In the genomic DNA of HBV, the core promoter [15] has been identified in a 3'-terminal part of the X gene. This promoter controls the transcription of the 3.5-kilobase messenger RNA (mRNA) (pregenome-core/polymerase mRNA), which acts as the pregenome and from which core protein and DNA polymerase-reverse transcriptase are translated [16]. The core promoter also controls the transcription of a second, slightly longer 3.5-kilobase mRNA (precore mRNA), which encodes the HBeAg precursor [17]. Mutations in the core promoter might affect transcription of either RNA (pregenome-core/polymerase mRNA or precore mRNA) and thus blot expression of these gene products. Specific mutations, however, might affect transcription of one type of mRNA more than the other and thus only block production of HBeAg or alternatively block production of core protein and DNA polymerase.

Hepatitis B virus DNA clones were propagated from 43 patients with fulminant hepatitis B and 14 patients with acute self-limited or severe hepatitis B and were sequenced within the core promoter (nucleotides 1742 to 1849) and the precore region (nucleotides 1814 to 1900). The results indicated a close association between fulminant hepatitis and HBV strains that do not produce HBeAg because of either mutations in the precore region that abort the translation of HBeAg or mutations in the core promoter that affect transcription of the HBeAg precursor. The functional role of mutations in the core promoter, however, must await experimental expression of precore mRNA and HBeAg in hepatoma cell lines transfected with mutant HBV.

Methods

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Patients

We studied 43 patients with fulminant hepatitis B, including 25 in whom the disease developed sporadically and 18 in whom it developed after transfusion. We used the diagnostic criteria for fulminant hepatitis by Trey and colleagues [18]: 1) the development of stage II to IV hepatic encephalopathy within 8 weeks of the onset of illness; 2) a prothrombin of less than 40%; and 3) no known history of previous liver diseases, which ruled out the possibility of chronic liver failure. We also studied 14 patients with acute hepatitis B who had never had a transfusion. Hepatitis was self-limited in 8 of these patients. In the other 6 patients, hepatitis was severe and, in some patients, accompanied by coagulopathy or stage I encephalopathy. We also tested the spouses of 2 patients with acute severe hepatitis who were carriers of HBV and 3 carriers suspected of transmitting HBV to 4 patients with fulminant hepatitis.

Hepatitis B virus infection was established as the cause of acute or fulminant hepatitis by the detection of high-titer IgM antibody to hepatitis B core antigen (anti-HBc), in the absence of any evidence for acute infection with hepatitis delta virus or hepatitis A virus.

Hepatitis B virus DNA clones were propagated from the patients' serum samples and were sequenced within the precore region and core promoter. The study was approved by each hospital's ethics committee; informed consent was obtained from patients or from family members serving as legal guardians if the patients could not make a decision. The precore region sequences of clones from 13 patients with sporadic fulminant hepatitis B, 8 patients with post-transfusion fulminant hepatitis, and 8 patients with acute hepatitis have been described [7, 9, 11].

Serologic Markers of Hepatitis Virus Infections

The presence of hepatitis B surface antigen (HBsAg) was determined by hemagglutination with commercial kits (MyCell, Institute of Immunology Co., Ltd., Tokyo, Japan). The presence of HBeAg and the corresponding antibody (anti-HBe) were determined by enzyme immunoassay with commercial kits (HBeAg/Ab EIA, Institute of Immunology Co., Ltd.). The presence of non-class-specific anti-HBc was determined by inhibition of hemagglutination [19], and IgM anti-HBc was determined by enzyme immunoassay [20]. The presence of antibody to hepatitis delta virus and IgM antibody to hepatitis A virus was determined with commercial assay kits (ANTI-DELTA RIA and HAVAB-M EIA, Abbott Laboratories, North Chicago, Illinois). The presence of antibody to hepatitis C virus (anti-HCV) was determined by enzyme immunoassay with commercial kits (EIA-I or EIA-II, Ortho Diagnostic Systems, Tokyo, Japan).

