracellular HBeAg and HBsAg expression. Intracellular HBeAg or HBsAg was evaluated by using whole-cell extracts from lysed transfected cells in buffer (120 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl [pH 8.0], 0.1 mM Pefablock [Boehringer, Mannheim, Germany], 0.1 mM dithiothreitol, 1% Triton X-100). Extracellular HBeAg or HBsAg was collected from the transfected-cell-medium supernatant. Two hundred-microgram aliquots were analyzed by commercial EIAs (Abbott Laboratories).

Preparation of nuclear extracts and in vitro-translated protein. HuH-7 nuclear extracts were prepared according to the method of Dignam et al. 1983 as described before (14).

Gel retardation assays. For gel retardation assays, nuclear extracts from HuH-7 cells or in vitro-translated protein was used. Three micrograms of nuclear extracts was used unless otherwise indicated. For binding assays, an oligonucleotide spanning the region of interest was used as a ³²P-labeled probe. Approximately 50 fmol, corresponding to 30,000 to 50,000 cpm, was used per binding reaction. The binding reaction was performed for 20 min on ice. Free DNA and DNA-protein complexes were resolved on a 6% polyacrylamide gel as described previously (37). The gels were dried and exposed for autoradiography.

Northern blot analysis. Northern blot analysis was performed as described before, according to standard procedures (38). In brief, total RNA was extracted from cells with the RNeasy kit (Qiagen Inc., Hilden, Germany) 2 to 3 days after transfection following the manufacturer's instructions. RNA samples were kept at 80°C until they were used. Twenty micrograms of total RNA was analyzed through a 1% MOPS [3-(N-morpholino)propanesulfonic acid] gel (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA; pH 6.8), followed by transfer to Hybond N⁺ nylon membrane (Amersham, Little Chalfont, England). For hybridization, a ³²P-labeled monomeric HBV DNA probe was used. Equal loading of RNA samples was verified by reprobing the membrane with a ³²P-labeled GAPDH (glyceraldehyde-3-phosphate dehydrogenase) probe.

Progeny HBV DNA. Progeny HBV DNA was isolated from transfected cells as described previously (25). HBV capsids were immunoprecipitated from cell lysates by polyclonal rabbit anti-HBcAg antiserum (DAKO, Hamburg, Germany) as described previously. Residual transfected plasmid HBV DNA was eliminated by Staphylococcus aureus nuclease treatment (100 U per 10⁶ cells; Boehringer). Viral progeny DNA was extracted from the capsids by using 500 µg of proteinase K (Sigma, Heidelberg, Germany)/ml-1% SDS at 56°C for 2 h. The DNA was purified by phenol extraction following separation on a 1% alkaline agarose gel (4). Southern blotting was performed on Hybond N⁺ nylon membranes.

SDS-PAGE and Western blot analysis. SDS-PAGE and Western blot analyses were performed with whole-cell nuclear extracts derived from HuH-7 cells transfected with the wt or mutant HBV replication-competent vector. Specific signals were detected by using polyclonal rabbit anti-core antiserum (DAKO) as described previously (5).

Quantification. Quantification of results was performed with a Fuji imager using the Tina-PCBAS software (Raytest, Straubenhardt, Germany) or by a densitometric evaluation as described before (6).

	RESULTS

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Distinct mutations in the enhancer I region derived from chronic HBV carriers decrease its activity. The region of the HBV genome containing most of the crucial regulatory elements for viral replication, the enhancer I and II regions, and the core promoter (nt 2229 to 162) was amplified by PCR and sequenced from sera of 10 patients with chronic HBV infection prior to OLT. Major mutational changes were found in the enhancer I region. Therefore, this region was subcloned and further analyzed to investigate its impact on HBV replication. As shown in Fig. 1B, all of the patients had mutated enhancer I sequences. In four patients (1, 2, 3, and 7), the mutations were localized in the so-called enhancer I core domain.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Transcriptional activity of the enhancer I mutants derived from patients 1 to 10. (A and C) The enhancer I sequences between nt 2364 and 2716 and between nt 2364 and 2768 derived from wt HBV and the HBV sequences isolated from patients 1 to 10 were cloned as depicted in combination with the SV40 promoter in the pGL2 vector (A) or in the enhancer I-X promoter constructs (C) with the luciferase reporter gene alone. (B and D) Transfection experiments were performed with the different enhancer I (B) or enhancer I-X promoter constructs (D) in HuH-7 cells. The cells were harvested 48 h after transfection, and luciferase activity was measured. The error bars indicate standard deviations. The relative luciferase activities of the different mutant constructs are shown in comparison to that of the wt constructs, which was set to 1 (dashed lines). The activities for patients 1 and 3 are highlighted.

