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PubMed1992

Hepadnavirus integration: mechanisms of activation of the N-myc2 retrotransposon in woodchuck liver tumors.

In persistent hepadnavirus infections, a distinctive feature of woodchuck hepatitis virus (WHV) is the coupling of frequent viral integrations into myc family genes with the rapid onset of primary liver tumors. We have investigated the patterns of WHV DNA insertion into N-myc2, a newly identified retroposed oncogene, in woodchuck hepatomas resulting from either natural or experimental infections. In both cases, integrated viral sequences were preferentially associated with the N-myc2 locus. In more than 40% of the woodchuck tumors analyzed, viral insertion sites were clustered in a 3-kb region upstream of N-myc2 or in the 3' noncoding region. Insertion of WHV sequences homologous to the human hepatitis B virus enhancers, either upstream or downstream of the N-myc2 coding domain, was associated with the production of normal N-myc2 mRNA or hybrid N-myc2-WHV transcripts, initiated at the normal N-myc2 transcriptional start site. Transient-transfection assays with different N-myc2-WHV constructs in HepG2 cells demonstrated that the viral enhancers could efficiently activate the N-myc2 promoter. These results, showing that cis activation of preferred cellular targets through enhancer insertion is a common strategy for tumor induction by WHV, emphasize the previously noted similarities between hepadnaviruses and nonacute oncogenic retroviruses.

Animals↗
PubMed1992

The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host.

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.

Animals↗
PubMed1993

Hepadnavirus infection requires interaction between the viral pre-S domain and a specific hepatocellular receptor.

To better define the molecules involved in the initial interaction between hepadnaviruses and hepatocytes, we performed binding and infectivity studies with the duck hepatitis B virus (DHBV) and cultured primary duck hepatocytes. In competition experiments with naturally occurring subviral particles containing DHBV surface proteins, these DNA-free particles were found to interfere with viral infectivity if used at sufficiently high concentrations. In direct binding saturation experiments with radiolabelled subviral particles, a biphasic titration curve containing a saturable component was obtained. Quantitative evaluation of both the binding and the infectivity data indicates that the duck hepatocyte presents about 10(4) high-affinity binding sites for viral and subviral particles. Binding to these productive sites may be preceded by reversible virus attachment to a large number of less specific, nonsaturable primary binding sites. To identify which of the viral envelope proteins is responsible for hepatocyte-specific attachment, subviral particles containing only one of the two DHBV surface proteins were produced in Saccharomyces cerevisiae. In infectivity competition experiments, only particles containing the large pre-S/S protein were found to markedly reduce the efficiency of DHBV infection, while particles containing the small S protein had only a minor effect. Similarly, physical binding of radiolabelled serum-derived subviral particles to primary duck hepatocytes was inhibited well only by the yeast-derived pre-S/S particles. Together, these results strongly support the notion that hepadnaviral infection is initiated by specific attachment of the pre-S domain of the large DHBV envelope protein to a limited number of hepatocellular binding sites.

Animals↗
PubMed1993

Analysis of the earliest steps of hepadnavirus replication: genome repair after infectious entry into hepatocytes does not depend on viral polymerase activity.

Hepadnaviruses contain a relaxed circular DNA genome (RC-DNA) with discontinuities in both strands. Upon infectious entry into a host cell, this genome is converted into a covalently closed superhelical form (CCC-DNA), which later serves as the template for transcription. Here we examined whether the viral polymerase activity is required for this repair reaction. Primary hepatocytes prepared from embryonated duck eggs were infected with the duck hepatitis B virus. Conversion of the RC-DNA into the CCC-DNA was then analyzed by a newly developed polymerase chain reaction technique. This method allows the efficient discrimination between the two DNA forms and is sensitive enough to monitor repair of the infecting viral DNA in the absence of replication and amplification. Thus, we were able to monitor this process in the presence of a potent inhibitor of the viral polymerase, the nucleoside analog 2',3'-dideoxyguanosine. The data show that inhibition of the viral polymerase activity has no influence on genome repair, suggesting that this enzymatic function is not required for conversion of the RC-DNA into the CCC-DNA. Consequently, antiviral drugs blocking the polymerase activity cannot prevent the infectious entry of the virus into a host cell.

Animals↗
PubMed1994

Evidence that hepatocyte turnover is required for rapid clearance of duck hepatitis B virus during antiviral therapy of chronically infected ducks.