Sequencing a Part of the X Gene and Precore Region

Hepatitis B virus DNA was extracted from serum, and a fragment of 263 base pairs corresponding to nucleotides 1679 to 1941 was amplified by polymerase chain reaction (PCR) with nested primers by a previously described method [5]. As shown in Figure 1, the fragment included the enhancer II [21] (nucleotides 1685 to 1773) and the core promoter [15] (nucleotides 1742 to 1849), as well as the precore region (nucleotides 1814 to 1900). Primers used for the first round of PCR were 5'-CATAAGAG GACTCTTGGACT-3' (sense; nucleotides 1653 to 1672) and 5'- AAAGAATTCAGAAGGCAAAAAAGA-3' (antisense; nucleotides 1949 to 1972); primers for the second round of PCR were 5'-AATGTCAACGACCGACCTTG-3' (sense; nucleotides 1679 to 1698 with a HincII site underlined) and 5'-TCCACAGAAGCTCCGAATTC-3' (antisense; nucleotides 1922 to 1941 with an EcoRI site underlined). Hepatitis B virus DNA amplified by PCR was digested with EcoRI and HincII (Takara Biochemicals, Kyoto, Japan). The digest of 242 base pairs corresponding to nucleotides 1685 to 1926 was ligated to the M13 phage vector that had been cleaved with EcoRI and HincII, and clones carrying the HBV DNA sequence were propagated. Hepatitis B virus DNA sequences were then determined by the dideoxy-chain termination method with the Sequenase DNA sequencing kit (7-deaza-dGTP edition, version 2.0, United States Biochemical Corp., Cleveland, Ohio) or the AutoRead DNA sequencing kit (Pharmacia LKB Biotechnology, Uppsala, Sweden).

View larger version (29K): [in this window] [in a new window]   Figure 1. A map of the X and precore-core genes and the fragment of hepatitis B virus (HBV) DNA sequenced. The initiation sites of two 3.5-kilobase messenger RNAs (mRNA) are indicated on the top. Boxes below represent liver-specific enhancer II, the basic core promoter, and a regulatory sequence located upstream of them. Positions of direct repeats (DR) are also shown. Sequences of HBV DNA amplified by the first or second round of polymerase chain reaction (PCR) are indicated at the bottom. nt = nucleotide.

Hepatitis B Virus Genotypes

Hepatitis B virus genotypes such as A, B, C, D, and E [22, 23] were determined by an intergenotypic divergence of more than 8.4% and an intragenotypic divergence of less than 3.0% within 659 base pairs of the HBV genome representing nucleotides 248 to 683 (436 base pairs) in the envelope gene and nucleotides 1699 to 1921 (223 base pairs) in the X gene and precore region.

Results

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Mutations in the Core Promoter and Precore Region of Hepatitis B Virus DNA Clones from Patients with Fulminant Hepatitis B

A part of the X gene bearing the core promoter (nucleotides 1742 to 1849) and the precore region (nucleotides 1814 to 1900) were sequenced in 878 HBV DNA clones from 43 patients with fulminant hepatitis B and 14 patients with acute hepatitis B. In Figure 1, the position of the target sequence is shown in relation to the X gene and precore region, as well as to the enhancer II [21] and core promoter with an upstream regulatory sequence [15].

Table 1 lists the outcome, HBeAg and anti-HBe status, and mutations in the core promoter and precore region in 387 HBV DNA clones from 25 patients with sporadic fulminant hepatitis. Mutations in the core promoter were detected in all 345 clones from 20 patients (80%), and the G-to-A point mutation at nucleotide 1896 that converted codon 28 in the precore region from tryptophan (TGG) to a stop codon (TAG) was observed in all 257 clones from 20 patients (80%). The mutation at nucleotide 1896 in 4 of the 20 patients was accompanied by another G-to-A point mutation at nucleotide 1899. Five patients (patients 1 to 5) had clones with mutations in the core promoter alone, and 5 other patients (patients 21 to 25) had clones with mutations in the precore region only.

Of the 25 patients with sporadic fulminant hepatitis, 11 survived and 14 died. No sex or age differences were noted between survivors and nonsurvivors. Hepatitis B e antigen was detected in only 2 patients and anti-HBe in only 1; neither HBeAg nor anti-HBe was detected in the remaining 22 (88%) patients. None of these 25 patients was positive for anti-HCV.

