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PubMed2003

Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver.

Hepatitis B virus (hepadnavirus) infections are maintained by the presence of a small and regulated number of episomal viral genomes [covalently closed circular DNA (cccDNA)] in the nuclei of infected cells. Although a number of studies have measured the mean copy number of cccDNA molecules in hepadnaviral-infected cells, the distribution of individual copy numbers have not been reported. Using a PCR-based assay, we examined the number of cccDNA molecules of the duck hepatitis B virus in single nuclei isolated from the liver of a chronically infected duck over the course of 131 days of infection. Nuclei were isolated from frozen serial biopsies and individually deposited into PCR microplates by flow sorting. Each nucleus was assayed by nested PCR for cccDNA and for cellular IFN-alpha genes as an internal control. We found that 90% of the nuclei assayed contained between 1 and 17 cccDNA molecules, with the remaining 10% containing more (90% confidence), and that differences in the mean number of copies and distribution of copy numbers occurred within the same animal at different times postinfection. Overall, the data suggest (i) that the number of cccDNA molecules per cell may fluctuate over time, and (ii) that, according to these fluctuations, a substantial fraction of cells may contain only one or a few copies. We infer from the results that infected hepatocytes express virus at different levels and that during cell division it is possible to segregate cells containing no cccDNA.

Animals↗
PubMed1990

Mechanism of translation of the hepadnaviral polymerase (P) gene.

Unlike many other reverse transcriptase genes, the polymerase (P) gene of the hepatitis B viruses is expressed by translational initiation from its own AUG codon rather than by ribosomal frameshifting during translation of the overlapping core gene (C). To explore the mechanism of its translation, we have fused the P gene of duck hepatitis B virus to the bacterial lacZ gene at a point 3' to the C-P overlap; this allows detection of the products of P translation with antisera to the lacZ-encoded protein. The C and P/Z coding regions were cloned downstream of a heterologous promoter and expressed in COS-7 cells. A single, bicistronic mRNA containing both C and P sequences is detected in these cells, and translational initiation occurs efficiently at the internally situated P AUG. Mutations affecting C translation have only minimal effects on P expression, in contrast to what would be expected from a modified scanning model for translation. We conclude that P translation depends on a mechanism other than scanning to allow internal entry of ribosomes to the region of the P initiator.

Animals↗
PubMed1992

Frequent amplification of c-myc in ground squirrel liver tumors associated with past or ongoing infection with a hepadnavirus.

Persistent infection with hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC) in humans. HCC has also been observed in animals chronically infected with two other hepadnaviruses: ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus (WHV). A distinctive feature of WHV is the early onset of woodchuck tumors, which may be correlated with a direct role of the virus as an insertional mutagen of myc genes: c-myc, N-myc, and predominantly the woodchuck N-myc2 retroposon. In the present study, we searched for integrated GSHV DNA and genetic alterations of myc genes in ground squirrel HCCs. Viral integration into host DNA was detected in only 3/14 squirrel tumors and did not result in insertional activation of myc genes, despite the presence of a squirrel locus homologous to the woodchuck N-myc2 gene. This suggests that GSHV may differ from WHV in its reduced ability to induce mutagenic integration events. However, the high frequency of c-myc amplification (6/14) observed in ground squirrel HCCs indicates that myc genes might be preferential effectors in the tumorigenic processes associated with rodent hepadnaviruses, a feature not reported so far in HBV-induced carcinogenesis. Together with previous observations, our results suggest that hepadnaviruses, despite close genetic and biological properties, may use different pathways in the genesis of liver cancer.

Animals↗
PubMed1993

Mutant woodchuck hepatitis virus genomes from virions resemble rearranged hepadnaviral integrants in hepatocellular carcinoma.