Duck hepatitis B virus (DHBV) DNA synthesis in congenitally infected ducks is inhibited by 2'-deoxycarbocyclic guanosine (2'-CDG). Three months of therapy reduces the number of infected hepatocytes at least 10-fold (W.S. Mason, J. Cullen, J. Saputelli, T.-T. Wu, C. Liu, W.T. London, E. Lustbader, P. Schaffer, A.P. O'Connell, I. Fourel, C.E. Aldrich, and A.R. Jilbert, Hepatology 19:393-411, 1994). The present study was performed to determine the kinetics of disappearance of infected hepatocytes and to evaluate the role of hepatocyte turnover in this process. Essentially all hepatocytes were infected before drug therapy. Oral treatment with 2'-CDG resulted in a prompt reduction in the number of infected hepatocytes. After 2 weeks, only 30 to 50% appeared to still be infected, and less than 10% were detectably infected after 5 weeks of therapy. To assess the possible role of hepatocyte turnover in these changes, 5-bromo-2'-deoxyuridine (BUdR) was administered 8 h before liver biopsy to label host DNA in hepatocytes passing through S phase, and stained nuclei were detected in tissue sections by using an antibody reactive to BUdR. The extent of nuclear labeling after 5 weeks was the same as that before therapy (ca. 1%). However, biopsies taken after 2 weeks of therapy showed a ca. 10-fold elevation in the number of nuclei labeled with BUdR. This result suggested that a rapid clearance of infected hepatocytes by 2'-CDG was caused not just by the inhibition of viral replication but also by an acceleration of the rate of hepatocyte turnover. To test this possibility further, antiviral therapy was carried out with another strong inhibitor of DHBV DNA synthesis, 5-fluoro-2',3'-dideoxy-3'-thiacytidine (524W), which did not accelerate hepatocyte turnover in ducks. 524W administration led to a strong inhibition of virus production but to a slower rate of decline in the number of infected hepatocytes, so that ca. 50% (and perhaps more) were still infected after 3 months of therapy. In addition, histopathologic evaluation of 2'-CDG-treated ducks revealed liver injury, especially at the start of therapy. No liver damage was observed during 524W therapy. These results imply that clearance of infected hepatocytes from the liver is correlated with hepatocyte turnover. Thus, in the absence of immune clearance or other sources for the accelerated elimination of infected hepatocytes, inhibitors of virus replication would have to be administered for a long period to substantially reduce the burden of infected hepatocytes in the liver.

Animals↗
PubMed1995

Illegitimate replication of linear hepadnavirus DNA through nonhomologous recombination.

Linear hepadnavirus DNA in primary hepatocyte cultures efficiently participates in intra- and intermolecular nonhomologous recombination at its ends. The products of this recombination are (i) monomeric covalently closed circular DNAs (cccDNAs) with deletions and insertions around the site of joining and (ii) oligomeric forms in which monomers are joined near the ends in random orientation. A fraction of monomeric cccDNAs can serve as intermediates in further DNA replication through at least five generations of nonhomologous recombination in a process we call illegitimate replication. We suggest that the monomeric and oligomeric linear DNAs produced by illegitimate replication may be precursors of the integrated and other high-molecular-weight hepadnaviral DNA forms seen in chronic infection.

Animals↗
PubMed1995

Susceptibility to duck hepatitis B virus infection is associated with the presence of cell surface receptor sites that efficiently bind viral particles.

To test the hypothesis that susceptibility of hepatocytes to duck hepatitis B virus (DHBV) infection requires cell surface receptors that bind the virus in a specific manner, we developed an assay for the binding of DHBV particles to monolayers of intact cells, using radiolabeled immunoglobulin G specific for DHBV envelope protein. Both noninfectious DHBV surface antigen particles and infectious virions bound to a susceptible fraction (approximately 60%) of Pekin duck hepatocytes. In contrast, binding did not occur to cells that were not susceptible to DHBV infection, including Pekin duck fibroblasts and chicken hepatocytes, and binding to Muscovy duck hepatocytes, which are only weakly susceptible (approximately 1% of cells) to DHBV infection, was virtually undetectable. Within a monolayer, individual Pekin duck hepatocytes appeared to differ markedly in the capacity to bind DHBV, which may explain difficulties that have been encountered in infecting 100% of cells in culture. We have also found that the loss of susceptibility to infection with DHBV that occurs when Pekin duck hepatocytes are maintained for more than a few days in culture correlates with a decline in the number of cells that bind virus particles efficiently. All of these results support the interpretation that the binding event detected by our assay is associated with the interaction between DHBV and specific cell surface receptors that are required for initiation of infection. Our assay may facilitate isolation and identification of hepatocyte receptors for this virus.

Animals↗
PubMed1996

Amino acids essential for RNase H activity of hepadnaviruses are also required for efficient elongation of minus-strand viral DNA.