Table 2 shows the findings in 18 patients with post-transfusion fulminant hepatitis B, of whom only 3 survived. Antibody to HBeAg was detected in 1 of the 16 patients tested; neither HBeAg nor anti-HBe was detected in the other 15 patients (94%). Two patients (patients 13 and 18) were positive for anti-HCV.

Mutations in the core promoter were detected in all 89 HBV DNA clones from 6 (33%) of the 18 patients. All 271 clones from 17 patients had A1896; clones from the remaining patient (patient 6) had either this mutation or an insertion of two nucleotides into the precore region, which induced a frameshift. The second G-to-A mutation at nucleotide 1899 was detected in clones from 6 patients. None of the patients was infected with HBV that carried mutations in the core promoter alone.

Mutations in the Core Promoter and Precore Region of Hepatitis B Virus DNA Clones from Patients with Acute Nonfulminant Hepatitis B

Findings for the six patients with acute severe hepatitis B and the eight patients with acute self-limited hepatitis B are shown in Table 3. Neither HBeAg nor anti-HBe was detected in any patient with acute severe hepatitis. All 69 HBV DNA clones from the six patients with acute severe hepatitis had either mutations in the core promoter or A1896 in the precore region, or both. Clones from three patients had mutations in the core promoter unaccompanied by A1896. Those from one patient had both mutations in the core promoter and A1896, and those from the remaining two patients had A1896 alone.

In contrast, neither mutations in the core promoter nor A1896 were detected in any of 140 clones from the eight patients with acute self-limited hepatitis. Four of them were positive for HBeAg, and the remaining four were positive for anti-HBe; none lacked detectable HBeAg or anti-HBe. Antibody to HCV was not detected in any of the 14 patients.

Patterns of Mutations in the Core Promoter of DNA Clones from Patients with Fulminant or Acute Severe Hepatitis B

Patterns of mutation detected in the core promoter of HBV DNA clones from 26 patients with fulminant hepatitis and 4 patients with acute severe hepatitis are shown in Figure 2. Hepatitis B virus DNA clones from all patients were of genotype B or C except those from one patient with fulminant hepatitis (patient 1; Table 1 who was infected with HBV genotype A. We therefore compared the sequences of our patients' HBV DNA clones with those of wild-type HBV of genotypes B and C, which have identical core-promoter sequences.

View larger version (19K): [in this window] [in a new window]   Figure 2. Point mutations in the core promoter of hepatitis B virus (HBV) DNA clones from 30 patients with fulminant or acute severe hepatitis B. The sequence of a wild-type HBV genotype B or C is indicated above. Three AT-rich regions are indicated by bars, and initiation sites for precore messenger RNA are indicated by arrows. Dashes represent nucleotides identical to those of wild-type HBV. Numbers of cases with mutations (a-k) are indicated on the right.

Of the 30 patients with mutations in the core promoter, 27 (90%) had the two point mutations, from A to T at nucleotide 1762 and from G to A at nucleotide 1764 (patterns a to i); the remaining 3 patients had either of the two mutations (patterns j and k). In addition, HBV DNA clones from some patients showed as many as six point mutations.

We recognized three AT-rich regions in the core promoter. The first AT-rich region (ATTA) spanned nucleotides 1752 to 1755, the second (TTAAA) spanned nucleotides 1758 to 1762, and the third (ATAAATT) spanned nucleotides 1789 to 1795. The first and second AT-rich regions were 26 to 28 base pairs upstream of the initiation sites for longer precore mRNAs (nucleotides 1783 and 1784) and shorter precore mRNAs (nucleotides 1789 to 1791), respectively. The third AT-rich region was 23 base pairs upstream of the pregenome-core/polymerase mRNA (nucleotide 1818). All mutations in the core promoter of HBV DNA clones from patients with fulminant hepatitis B, except for pattern k, converted the last A in the second AT-rich region to T.

