Hepatitis B virus and hepatocellular carcinoma

Hepatitis B is a very important public health problem. Epidemiologic studies have shown a relationship between the hepatitis B virus (HBV) chronic carrier state and the development of hepatocellular carcinoma. HBV belongs to the Hepadna viruses family which includes the woodchuck hepatitis virus (WHV), Woodchucks infected with WHV represent a good experimental model to study the viral oncogenesis. In 85% of the studied cases, WHV acts by insertional mutagenesis in a gene of the myc family, mostly the N-myc2 gene. Expression of the myc genes is increased, suggesting the role of the viral enhancer. Study of transgenic mice have shown the liver specificity of the WHV action. In man, the liver oncogenesis is not explained. Studies are in progress to detect inactivation of tumor suppressor genes.     

Apoptosis is induced by N-myc expression in hepatocytes, a frequent event in hepadnavirus oncogenesis, and is blocked by insulin-like growth factor II

Induction of hepatocellular carcinoma in woodchucks by woodchuck hepatitis virus is associated with the activation of N-myc gene expression, usually by viral DNA integration in cis to the N-myc locus. We have examined the consequences of N-myc up-regulation in rodent hepatic cells in culture. Mouse alpha ML hepatocytes infected with a retroviral vector overexpressing the woodchuck N-myc2 gene display a higher proliferation rate than parental alpha ML cells but are morphologically unchanged and do not form colonies in soft agar. However, they display an increased propensity to undergo apoptosis, an effect that is markedly augmented by serum deprivation. Expression of the woodchuck hepatitis virus X gene in alpha ML cells does not alter the growth phenotype of the cells and has no effect upon N-myc-dependent apoptosis. However, apoptosis in N-myc2-expressing alpha ML cells is strongly inhibited by insulin-like growth factor II (IGF II). IGF II gene expression is also strongly up-regulated during hepatic carcinogenesis in vivo in virally infected animals and has been speculated to be part of an autocrine growth-stimulatory pathway. Our results suggest that IGF II may play another role in the development of virus-induced hepatoma: the prevention of programmed cell death triggered by deregulated N-myc expression.     

Covalently closed circular viral DNA formed from two types of linear DNA in woodchuck hepatitis virus-infected liver

We found that livers from woodchucks chronically infected with woodchuck hepatitis virus (WHV) contained covalently closed circular DNA (cccDNA) molecules with deletions and insertions indicative of their formation from linear viral DNA by nonhomologous recombination, as we previously described for the duck hepatitis B virus (W. Yang and J. Summers, J. Virol. 69:4029-4036, 1995). However, evidence for two different types of linear precursors was obtained by analysis of the recombination joints in WHV cccDNA. Type 1 linear precursors possessed the structural properties that correspond to those of in situ-primed linear DNA molecules, which constitute between 7 and 20% of all viral DNA replicative intermediates synthesized in the liver. Type 2 linear precursors are hypothetical species of linear DNAs with a terminal duplication of the cohesive-end region, between DR1 and DR2. This type of linear DNA has not been previously described and was not detected among the DNA species present in nucleocapsids. A fraction of cccDNAs formed from both type 1 and type 2 linear DNAs are predicted to be functional for further DNA synthesis, and some evidence for the formation of two or more generations of cccDNA from linear DNA was observed.     

Efficacy of the carbocyclic 2'-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection

Daily oral treatment with the cyclopentyl 2'-deoxyguanosine nucleoside BMS-200475 at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduced the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks as measured by reductions in serum WHV DNA levels and endogenous hepadnaviral polymerase activity. Within 4 weeks of daily therapy with 0.5 or 0.1 mg of BMS-200475 per kg, endogenous viral polymerase levels in serum were reduced about 1,000-fold compared to pretreatment levels. Serum WHV DNA levels determined by a dot blot hybridization technique were comparably decreased in these treated animals. In the 3-month study, the sera of animals that had undetectable levels of WHV DNA by the dot blot technique were further analyzed by a highly sensitive semiquantitative PCR assay. The results indicate that BMS-200475 therapy reduced mean WHV titers by 10(7)- to 10(8)-fold, down to levels as low as 10(2) to 10(3) virions/ml of serum. Southern blot hybridization analysis of liver biopsy samples taken from animals during and after BMS-200475 treatment showed remarkable reductions in the levels of WHV DNA replicative intermediates and in the levels of covalently closed circular viral DNA. WHV viremia in BMS-200475-treated WHV carriers eventually returned to pretreatment levels after therapy was stopped. These results indicate that BMS-200475 should be evaluated in clinical trials for the therapy of chronic human hepatitis B virus infections.     