The hepadnavirus P gene contains amino acid sequences which share homology with all known RNases H. In this study, we made four mutants in which single amino acids of the duck hepatitis B virus (DHBV) RNase H region were altered. In two of them, amino acids at locations comprising the putative catalytic site were changed, while the remaining mutants had alterations at amino acids conserved among hepadnaviruses. Transfection of these mutant genomes into permissive cells resulted in synthesis of several discrete viral nucleic acid species, ranging in apparent sizes from approximately 500 to 3,000 bp, numbered I, II, III, IV, and V. While the locations of the species were similar in all mutants, the proportions of the species varied among the mutants. Analysis of the nucleic acid species revealed that they were hybrid molecules of RNA and minus-strand DNA, indicating that the RNase H activity was missing or greatly reduced in these mutants. Primer extension experiments showed that the mutant viruses initiated minus-strand viral DNA synthesis normally. The 3' termini of minus-strand DNA in species II, III, and IV were mapped just downstream of nucleotides 1659, 1220, and 721, respectively. Species V contained essentially full-length minus-strand viral DNA. A parallel amino acid change in the putative catalytic site of the HBV RNase H domain resulted in accumulation of low-molecular-weight hybrid molecules consisting of RNA and minus-strand DNA and similar in size and pattern to those seen with DHBV. These studies demonstrate experimentally the involvement of the C-terminal portion of the P gene in RNase H activity in both DHBV and human hepatitis B virus and indicate that the amino acids essential for RNase H activity of hepadnavirus P protein are also important for the efficient elongation of minus-strand viral DNA.

Amino Acid Sequence↗
PubMed1997

Interaction of the UV-damaged DNA-binding protein with hepatitis B virus X protein is conserved among mammalian hepadnaviruses and restricted to transactivation-proficient X-insertion mutants.

We carried out a comparative analysis of several proposed host protein partners of the human hepatitis B virus X protein (HBx) using both the GAL4- and the LexA-based yeast two-hybrid system. We showed that the interaction of HBx with the UV-damaged DNA-binding protein (UVDDB) is positive in both yeast systems, detectable in cotransfected human cells, conserved by rodent hepadnavirus X proteins (known to transactivate in human cells), and tightly correlated with the transactivation proficiency of X-insertion mutants. Taken together, our results strongly suggest that UVDDB is involved in X-mediated transactivation.

Binding Sites↗
PubMed1998

Protective efficacy of DNA vaccines against duck hepatitis B virus infection.

The efficacy of DNA vaccines encoding the duck hepatitis B virus (DHBV) pre-S/S and S proteins were tested in Pekin ducks. Plasmid pcDNA I/Amp DNA containing the DHBV pre-S/S or S genes was injected intramuscularly three times, at 3-week intervals. All pre-S/S and S-vaccinated ducks developed total anti-DHBs and specific anti-S antibodies with similar titers reaching 1/10,000 to 1/50,000 and 1/2,500 to 1/4,000, respectively, after the third vaccination. However, following virus challenge, significant differences in the rate of virus removal from the bloodstream and the presence of virus replication in the liver were found between the groups. In three of four S-vaccinated ducks, 90% of the inoculum was removed between <5 and 15 min postchallenge (p.c.) and no virus replication was detected in the liver at 4 days p.c. In contrast, in all four pre-S/S-vaccinated ducks, 90% of the inoculum was removed between 60 and 90 min p.c. and DHBsAg was detected in 10 to 40% of hepatocytes. Anti-S serum abolished virus infectivity when preincubated with DHBV before inoculation into 1-day-old ducklings and primary duck hepatocyte cultures, while anti-pre-S/S serum showed very limited capacity to neutralize virus infectivity in these two systems. Thus, although both DNA vaccines induced high titers of anti-DHBs antibodies, anti-S antibodies induced by the S-DNA construct were highly effective in neutralizing virus infectivity while similar levels of anti-S induced by the pre-S/S-DNA construct conferred only very limited protection. This phenomenon requires further clarification, particularly in light of the development of newer HBV vaccines containing pre-S proteins and a possible discrepancy between anti-HBs titers and protective efficacy.

Animals↗
PubMed1998

Avian hepatitis B virus infection is initiated by the interaction of a distinct pre-S subdomain with the cellular receptor gp180.