Mutations in the enhancer I core domain result in lack of activation through decreased binding of liver-specific transcription factors. The enhancer I sequences isolated from patient 1 and patient 3 showed the most prominent reduction in enhancer I activity compared to that of wt HBV. We therefore decided to characterize these two HBV variants in more detail. The mutation derived from patient 1 contained a 2-nt deletion between nt 2531 and 2532 and a 2-nt insertion between nt 2535 and 2536 in the enhancer I HNF3 element (HNF3; AAGTA was changed to GTGCA in the HNF3 motif). The mutation derived from patient 3 consisted of a single point mutation at nucleotide 2546 in the enhancer I HNF4 motif (HNF4). Other enhancer I mutations found in the HBV variants of patients 1 and 3 are not located at a known protein-binding site of the enhancer I region (Fig. 1B). Analysis of the other mutants shown in Fig. 1B showed they were not located in known DNA binding sites controlling enhancer I activity.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Mutations in the HBV enhancer I core domain decrease binding of HNF3 and HNF4. (A) 32P-labeled wt (left, lane 2) or mutant (left, lane 3) HNF3 oligonucleotides were used in gel shift experiments. The HNF3 oligonucleotides were incubated with HuH-7 nuclear extracts. The positions of the HNF3 complexes are marked. In vitro-translated (ivt) HNF3, -, and - proteins were incubated with wt (right, lanes 3 to 5) or mutant (right, lanes 6 to 8) HNF3 oligonucleotides. Incubation of reticulocyte (RL) extracts with wt HNF3 oligonucleotide is shown in lane 2. The positions of HNF3 complexes and free probe are depicted on the left. (B) HNF4 wt (left, lane 2) or mutant (left, lane 3) 32P-labeled oligonucleotides were incubated with in vitro-translated HNF4 protein. HNF4 was supershifted with polyclonal anti-HNF4 antibodies (left, lane 4). Competition experiments were performed with cold wt HNF4 (right, lanes 2 to 5) or mutant HNF4 (right, lanes 6 to 9) in the indicated molar excess in the incubation assay. (C) HuH-7 cells were transfected with wt, HNF3, and HNF4 enhancer I reporter gene constructs. Cotransfection experiments were performed with expression vectors for HNF3 (left) and HNF4 (right). The constructs were cotransfected with increasing amounts (0, 50, 150, 300, or 600 ng) of expression vectors for HNF3 (left) or HNF4 (right). The cells were harvested 48 h after transfection, and the luciferase activity was determined. The error bars indicate standard deviations. The activities of the samples in which only the luciferase reporter construct was transfected were set to 1. Note that dramatic changes were found in the basal activity with HNF3 and -4 compared to that with the wt HBV construct (Fig. 2). The changes are shown as fold activation.

The HNF3 and HNF4 mutants result in decreased replication efficiency of HBV. From our initial experiments, we conclude that the described mutations in the enhancer I core domain result in decreased binding of HNF3 and -4 to this regulatory element. The functional unresponsiveness of the enhancer I core domain is responsible for the significantly reduced transcription of the different luciferase constructs tested. In order to determine the impacts of these mutations on HBV replication, we generated replication-competent HBV constructs containing the mutated enhancer I regions of patients 1 and 3 (HNF3 and HNF4) (Fig. 1B).

View larger version (33K): [in this window] [in a new window]   FIG. 4.   Enhancer I mutants reduce expression of viral gene products. (A) Northern blot analysis was performed with total RNA derived from HuH-7 cells transfected with an empty control vector (mock; pBluescript) (lane 1) or HBV replication-competent vectors for 2 days. The wt (lane 2) and two clones of the enhancer I HNF4 (lanes 3 and 4) and HNF3 (lanes 5 and 6) vectors are shown. Twenty micrograms of RNA was separated on a 1% gel and transferred to nylon membranes. HBV-specific RNA was detected with a 32P-labeled HBV probe. The positions of the HBV-specific RNAs are shown at the right. Differences in the RNA concentrations were visualized by quantification of RNA bands. Pregenomic RNAs, pre-S and S mRNAs, and X mRNA are shown. The intensity of RNA expression found with the wt HBV replication-competent construct was set to 100%, and changes found with the mutant HNF3 and HNF4 constructs were calculated accordingly. The results of three independent experiments were quantified for this analysis. The error bars indicate standard deviations. (B) The amount of intra- and extracellular HBeAg or HBsAg was determined in whole-cell extracts or the supernatant 6 days after transfection by EIA. The expression of the wt HBV construct was set to 100%, and the other samples (mock, HNF3, and HNF4) were calculated accordingly. The error bars indicate standard deviations. The average of three independent experiments is shown. rel. conc., relative concentration.