Structure and glycosylation patterns of surface proteins from woodchuck hepatitis virus

Woodchucks chronically infected with woodchuck hepatitis virus (WHV) are a valuable model for human hepatitis B virus (HBV) in studies of pathogenesis, immunity, and antiviral therapy. For this reason, substantial efforts to characterize both the similarities and the differences between HBV and WHV are being made. The structure of the WHV surface proteins (WHs proteins) has not yet been adequately elucidated. The bands that would be expected for glycosylated and nonglycosylated small (S) WHs protein are found by sodium dodecyl sulfate gel electrophoresis of purified WHs protein, but the bands corresponding to the middle (M) and large (L) WHs proteins of HBV are not seen at the expected sizes, even though the sequences of the WHV and HBV surface protein genes are 60% homologous. By amino-terminal sequencing we have identified two bands at 41 and 45 kDa as the MWHs proteins, 8 kDa larger than expected. We have also confirmed that two bands at 24 and 27 kDa are SWHs proteins. A protein of 49 kDa was blocked at the N terminus, which using immunoblotting with an antiserum against WHV pre-S1 (positions 126 to 146) was identified, together with a part of the 45-kDa protein, as glycosylated and nonglycosylated LWHs protein of the expected size. Sialidase and O-glycosidase digestion showed that the larger size of MWHs protein results from the presence of O glycoside groups which are probably in the pre-S2 domain of MWHs protein. Since the pre-S2 domains of HBV and WHV have similar numbers of potential O glycosylation sites, it appears to be likely that the glycosyltransferases act differently on the viral proteins in woodchucks and humans.     

Insulin-like growth factor II blocks apoptosis of N-myc2-expressing woodchuck liver epithelial cells

N-myc2 and insulin-like growth factor II (IGF-II) are coordinately overexpressed in the great majority of altered hepatic foci, which are the earliest precancerous lesions observed in the liver of woodchuck hepatitis virus carrier woodchucks, and these genes continue to be overexpressed in hepatocellular carcinomas (HCCs). We have investigated the function of these genes in woodchuck hepatocarcinogenesis by using a woodchuck liver epithelial cell line (WC-3). WC-3 cells react positively with a monoclonal antibody (12.8.5) against woodchuck oval cells, suggesting a lineage relationship with oval cells. Overexpression of N-myc2 in three WC-3 cell lines caused their morphological transformation and increased their growth rate and saturation density in medium containing 10% serum. Removal of serum from the medium increased cell death of the N-myc2-expressing lines, whereas cell death in control lines was minimal. The death of N-myc2-expressing WC-3 cells was accompanied by nucleosomal fragmentation of cellular DNA, and DAPI (4',6-diamidino-2-phenylindole) staining revealed condensation and fragmentation of the nuclei, suggesting that N-myc2-expressing WC-3 cells undergo apoptosis in the absence of serum. In colony regression assays, conducted in the absence of serum, control colonies were stable, while N-myc2-expressing colonies regressed to various degrees. Addition of recombinant human IGF-II to the serum-free medium blocked both cell death and colony regression in all the N-myc2-expressing lines. Therefore, coordinate overexpression of N-myc2 and IGF-II in woodchuck altered hepatic foci may allow cells which otherwise might die to survive and progress to hepatocellular carcinoma.     

Absence of mutations in the p53 tumor suppressor gene in woodchuck hepatocellular carcinomas associated with hepadnavirus infection and intake of aflatoxin B1

Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered major risk factors in the development of hepatocellular carcinoma (HCC) in humans and in animals. A high rate of mutations in the p53 tumor suppressor gene in hepatocellular carcinomas of predominantly hepatitis B virus (HBV) carrier patients has been recently related to dietary aflatoxin. Another member of the hepadnavirus family, the woodchuck hepatitis virus (WHV), infects woodchucks in a manner similar to that of HBV in humans. Therefore, it was of particular interest to determine whether the p53 gene in woodchuck HCCs associated with hepadnavirus infection and with exposure to AFB1 is affected in the same manner as in human HCCs. By direct PCR-sequencing, we analyzed exons 4-9 of the p53 gene in 13 HCCs from 12 woodchucks (two uninfected, ten WHV carriers). Six WHV carrier and two uninfected woodchucks were treated with AFB1. None of the analyzed HCC samples exhibited mutations, either in p53 gene exons 4-9, or in splicing donor-acceptor sites. The present data are consistent with our previous study that indicated a low rate of p53 mutations in HCCs of AFB1-treated ground squirrels, either infected or not infected with ground squirrel hepatitis virus, and in WHV carrier woodchucks not exposed to AFB1. Overall, our findings indicate that in woodchucks and in ground squirrels exposure to aflatoxin may affect the development of p53 mutations less than in humans.     