Functionally relevant hepadnavirus-cell surface interactions were investigated with the duck hepatitis B virus (DHBV) animal model by using an in vitro infection competition assay. Recombinant DHBV pre-S polypeptides, produced in Escherichia coli, were shown to inhibit DHBV infection in a dose-dependent manner, indicating that monomeric pre-S chains were capable of interfering with virus-receptor interaction. Particle-associated pre-S was, however, 30-fold more active, suggesting that cooperative interactions enhance particle binding. An 85-amino-acid pre-S sequence, spanning about half of the DHBV pre-S chain, was characterized by deletion analysis as essential for maximal inhibition. Pre-S polypeptides from heron hepatitis B virus (HHBV) competed DHBV infection equally well despite a 50% difference in amino acid sequence and a much-reduced infectivity of HHBV for duck hepatocytes. These observations are taken to indicate (i) that the functionality of the DHBV pre-S subdomain, which interacts with the cellular receptor, is determined predominantly by a defined three-dimensional structure rather than by primary sequence elements; (ii) that cellular uptake of hepadnaviruses is a multistep process involving more than a single cellular receptor component; and (iii) that gp180, a cellular receptor candidate unable to discriminate between DHBV and HHBV, is a common component of the cellular receptor complex for avian hepadnaviruses.

Amino Acid Sequence↗
PubMed1998

Infection of ducklings with virus particles containing linear double-stranded duck hepatitis B virus DNA: illegitimate replication and reversion.

Double-stranded linear DNA is synthesized as a minor viral DNA species by all hepadnaviruses. In a previous study (W. Yang and J. Summers, J. Virol. 69:4029-4036, 1995) we showed that virus particles containing linear DNA of the duck hepatitis B virus (DHBV) could initiate an infection of primary duck hepatocytes. In cells infected by linear DNA containing viruses the transcriptional template, covalently closed circular DNA, was formed by circularization of linear DNA by nonhomologous recombination between the two ends. This process was shown to result in viral DNA replication through multiple generations of linear DNA intermediates, a process we called illegitimate replication. In this study we showed that viruses containing linear DHBV DNA produced by engineered insertions in the r sequence, which encodes the 5' end of the pregenome, could infect hepatocytes in vivo, and these hepatocytes proceeded to carry out illegitimate replication. Nonhomologous recombination quickly produced revertants and partial revertants in which all or part of the insertion was deleted. One such partial revertant that replicated primarily through circular DNA intermediates, but which synthesized elevated levels of linear DNA, could be sustained for several days as the predominant genotype in vivo, but this mutant was eventually displaced by variants showing full reversion to legitimate replication and that synthesized normal low levels of linear DNA. Full revertants did not necessarily contain the wild-type r sequence. The results suggest that the linear DNA produced during DHBV infection initiates cycles of illegitimate replication by generating mutants with altered r sequences. Some r sequence mutants carry out a mixture of legitimate and illegitimate replication that can contribute to elevated production of linear DNA in individual cells.

Animals↗
PubMed1998

Glucagon treatment interferes with an early step of duck hepatitis B virus infection.

The effect of glucagon on the establishment of hepadnavirus infection was studied in vitro with the duck hepatitis B virus (DHBV) model. The presence of the peptide hormone throughout infection or starting up to 8 h after virus uptake resulted in a dose-dependent reduction in the levels of intra- and extracellular viral gene products and of secreted virions. Treatment with forskolin or dibutyryl-cyclic AMP, two drugs that also stimulate the cyclic AMP (cAMP) signal transduction pathway, resulted in comparable inhibition, suggesting that the inhibitor effect is related to changes in the activity of protein kinase A. In persistently infected hepatocytes, only a slight, but continuous, decrease in viral replication was observed upon prolonged drug treatment. Time course analysis, including detection of DHBV covalently closed circular (ccc) DNA templates, revealed that glucagon acts late during the establishment of infection, at a time when the virus is already internalized, but before detectable ccc DNA accumulation in the nucleus. These data suggest that nuclear import (and reimport) of DHBV DNA genomes from cytosolic capsids is subject to cAMP-mediated regulation by cellular factors responding to changes in the state of the host cell.

Animals↗
PubMed1999

Integration of hepadnavirus DNA in infected liver: evidence for a linear precursor.

DNA of the avian hepadnavirus, duck hepatitis B virus, was found to be integrated at low abundance into the cellular DNA extracted from the livers of infected ducklings. The frequency of integration was estimated to be at least one viral genome per 10(3) to 10(4) cells by 6 days postinfection. The structures of virus-cell junctions determined by sequencing were compared with those of virus-virus junctions formed by nonhomologous recombination between the ends of linear viral DNA forms. This comparison allowed us to conclude that linear viral DNA was the preferential form used as an integration substrate. Potential factors promoting viral DNA integration during chronic infection are discussed.

Animals↗
PubMed1999

Residues critical for duck hepatitis B virus neutralization are involved in host cell interaction.