Although hepadnaviruses are implicated in the etiology of hepatocellular carcinoma, the pathogenic mechanisms involved remain uncertain. Clonally propagated integrations of hepadnaviral DNA into cellular DNA can be demonstrated in most virally induced hepatocellular carcinomas. Integration occurs at random sites in cellular DNA, but the highly preferred sites in viral DNA are adjacent to the directly repeated sequence DR1, less often DR2, or in the cohesive overlap region. Integrants invariably contain simple deletions or complex rearrangements that have been thought to occur after integration. We report here the detection of mutant woodchuck hepatitis virus (WHV) genomes cloned from virions in serum that are strikingly similar to the rearranged hepadnaviral genomes found previously as integrated sequences in cellular DNA. Of 102 cloned genomes studied, 2 had large inverted duplications, 1 a 219-nucleotide direct duplication, and 1 a 219-nucleotide deletion. Virus-virus DNA junctions occurred either adjacent to DR1 or DR2 or in the cohesive overlap region at preferred topoisomerase I cleavage sites. Since these sites are located in the single-stranded regions of the genome, cleavage by topoisomerase I would produce linear molecules that would be expected to be highly recombinogenic since this enzyme, possessing nicking and ligating activities, would remain covalently attached. Sucrose density gradient centrifugation coupled with polymerase chain reaction studies confirmed that the mutant WHV DNA forms resided in virions and did not represent free viral DNA released from infected cells or were unlikely to be an artifact of the cloning process. Thus, the finding in virions of mutant WHV DNA similar to WHV DNA integrated into cellular DNA suggests that the processes of mutation and integration are linked in some instances. Furthermore, the mutant genomes that are preferentially integrated into cellular DNA may have an etiologic role in hepatocarcinogenesis.

Animals↗
PubMed1995

The pre-S domain of the large viral envelope protein determines host range in avian hepatitis B viruses.

In addition to their well-recognized hepatotropism, all hepatitis B viruses (HBVs) display marked species specificity, growing poorly or not at all in species other than those closely related to their natural hosts. We have examined the molecular basis for this narrow host range, using duck HBV (DHBV) and heron HBV (HHBV) as a model system. HHBV virions will not infect ducks in vivo and infect cultured duck hepatocytes extremely inefficiently in vitro. Mutant HHBV genomes lacking all viral envelope proteins (HHBV env-) can be complemented in trans with DHBV envelope proteins; the resulting pseudotyped virions can efficiently infect duck hepatocytes. Further complementation analysis reveals that of the two viral surface proteins (L and S), it is the L protein that determines host range. Pseudotyping of HHBV env- with DHBV/HHBV chimeric envelope proteins reveals that replacement of as few as 69 amino acids of the pre-S domain of the HHBV L protein by their DHBV counterparts is sufficient to permit infection of duck hepatocytes. These studies indicate that the species-specificity of hepadnaviral infection is determined at the level of virus entry and is governed by the pre-S domain of the viral L protein.

Amino Acid Sequence↗
PubMed1998

Liver repopulation with xenogenic hepatocytes in B and T cell-deficient mice leads to chronic hepadnavirus infection and clonal growth of hepatocellular carcinoma.

To investigate host and viral mechanisms determining hepadnaviral persistence and hepatocarcinogenesis, we developed a mouse model by transplanting woodchuck hepatocytes into the liver of mice that contain the urokinase-type plasminogen activator transgene (uPA) and lack mature B and T lymphocytes due to a recombination activation gene 2 (RAG-2) gene knockout. The woodchuck hepatocytes were transplanted via intrasplenic injection and were found to integrate into the recipient mouse liver cord structure. Normal adult woodchuck hepatocytes proliferated and reconstituted up to 90% of the uPA/RAG-2 mouse liver. uPA/RAG-2 mice containing woodchuck hepatocytes were infectable with woodchuck hepatitis virus (WHV) and showed WHV replication for at least 10 months with titers up to 1 x 10(11) virions per ml in the peripheral blood. WHV-infected hepatocytes from chronic carrier woodchucks also established a persistent infection in uPA/RAG-2 mice after an 8- to 12-week lag period of viremia. Although WHV envelope, core, and X proteins were produced in the uPA/RAG-2 mice, no inflammatory host immune response was observed in the liver of WHV-replicating mice. A first antiviral test demonstrated a greater than four orders of magnitude drop in WHV titer in response to interferon alpha treatment. WHV replication was up-regulated by dexamethasone treatment. Comparison of precancerous lesions in donor woodchucks versus recipient uPA/RAG-2 mice revealed an enrichment of dysplastic precancerous hepatocytes in transplanted mice. Clonal amplification of hepatocytes from a woodchuck with hepatocellular carcinomas was demonstrated by the detection of unique WHV DNA integration patterns in hepatocellular carcinomas that arose in uPA/RAG-2 mice. In the absence of B or T cell-mediated immune responses, WHV establishes a persistent noncytotoxic infection of woodchuck hepatocytes in uPA/RAG-2 chimeric mouse livers. Further studies of the kinetics of hepadnavirus infection and replication in quiescent and proliferating hepatocytes should increase our understanding of hepadnavirus spread and aid in the design of therapies to block or cure persistent infection.