The GDPAL region of the pre-S1 envelope protein is important for morphogenesis of woodchuck hepatitis virus

The pre-S envelope protein of duck hepatitis B virus (DHBV) contains a region, Asp-Asp-Pro-Leu-Leu (DDPLL), that is specifically required for virus assembly and secretion (Lenhoff and Summers, J Virol 1994;68:4565-4571). We found that amino acids 201 to 205 of the pre-S envelope protein of woodchuck hepatitis virus (WHV) form a conserved amino acid cluster, Gly-Asp-Pro-Ala-Leu (GDPAL), which resembles the DDPLL sequence of DHBV. To determine whether the GDPAL region was functionally equivalent to the DDPLL region, we deleted this region from the pre-S protein of WHV or mutated individual amino acids within the region. The mutant DNA was transfected into human hepatoma cell line Huh7, and the medium was assayed for virion production by immunoprecipitation and Southern blot analysis. We found that an in-frame deletion of this small region inhibited virion formation, suggesting that the GDPAL region of the pre-S envelope protein was required for virus assembly and/or secretion of WHV. Individual replacement of alanine 204, leucine 205, or serine 206 with other amino acid residues did not affect virus production. However, substitution of either aspartic acid 202 with valine or proline 203 with leucine dramatically inhibited WHV production. Furthermore, the GDPAL mutants were individually tested for their abilities to complement a pre-S1 defective genome. The results showed that the GDPAL region functioned as part of the pre-S1 protein but was not required to function as part of the pre-S2 protein.     

Urea denaturation of staphylococcal nuclease monitored by Fourier transform infrared spectroscopy

Hepadnavirus invasion in woodchucks has been identified as a potent inducer of autoantibodies against asialoglycoprotein receptor (anti-ASGPR), a molecule essentially unique to hepatocytes that mediate clearance of desialylated serum proteins. We evaluated the possible pathogenetic importance of anti-ASGPR triggered by woodchuck hepatitis virus (WHV), using anti-ASGPR-reactive serum immunoglobulins (Igs) from five animals with different stages of WHV hepatitis or self-limited WHV infection and isolated woodchuck hepatocytes or HepG2 cells as targets. The results revealed that WHV-induced anti-ASGPR can specifically inhibit asialoglycoprotein recognition by both homologous and heterologous liver cells, as tested in an asialofetuin (ASFN)-binding radioassay. However, the extent of the interference significantly varied (from 85% inhibition to none) for anti-ASGPR with similar titer from different animals, indicating a high degree of heterogeneity in the ASGPR epitope specificity and in the potential biological effects of these autoantibodies. The WHV-triggered anti-ASGPR also induced complement-mediated hepatocytolysis in a microculture tetrazolium (MTT) assay, which ranged from 8.9% +/- 0.3% to 33.6% +/- 3.6% (mean +/- SD) for different animals and target cell numbers. This cytopathic effect was strictly ASGPR-specific, complement-dependent, and was not related to the anti-ASGPR ability to inhibit ligand-hepatocyte binding. Our findings indicate that among pathways by which anti-ASGPR autoimmunity could cause liver damage, hepadnavirus-induced anti-ASGPR might impair hepatocytes by both disrupting clearance of desialylated proteins and activation of the complement-mediated cytolysis. These cytopathic effects might contribute to the pathogenesis, aggravate severity, and prolong recovery from liver injury in viral hepatitis.     

T-Cell response to woodchuck hepatitis virus (WHV) antigens during acute self-limited WHV infection and convalescence and after viral challenge

The infection of woodchucks with woodchuck hepatitis virus (WHV) provides an experimental model to study early immune responses during hepadnavirus infection that cannot be tested in patients. The T-cell response of experimentally WHV-infected woodchucks to WHsAg, rWHcAg, and WHcAg peptides was monitored by observing 5-bromo-2'-deoxyuridine and [2-3H]adenine incorporation. The first T-cell responses were directed against WHsAg 3 weeks after infection; these were followed by responses to rWHcAg including the immunodominant T-cell epitope of WHcAg (amino acids 97 to 110). Maximal proliferative responses were detected when the animals seroconvered to anti-WHs and anti-WHc (week 6). A decrease in the T-cell response to viral antigens coincided with clearance of viral DNA. Polyclonal rWHcAg-specific T-cell lines were established 6, 12, 18, and 24 weeks postinfection, and their responses to WHcAg peptides were assessed. Five to seven peptides including the immunodominant epitope were recognized throughout the observation period (6 months). At 12 months after infection, T-cell responses to antigens and peptides were not detected. Reactivation of T-cell responses to viral antigens and peptides occurred within 7 days after challenge of animals with WHV. These results demonstrate that a fast and vigorous T-cell response to WHsAg, rWHcAg, and amino acids 97 to 110 of the WHcAg occurs within 3 weeks after WHV infection. The peak of this response was associated with viral clearance and may be crucial for recovery from infection. One year after infection, no proliferation of T cells in response to antigens was observed; however, the WHV-specific T-cell response was reactivated after challenge of woodchucks with WHV and may be responsible for protection against WHV reinfection.     

Role of surgical interventions in the treatment of patients with trophoblastic tumor resistant to chemotherapy

A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.