To date, no detailed analysis of the neutralization properties of duck hepatitis B virus (DHBV) has been reported, and it is not clear whether any of the known neutralization epitopes correspond to the viral receptor binding site or to sequences involved in the cell entry pathway. We demonstrate here that antibodies directed against two overlapping peptides (amino acids 83 to 97 and 93 to 107), covering the sequences of most DHBV pre-S neutralizing epitopes, both inhibit virus binding to primary duck hepatocytes and neutralize virus infectivity. An extensive mutagenesis of the motif 88WTP90, which is the shortest sequence of the epitope recognized by the virus-neutralizing monoclonal antibody (MAb) 900 was performed in order to define the amino acids involved in these interactions. Single point mutations within this epitope affected neither virus replication nor infectivity but abolished virus neutralization by MAb 900 completely. Interestingly, mutants with two and three consecutive residue replacements (SIP and SIH) within this epitope retained replication competence but were no longer infectious. The loss of infectivity of SIH and SIP mutant particles was associated with significantly reduced binding to primary duck hepatocytes and could be rescued by trans complementation with wild-type pre-S protein. Taken together, these results indicate that each amino acid of the DHBV pre-S sequence 88WTP90 is critical for recognition by the neutralizing MAb 900 and that replacement of the first two or all three residues strongly reduces virus interaction with hepatocytes and abrogates infectivity. These data imply that the motif 88WTP90 contains key residues which are critical for interaction with both the neutralizing MAb and the host cell.

Animals↗
PubMed1999

Enrichment of a precore-minus mutant of duck hepatitis B virus in experimental mixed infections.

A precore-deficient mutant of duck hepatitis B virus (DHBV) produced by site-directed mutagenesis was tested for its ability to compete with wild-type virus in a mixed infection of 3-day-old ducklings. The mutation was shown to produce a cis-acting defect, resulting in a replication rate that was about one-half that of wild-type virus. Accordingly, wild-type virus was rapidly selected during the spread of infection. During the chronic phase of the infection, however, two selection patterns were seen. In 4 of 10 ducks, the wild-type virus slowly replaced the precore mutant. In another four ducks, the precore mutant virus slowly replaced the wild-type virus. In the remaining two ducklings, ratios of wild-type and precore mutant virus fluctuated, with wild-type virus slowly predominating. The replacement of wild-type virus was not due to the emergence of a rapidly replicating variant of the precore mutant, since genomes cloned from the infected ducks retained their original replication defect. Replacement of wild-type virus, however, correlated with elevated anti-core antibody titers, which continued to increase with time. The selection of a precore-negative strain of DHBV may be analogous to the selection for precore mutants of HBV during chronic hepatitis in humans.

Animals↗
PubMed2000

Maternally transferred antibodies from DNA-immunized avians protect offspring against hepadnavirus infection.

The outcome and protective efficacy of maternal antibodies elicited by DNA immunization to the large (L) hepadnavirus envelope protein were studied using the duck hepatitis B virus (DHBV) model. Following genetic immunization of breeding ducks with a DHBV L protein gene-bearing plasmid, specific and highly neutralizing antibodies were transferred from the sera of immunized ducks, via the egg yolk, to the progeny of vaccinees. Interestingly, large amounts (60 to 100 mg/egg) of high-titer and L protein-specific yolk immunoglobulins (immunoglobulin Y) accumulated in the egg yolk. These results suggest that eggs from genetically immunized avians may represent a potent source of DNA-designed antibodies specific to viral antigen. Importantly, these antibodies are vertically transmitted and protect offspring against high-titer DHBV challenge.

Animals↗
PubMed2000

Low dynamic state of viral competition in a chronic avian hepadnavirus infection.

The dynamic state of infection of 11 ducks with the duck hepatitis B virus was investigated. Chronic infections were established in newly hatched ducklings by inoculation with a mixture of wild-type virus and a mutant virus with a partial replication defect. As expected, the wild-type virus was rapidly enriched in the virus population during the spread of infection. Enrichment thereafter was correlated with normal growth of the liver, with the average mutant-to-wild-type ratio stabilizing for at least 2 months beyond the time at which the liver mass stabilized. Using experimentally determined growth rates for the mutant and wild-type viruses, we estimated that after the spread of infection, competition between the two virus strains was limited by the amount of replication required to infect new hepatocytes in the growing livers. The results suggest that, in a chronically infected liver, the selection of variants with a replication rate advantage is inefficient and that the emergence of such variants would depend on induced liver cell turnover, such as that occurring during chronic hepatitis.

Animals↗