Mutations in the Core Promoter and Precore Region in DNA Clones from Five Carriers Who Were Implicated in Transmitting HBV to Six Patients with Fulminant or Acute Severe Hepatitis

Serum samples from five carriers suspected of transmitting HBV to six patients were available for analysis. Mutations in HBV DNA clones from implicated carriers and those from patients are shown in Table 4. In all clones from the six patients, mutation patterns in the core promoter were identical to those in implicated carriers. Mutation in nucleotide 1896 in the precore region was seen in all clones from the carrier and two patients in case 5. It was seen only in some clones from the carrier and patient in case 4 and the carrier in case 2.

Discussion

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Many studies suggest a close association between fulminant hepatitis B and HBV strains with mutations in the precore region, typified by the G-to-A mutation at nucleotide 1896, which aborts the synthesis and secretion of HBeAg [4-12]. However, some patients with fulminant hepatitis B have HBV DNA clones that do not show any such mutations in the precore region [6, 7, 13, 14]. Because HBV strains with precore mutations are common in patients with fulminant hepatitis who are from Israel [10], southern Mediterranean countries [6], and Japan [7-9, 11, 12], but rarely in those who are from the United States [13] or France [14], a different epidemiologic distribution has been proposed for the fulminant HBV strains with precore mutations [13, 14].

We found mutations in the core promoter, with or without precore mutations, in HBV DNA clones from most patients with fulminant hepatitis B in Japan. Because direct sequencing may fail to show small populations of mutant HBV [25], substantial HBV DNA clones were propagated from each serum sample (mean, 15.4 clones; range, 3 to 54 clones) and sequenced individually. Two point mutations in the core promoter, from A to T at nucleotide 1762 and from G to A at nucleotide 1764, were detected in all 434 HBV DNA clones from 20 (80%) of 25 patients with sporadic fulminant hepatitis and 6 (33%) of 18 patients with post-transfusion fulminant hepatitis. All 282 clones from the 18 patients with post-transfusion fulminant hepatitis had a nonsense mutation at codon 28 (A1896) or a 2-base pair insertion in the precore region, which prohibits the expression of HBeAg. Mutation at nucleotide 1896 was found in all 257 clones from only 20 (80%) of 25 patients with sporadic fulminant hepatitis. However, all 130 clones from the remaining 5 patients had HBV strains with mutations in the core promoter.

Thus, all 43 patients with sporadic or post-transfusion fulminant hepatitis B were infected with HBV strains that had mutations in either the core promoter or the precore region, or both, and neither HBeAg nor anti-HBe was detected in 38 (93%) of the 41 patients tested. Mutations in the core promoter or the precore region, or both, were also detected in all 69 clones from the six patients with or acute severe hepatitis, none of whom had detectable HBeAg or anti-HBe in serum. In sharp contrast, we did not observe such mutations in any of 140 clones from the eight patients with acute self-limited hepatitis B whose serum contained either HBeAg or anti-HBe. Five carriers suspected of transmitting infection to six patients without fulminant or acute severe hepatitis had HBV strains with mutation patterns in the core promoter that were identical to those in corresponding patients, in the presence or absence of A1896.

These results indicate that mutations in the core promoter and those in the precore region, together or independently, are associated with fulminant or acute severe hepatitis, and that HBV strains with such mutations could not direct the production of HBeAg. A low prevalence of HBeAg or anti-HBe in patients with fulminant hepatitis B has been noted. Only 4 (24%) of 17 patients in England with fulminant hepatitis B were positive for HBeAg or anti-HBe compared with 15 (88%) of 17 patients with acute uncomplicated hepatitis [26]. The failure to detect anti-HBe is remarkable in view of the heightened immune responses to HBV-related antigens in patients with fulminant hepatitis B [27, 28]. Circulating HBeAg has been proposed as a viral strategy to induce immunotolerance [29]. Its absence, therefore, would accelerate inflammatory activity in the liver [30], which could be relevant to the pathogenesis of fulminant hepatitis.