Animals↗
PubMed1998

Isolation of a hepadnavirus from the woolly monkey, a New World primate.

Hepatitis B virus (HBV) infections are a major worldwide health problem with chronic infections leading to cirrhosis and liver cancer. Viruses related to human HBV have been isolated from birds and rodents, but despite efforts to find hepadnaviruses that infect species intermediate in evolution between rodents and humans, none have been described. We recently isolated a hepadnavirus from a woolly monkey (Lagothrix lagotricha) that was suffering from fulminant hepatitis. Phylogenetic analysis of the nucleotide sequences of the core and surface genes indicated that the virus was distinct from the human HBV family, and because it is basal (ancestral) to the human monophyletic group, it probably represents a progenitor of the human viruses. This virus was designated woolly monkey hepatitis B virus (WMHBV). Analysis of woolly monkey colonies at five zoos indicated that WMHBV infections occurred in most of the animals at the Louisville zoo but not at four other zoos in the United States. The host range of WMHBV was examined by inoculation of one chimpanzee and two black-handed spider monkeys (Ateles geoffroyi), the closest nonendangered relative of the woolly monkey. The data suggest that spider monkeys are susceptible to infection with WMHBV and that minimal replication was observed in a chimpanzee. Thus, we have isolated a hepadnavirus with a host intermediate between humans and rodents and establishes a new animal model for evaluation of antiviral therapies for treating HBV chronic infections.

Animals↗
PubMed2002

Role of p50/CDC37 in hepadnavirus assembly and replication.

The cellular chaperone Hsp90 has been shown to associate with the reverse transcriptase (RT) of the duck hepatitis B virus and is required for RT functions. However, the molecular basis for the specific interaction between the RT and Hsp90 remains unknown. Comparison of protein compositional properties suggests that the RT is highly related to the protein kinase c-Raf, which interacts with Hsp90 via the cochaperone p50 (CDC37). We tested whether the RT, like c-Raf, is specifically recognized by p50. Immunoprecipitation and pull-down assays showed that p50 or p50deltaC, a p50 mutant defective in Hsp90 binding, could interact specifically with the RT both in vitro and in vivo, indicating that p50 can bind the RT independently of Hsp90. Furthermore, purified p50 and p50deltaC interacted directly with purified RT. The importance of p50-RT interaction for RT functions was underscored by 1) inhibition of protein-primed initiation of reverse transcription by p50deltaC in vitro and 2) stimulation of viral DNA replication and RNA packaging by p50 and their inhibition by p50deltaC in transfected cells. These results suggest that p50 can function as a cellular cofactor for the hepadnavirus RT by mediating the interaction between the RT and Hsp90.

Animals↗
PubMed2004

6-[2-phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines: a new class of acyclic pyrimidine nucleoside phosphonates with antiviral activity.

Acyclic nucleoside phosphonate derivatives containing a pyrimidine base preferably bearing amino groups at C-2 and C-4 (DAPym), and linked at the C-6 position to (S)-[3-hydroxy-2-(phosphonomethoxy)propoxy] (HPMPO), 2-(phosphonomethoxy) ethoxy (PMEO) or (R)-[2-(phosphonomethoxy)propoxy] (PMPO), display an antiviral sensitivity spectrum that closely mimic that of the parental (S)-HPMP-, PME- and (R)-PMP-purine derivatives. Several PMEO-DAPym derivatives proved as potent as PMEA (adefovir) and (R)-PMPA (tenofovir) in inhibiting Moloney murine sarcoma virus (MSV)-induced tumor formation in newborn NMRI mice. The HPMPO-, PMEO- and PMPO-DAPym derivatives represent a novel well-defined subclass among the acyclic nucleoside phosphonates endowed with potent and selective antiviral activity.

Adenine↗
PubMed1998

Treatment of duck hepatitis B virus by antisense poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotides.

The poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotide (poly-DNP-RNA) with antisense sequence 5'ggguguauggaaaagccguc-3' was designed to target the sequence 2468-2487 in the polymerase gene of duck hepatitis B virus (DHBV). The stereochemically pure RNA was synthesized by using T7 RNA polymerase with synthetic DNA template and subsequently derivatized with 2,4-dinitrofluorobenzene in mild basic conditions to make the poly-DNP-RNA with an average DNP/base ratio of 0.7. In vitro studies showed that this antisense poly-DNP-RNA can hybridize with sense DNA and has high resistance to RNase A digestion. These poly-DNP-RNA were also found to be potent sequence-independent inhibitors of the reverse transcriptase activity of DHBV DNA-polymerase. For in vivo studies, DHBV-infected ducks were treated with antisense, sense, and random noncomplementary sequence poly-DNP-RNA, respectively. The data showed that the antisense poly-DNP-RNA completely inhibited the duck viremia in all nine ducks that had been treated with a dose of 1 mg/kg (i.v.) per day for 25 days. The viremia did not come back after 10 months recession. In the sense group, three of the four ducks showed no inhibition, and in the random group, both ducks maintained their viremia. After 45 days of treatment with the antisense poly-DNP-RNA, followed by 2 weeks of recession, PCR as well as QC-PCR assay and microscopic examination showed that viral DNA had disappeared in liver and that the histology of the damaged liver (filled with fat granules) had returned to normal.

Animals↗
PubMed2000

The complexities of genome analysis, the Retroid agent perspective.

MOTIVATION: The sequences of Retroid agents from a wide diversity of organisms constitute the largest set of complete genomes currently available for the study of genomic architecture and the transfer of information within and between organisms. These agents are ubiquitous in Eukaryotes, comprising 50-90% of the genomic information in some cases. RESULTS: Analyses conducted for over a decade illustrate that Retroid agents are engaged in a wide spectrum of molecular evolutionary events. A description of these complexities is presented as a three parameter conceptual framework that considers type, size, and mechanism of events that contribute to the evolution of genes, genomes, and organisms. The results of new data mining studies further illustrate the complexity of the network of relationships among and between Retroid agents and other organisms. A hidden Markov model construction strategy is presented that generates a multiple alignment more similar to those refined by human experts. CONTACT: mars@parvati.msu.montana. edu

Animals↗
PubMed1989

In vitro production of infectious woodchuck hepatitis virus by lipopolysaccharide-stimulated peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) from a wild-caught woodchuck (WC192) chronically infected with woodchuck hepatitis virus (WHV) carried low levels of nonreplicating WHV genomes. In a previous study, these WHV genomes were induced to replicate and intact WHV particles were released when these PBLs were cultured in the presence of the generalized mitogen, lipopolysaccharide (LPS). To determine whether the culture-derived WHV particles were infectious, adult woodchucks were inoculated with cell-free culture medium from either LPS-stimulated or unstimulated WC192 PBLs. None of three animals inoculated with medium from the unstimulated PBL cultures developed positive WHV serologic markers or elevated liver enzyme levels during a 42-w observation period. In contrast, all three animals that received medium from the LPS-stimulated cultures displayed serologic markers of acute WHV infections 8-10 w after inoculation at up to a 100-fold dilution of the original culture medium. One of the three infected animals developed an acute hepatitis coincident with the appearance of the WHV markers. These results demonstrate that the WHV particles released from LPS-stimulated WC192 PBLs in culture are mature, infectious virions that also cause liver disease. Thus the LPS-induced replication of WHV in these cell cultures represents a transition from a latent to productive viral infection.

Animals↗
PubMed1992

Immunosuppression with cyclosporine during the incubation period of experimental woodchuck hepatitis virus infection increases the frequency of chronic infection in adult woodchucks.

The immunosuppressive agent cyclosporine was given to adult woodchucks during acute experimental infection with woodchuck hepatitis virus (WHV). All 17 woodchucks given WHV alone or with a vehicle resolved the infection (i.e., zero chronicity), but when cyclosporine was given throughout the incubation and acute phases of infection (0-12 or 14 weeks; n = 12), the rate of chronic infection increased to 92%. When cyclosporine was given only during the incubation period (0-4 weeks; n = 10) or only during the acute phase of infection (2-12 weeks; n = 9), the rates increased to 50% and 55%, respectively. However, when the drug was given after the acute phase (8-18 weeks; n = 9), the chronic infection rate (11%) did not differ from that in untreated and vehicle controls. Immune responses inhibited by cyclosporine are important in resolution of acute WHV infection and occur mainly during the first 8 weeks. Immunosuppression of these responses for even short intervals during incubation (e.g., 0-4 weeks) increases the risk of chronicity.