View larger version (23K): [in this window] [in a new window]   FIG. 5.   The replication competence of enhancer I mutants is impaired. HuH-7 cells were transfected with the empty vector control (pBS; mock), the wt, and the enhancer I HNF3 and HNF4 HBV replication-competent constructs. (A and B) Quantification of intracellular core protein expression. (A) Core protein was precipitated by monoclonal mouse anti-HBcAg-specific antibodies. The precipitated protein was run on SDS-PAGE and transferred to nylon membranes. Filters were probed with polyclonal rabbit anti-HBcAg antiserum. Either 105 or 106 cells were used in the experiments as indicated. In lane 1, recombinant core protein was used. (B) The relative amounts of HBcAg were determined by Fuji phosphoimager or densitometric evaluation. The amount found with the wt HBV construct was set to 100%. The error bars indicate standard deviations. (C and D) The amount of newly synthesized progeny DNA was determined from cytoplasmic HBV core particles by using anti-HBc antibodies for immunoprecipitation from 106 cells. (C) HBV DNA was separated on a 1% alkaline agarose gel and visualized by Southern blot analysis as described previously (4). A 3.2-kb linear HBV DNA was used as a reference (lane 1). (D) The concentration of progeny HBV DNA was quantified; wt values were set to 100%, and the amounts of the HNF3 and HNF4 mutants were calculated. The error bars indicate standard deviations.

The occurrence of the HNF4 and HNF3 enhancer I mutants correlates with reduced HBV replication. The HNF4 and HNF3 enhancer I core domain mutants were isolated from sera derived immediately before liver transplantation (OLT). Here, we studied HBV replication in both patients at time points before and after liver transplantation (Fig. 6).

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Clinical course of patients 1 (HNF3) and 3 (HNF4). Virological parameters (HBV DNA [pg/ml], HBsAg, HBeAg, and anti-HBs) of patient 1 (A) and patient 3 (B) were determined at different time points before and after liver transplantation. The time points are indicated based on the day of OLT, which was set to 0. Sequencing of the HBV DNAs at different time points revealed the dominant DNA strains as indicated. +, present; , absent; LTx, liver transplantation.

	DISCUSSION

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Chronic HBV infection reflects the permanent interaction of the immune system with viral epitopes (12, 32). Escape mutants, which are selected in the viral genome during the course of infection, contribute to the persistence of the virus and may also result in a more progressive form of liver disease. During chronic HBV infection, two stages of viremia can frequently be distinguished. Directly after HBV infection, high HBV DNA levels are found associated with a strong inflammatory response in the liver. Later, during infection, HBV replication decreases and may be associated with seroconversion from HBeAg to anti-HBe (28). However, seroconversion from HBeAg to anti-HBe does not necessarily indicate that HBV DNA levels have decreased. For example, selection of a stop mutation in the pre-C region results in a lack of HBeAg expression, leading to an anti-HBe status and increased viral replication (20, 25).

The switch between a high- and a low-replicative period during chronic HBV infection has not been addressed so far. This clinical observation led us to hypothesize that mutations resulting in decreased HBV replication may also be able to escape the host's immune response. Ideally, an escape mutation would lead to both decreased expression of viral epitopes and diminished replication of the virus. Such a mutation would most likely affect the regulation of several viral genes rather than affecting the function of a single protein. A candidate region for this type of mutation is the enhancer I sequence within the HBV genome. Originally identified as a regulatory motif able to influence the expression of different viral genes (34), the enhancer I region may be a potential target for mutations leading to escape from the immune system.

We therefore sequenced the enhancer I regions of 10 patients with chronic HBV infection prior to OLT. In the majority of these viral sequences, the enhancer I region was mutated, resulting in decreased transcriptional activity of viral and heterologous promoters. The lowest activity was detected when enhancer I sequences with mutations in the previously described HNF3 and -4 transcription factor binding sites were tested (11, 18, 30).