The point mutation at nucleotide 1896 that converts codon 28 in the precore region from tryptophan (TGG) to a stop codon (TAG) was observed in 38 patients (88%) with fulminant hepatitis and 3 patients (50%) with acute severe hepatitis. This inhibits the production of HBeAg by affecting the translation of the HBeAg precursor [4, 5]. Hepatitis B virus strains in the remaining 8 patients with fulminant or acute severe hepatitis did not have A1896; however, they all had mutations in the core promoter. It is tempting to speculate that the observed mutations in the core promoter would influence the transcription of precore mRNAs toward a decreased expression of HBeAg. Recently, Laskus and colleagues [31] described HBV strains with various mutations involving the core promoter and proposed that such mutation would affect the transcription of precore mRNAs and prohibit the production of HBeAg. Preisler-Adams and colleagues [32] reported a low in vitro expression of HBeAg by mutant HBV with a point mutation in enhancer II or with deletion in the X gene.

As shown in Figure 1, HBeAg is translated from 3.5-kilobase precore mRNAs [16], which are influenced by enhancer II [21], a core promoter, and an upstream regulatory sequence [15]. The A-to-T point mutation at nucleotide 1762, detected in the core promoter of HBV DNA clones from patients with fulminant hepatitis B, affects the last A of the second AT-rich region (TTAAA). It is 26 to 28 nucleotides upstream of the start points of slightly shorter 3.5-kilobase precore mRNAs.

The AT-rich regions are identified approximately 25 base pairs upstream of the mRNA start points in cellular and viral promoters [33]. Point mutations in the AT-rich region, represented by the TATA sequence motif in eukaryotic promoters, drastically decrease the transcription of mRNA [34]. Mutations in the AT-rich region would prohibit its coupling with TATA-binding protein, which interacts with RNA polymerase II for transcribing mRNAs [35]. It is therefore reasonable to presume that the A-to-T mutation in the second AT-rich region of the core promoter in HBV strains from patients with fulminant hepatitis B would blot the transcription of precore mRNAs and affect the production of HBeAg. The validity of this hypothesis could be evaluated by transfecting hepatoma cell lines with HBV strains that have mutations in the core promoter and then by determining levels of various mRNAs and HBeAg.

In view of the high level of HBV replication in patients with fulminant hepatitis, such mutations would have little effect on the transcription of pregenome-core/polymerase mRNA. It is remarkable that the third AT-rich region (ATAAATT) at 23 nucleotides upstream, which is implicated in the transcription of HBV pregenome [17], was kept intact in all HBV mutants from patients with fulminant hepatitis B. This would explain the separate controls of pregenome-core/polymerase mRNA and precore mRNA, which we postulate may occur in HBV strains that have mutations in the core promoter and cannot direct the production of HBeAg.

Mutations in the core promoter that are unaccompanied by precore mutations, identified in the eight patients with fulminant hepatitis B or acute severe hepatitis, may explain the fact that no precore mutations in HBV DNA clones were reported in some patients with fulminant hepatitis. It would be worthwhile to search for mutations in the core promoter in HBV DNA clones from patients with fulminant hepatitis B in the United States, England, and France who are infected with HBV strains that do not have precore mutations [6, 13, 14].

In France, the point mutation at nucleotide 1896 is rarely seen in HBV DNA clones from carriers, which is ascribed to the predominance of HBV genotype A there [36]. Theoretically, such a mutation converts a pairing between C, making the third letter of codon 15 for proline (CCC) in genotype A, and G as the second letter of codon 28 for tryptophan (TGG) in the precore region to a C-A pair that is incompatible. The mutation would destabilize the stem and loop structure required for the viral encapsidation [37]. In contrast, in HBV genomes of genotype B or C with codon 15 for proline (CCT), A1896 would stabilize the stem and loop structure by creating another Watson-Crick (T-A) pair [38]. Such differences may explain why A1896 is not common in patients with fulminant hepatitis in France or the United States, where HBV of genotype A predominates. Support for this hypothesis is shown by the fact that HBV DNA clones of genotype A from the one patient with fulminant hepatitis in our study lacked A1896; however, all clones had mutations in the core promoter. This suggests that HBV strains with mutations in the core promoter may be a significant cause of fulminant hepatitis B in locations where genotype A is prevalent.