Animals↗
PubMed1993

Chemical disinfection of duck hepatitis B virus: a model for inactivation of infectivity of hepatitis B virus.

The susceptibility of duck hepatitis B virus (DHBV) to the virucidal effects of sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) was compared to hepatitis B virus (HBV) with the aim of using the duck as a model for studying HBV disinfection. Using viral DNA polymerase (DNAP) as a target, inhibition of DNAP activity by chlorine disinfectants was found to be concentration-dependent but independent of contact time. Two minute exposure of minimal effective concentrations of sodium hypochlorite (domestic bleach: 3600 ppm and industrial bleach: 3180 ppm) and sodium dichloroisocyanurate (3000 ppm available chlorine) to DHBV- and HBV-rich plasma totally inhibited DNA polymerase activity. DHBV particles in DHBV-carrier duck plasma (10(4.5) ID50/mL) were treated with these concentrations and inoculated intravenously into 18 one-day old ducklings (six animals/disinfectant). Analysis of plasma (0, 7 and 14 days post-infection) and post-mortem liver (14 days post-infection) by DNA hybridization techniques showed that DHBV DNA was undetectable in samples from all animals inoculated with disinfected virus particles. However, post-inoculation plasma and liver of 18 of 18 control ducklings inoculated with untreated virions were positive for DHBV DNA. These results show for the first time that total inhibition in vitro of hepadnavirus DNA polymerase activity by chemical disinfectants is predictive of inactivation of infectivity in vivo.

Animals↗
PubMed2000

Inactivation of a hepadnavirus by electrolysed acid water.

Glutaraldehyde is used as a disinfectant for endoscopes, but is an irritant and so should be replaced by an alternative. Electrolysed acid water (EAW) has a bactericidal effect, and an endoscopic washing device using EAW has been developed in Japan. To investigate the effect of EAW on the infectivity of viruses, we treated duck hepatitis B virus (DHBV), which has similar properties to hepatitis B virus, with EAW, and determined the number of remaining infectious virus particles in a bioassay system. One-day-old Pekin ducks were inoculated with duck serum containing 10(5.5) ID(50) DHBV; the serum had previously been incubated with 100 volumes of EAW or ion-exchanged water at room temperature for 7 min. DHBV infection was indicated by detection of viral DNA in duck serum samples 1-8 weeks after inoculation. Treatment of serum with EAW diminished DHBV infectivity whereas treatment with ion-exchanged water did not. The virus load was estimated to have been reduced to 10(1)-10(3) ID(50) during the first 1 min and to <10(0.5) ID(50) in the next 6 min of incubation when compared with the control. Thus, EAW directly inactivates DHBV and its clinical application is recommended.

Animals↗
PubMed2005

Translation of stable hepadnaviral mRNA cleavage fragments induced by the action of phosphorothioate-modified antisense oligodeoxynucleotides.

Phosphorothioate-modified antisense oligodeoxynucleotides (ASOs) are used to suppress gene expression by inducing RNase H-mediated cleavage with subsequent degradation of the target mRNA. However, previous observations suggest that ASO/RNase H can also result in the generation of stable mRNA cleavage fragments and expression of truncated proteins. Here, we addressed the underlying translational mechanisms in more detail using hepadnavirus-transfected hepatoma cells as a model system of antisense therapy. Generation of stable mRNA cleavage fragments was restricted to the ASO/RNase H pathway and not observed upon cotransfection of isosequential small interfering RNA or RNase H-incompetent oligonucleotides. Furthermore, direct evidence for translation of mRNA fragments was established by polysome analysis. Polysome-associated RNA contained cleavage fragments devoid of a 5' cap structure indicating that translation was, at least in part, cap-independent. Further analysis of the uncapped cleavage fragments revealed that their 5' terminus and initiation codon were only separated by a few nucleotides suggesting a 5' end-dependent mode of translation, whereas internal initiation could be ruled out. However, the efficiency of translation was moderate compared to uncleaved mRNA and amounted to 13-24% depending on the ASO used. These findings provide a rationale for understanding the translation of mRNA fragments generated by ASO/RNase H mechanistically.

5' Untranslated Regions↗