These sites are located in the enhancer I core domain, which has the strongest impact on transcriptional activation of HBV genes. To characterize the molecular mechanism responsible for this loss of activity in more detail, we examined the abilities of HNF3 and -4 to bind to these mutated sequences.

Surprisingly, the minor sequence changes we detected in the enhancer I regions of these two patients almost entirely abolished the binding of these transcription factors. In agreement with the loss of transcriptional activity, we detected dramatically decreased levels of pregenomic RNA (40% for HNF3 and 20% for HNF4) and an even stronger reduction in X mRNA expression (25% for HNF3 and 18% for HNF4). A less pronounced effect was evident for the pre-S and S mRNAs (70% for HNF3 and 60% for HNF4), supporting the observation that an element outside of the enhancer I region is essential for the control of the two S promoters (47). Not surprisingly, these remarkable reductions in transcriptional activity resulted in significant changes in the expression of core protein levels (HNF3, 30%, and HNF4, 15%) and nucleocapsid assembly (25% progeny DNA for HNF4 and 8% for the HNF3 mutant) compared with a wt HBV genome.

Analyzing the mutated enhancer I sequence within the background of a wt HBV genome helped us to specifically determine the roles of mutations in this region. The coding sequence of the viral polymerase, however, overlaps the enhancer I sequence (17). The mutations we described result in amino acid changes in the corresponding polymerase sequences for HNF3 (Leu to Pro and Ser to Cys) and for the single HNF4 point mutation (Met to Ile). These mutations were located in the reverse-transcription domain of the viral polymerase. These polymerase mutations may lead to a reduction in HBV polymerase activity. However, our results as shown in Fig. 5 indicate that in the mutated viral strains the amounts of viral RNA, viral capsids, and HBV progeny DNA are significantly reduced, leading to a comparable decrease. While the expression of viral RNA and HBV core protein is independent of the HBV polymerase activity, our results also indicate that the capsid-to-progeny DNA ratio is not changed between the wt and the mutant strains. Therefore, these results revealed that the encapsidated wt and mutant HBV polymerase seem to have comparable activities, and thus the change in the polymerase has no major influence on the replication level of the mutant viruses.

The in vitro studies presented here illustrate the effects HNF3 and HNF4 mutations have on the expression of viral proteins and the assembly of virus particles. They are important for understanding the molecular mechanisms which may be responsible for the decreased replication and immunogenicity of HBV in chronic carriers. The clinical significance of the enhancer mutations, however, is very difficult to assess. When we first detected the HNF3 and HNF4 mutations, the amount of HBV DNA was measured as 3 pg/ml in patient 1 and 15 pg/ml in patient 3, possibly reflecting the effects of these mutations on virus replication in vivo. After liver transplantation, however, both patients were reinfected with HBV. Patient 1 showed a 240-fold increase and patient 3 showed a 40-fold increase in HBV DNA levels. Sequencing of the enhancer I regions in both patients revealed the wt HBV sequence. In general, in patients undergoing liver transplantation HBV replication increases compared to the pre-OLT level (36). These patients receive lifelong immunosuppressive therapy, and it has been demonstrated that the glucocorticoids especially increase HBV replication through a glucocorticoid-responsive element in the HBV genome (42, 43). At the time of reinfection, however, both patients received only very low doses (2.5 and 5 mg, respectively) of this immunosuppressive drug. Thus, the increase in HBV replication after OLT cannot simply be explained by glucocorticoid responsiveness.

All 10 patients with chronic active HBV infection investigated in this study had mutations in the enhancer I region. We believe that these mutations may influence replication and immunogenicity of the virus to different degrees. While mutations like HNF3 and HNF4 abolish the binding of important transcription factors, other mutations might only exert an effect in association with mutations in the enhancer I region or elsewhere in the HBV genome. Mutating regulatory sequences in the HBV genome allows the virus to affect different functions, such as protein expression, virus assembly, and expression of epitopes, simultaneously, which under the selective pressure of the host's immune system might provide an efficient escape mechanism.

In summary, our experiments describe a new form of escape mechanism which controls the level of HBV replication. Therefore, enhancer I mutants seem to be involved in explaining the clinical observation that two stages of viremia during chronic HBV infection can be differentiated: a high- and a low-replicative period.