Duck Hepatitis B Virus Core Research Articles

Therapeutic Antiviral Effect of the Nucleic Acid Polymer REP 2055 against Persistent Duck Hepatitis B Virus Infection.

Previous studies have demonstrated that nucleic acid polymers (NAPs) have both entry and post-entry inhibitory activity against duck hepatitis B virus (DHBV) infection. The inhibitory activity exhibited by NAPs prevented DHBV infection of primary duck hepatocytes in vitro and protected ducks from DHBV infection in vivo and did not result from direct activation of the immune response. In the current study treatment of primary human hepatocytes with NAP REP 2055 did not induce expression of the TNF, IL6, IL10, IFNA4 or IFNB1 genes, confirming the lack of direct immunostimulation by REP 2055. Ducks with persistent DHBV infection were treated with NAP 2055 to determine if the post-entry inhibitory activity exhibited by NAPs could provide a therapeutic effect against established DHBV infection in vivo. In all REP 2055-treated ducks, 28 days of treatment lead to initial rapid reductions in serum DHBsAg and DHBV DNA and increases in anti-DHBs antibodies. After treatment, 6/11 ducks experienced a sustained virologic response: DHBsAg and DHBV DNA remained at low or undetectable levels in the serum and no DHBsAg or DHBV core antigen positive hepatocytes and only trace amounts of DHBV total and covalently closed circular DNA (cccDNA) were detected in the liver at 9 or 16 weeks of follow-up. In the remaining 5/11 REP 2055-treated ducks, all markers of DHBV infection rapidly rebounded after treatment withdrawal: At 9 and 16 weeks of follow-up, levels of DHBsAg and DHBcAg and DHBV total and cccDNA in the liver had rebounded and matched levels observed in the control ducks treated with normal saline which remained persistently infected with DHBV. These data demonstrate that treatment with the NAP REP 2055 can lead to sustained control of persistent DHBV infection. These effects may be related to the unique ability of REP 2055 to block release of DHBsAg from infected hepatocytes.

Regulation of Multiple Stages of Hepadnavirus Replication by the Carboxyl-Terminal Domain of Viral Core Protein in trans

Mutational analyses have indicated that the carboxyl-terminal domain (CTD) of hepadnavirus core protein and its state of phosphorylation are critical for multiple steps in viral replication. Also, CTD interacts with host proteins in a phosphorylation state-dependent manner. To ascertain the role of CTD in viral replication without perturbing its sequence and the role of CTD-host interactions, CTD of the human hepatitis B virus (HBV) or duck hepatitis B virus (DHBV) core protein, either the wild type (WT) or with alanine or glutamic acid/aspartic acid substitutions at the phosphorylation sites, was expressed in cells replicating DHBV with the WT core protein. A dramatic decrease in phosphorylation of the DHBV core protein (DHBc) was observed when the WT and most HBV core protein CTD (HCTD) variants were coexpressed in trans, which was accompanied by a profound reduction of viral core DNA and, in particular, the double-stranded DNA. One HCTD variant that failed to change DHBc phosphorylation also had no effect on DHBV core DNA. All WT and variant HCTDs and DHBc CTDs (DCTDs) decreased the DHBV covalently closed circular (CCC) DNA. Identification of CTD-host interactions indicated that CDK2 binding by CTD may mediate its inhibitory effect on DHBc phosphorylation and reverse transcription via competition with DHBc for the host kinase, whereas importin α binding by CTD may contribute to inhibition of CCC DNA production by competitively blocking the nuclear import of viral nucleocapsids. These results suggest the possibility of blocking multiple steps of viral replication, especially CCC DNA formation, via inhibition of CTD functions. Mutational analyses have suggested that the carboxyl-terminal domain (CTD) of hepadnavirus core protein is critical for viral replication. However, results from mutational analyses are open to alternative interpretations. Also, how CTD affects virus replication remains unclear. In this study, we took an alternative approach to mutagenesis by overexpressing CTD alone in cells replicating the virus with the wild-type core protein to determine the roles of CTD in viral replication. Our results revealed that CTD can inhibit multiple stages of viral replication, and its effects may be mediated at least in part through specific host interactions. They suggest that CTD, or its mimics, may have therapeutic potential. Furthermore, our experimental approach should be broadly applicable as a complement to mutagenesis for studying protein functions and interactions while at the same time providing a means to identify the relevant interacting factors.

Development and application of a universal Taqman real-time PCR for quantitation of duck hepatitis B virus DNA

To develop a quantitative assay for universal detection of duck hepatitis B virus (DHBV) DNA, a Taqman real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) assay was developed using primers and probes based on genomic sequences located at nucleotide 241–414 of the DHBV Core region which possesses the highest homology among the 44 DHBV genomes available in Genbank. The DHBV Core gene cloned in pGEM-T was used to generate DHBV DNA standard. The assay had a lowest detection limit of 103 copies/ml and a good linear standard curve (Y=−3.989X+49.086, r2=0.9993) over a wide range of input DHBV DNA (103 to 1010 copies/ml). The standard deviation of intra- and inter-assay was 0.01–0.06 and 0.05–0.16, respectively, and the coefficient of variation was 1.3–1.8%. The specificity of the assay was validated using duck hepatitis virus type 1, hepatitis B virus, and E. coli DNA. Comparison of ABI 7300 and Bio-Rad iQ5 PCR instruments yielded highly consistent results. The assay showed a positive rate of 63.8% (51/80) DHBV DNA in peripheral blood and liver tissue from ducks from Xi’an, China. The FQ-PCR developed is highly sensitive, specific, reproducible and versatile, and may be used to universally detect DHBV DNA of different DHBV strains.

In vivoinfectivity of liver extracts after resolution of hepadnaviral infection following therapy associating DNA vaccine and cytokine genes

DNA-based vaccination appears of promise for chronic hepatitis B immunotherapy, although there is an urgent need to increase its efficacy. In this preclinical study, we evaluated the therapeutic benefit of cytokine (IL-2, IFN-γ) genes co-delivery with DNA vaccine targeting hepadnaviral proteins in the chronic duck hepatitis B virus (DHBV) infection model. Then, we investigated the persistence of replication-competent virus in the livers of apparently resolved animals. DHBV carriers received four injections of plasmids encoding DHBV envelope and core alone or co-delivered with duck IL-2 (DuIL-2) or duck IFN-γ (DuIFN-γ) plasmids. After long-term (8 months) follow-up, viral covalently closed circular (ccc) DNA was analysed in duck necropsy liver samples. Liver homogenates were also tested for in vivo infectivity in neonatal ducklings. Co-delivery of DuIFN-γ resulted in significantly lower mean viremia starting from week 21. Viral cccDNA was undetectable by conventional methods in the livers of 25% and 57% of animals co-immunized with DuIL-2 and DuIFN-γ, respectively. Interestingly, inoculation of liver homogenates from 7 such apparently resolved animals, exhibiting cccDNA undetectable in Southern blotting and DHBV expression undetectable or restricted to few hepatocytes, revealed that three liver homogenates transmitted high-titre viremia (3-5×10(10) vge/mL) to naïve animals. In conclusion, our results indicate that IFN-γ gene co-delivery considerably enhances immunotherapeutic efficacy of DNA vaccine targeting hepadnaviral proteins. Importantly, we also showed that livers exhibiting only minute amounts of hepadnaviral cccDNA could induce extremely high-titre infection, highlighting the caution that should be taken in occult hepatitis B patients to prevent HBV transmission in liver transplantation context.

Pre-P Is a Secreted Glycoprotein Encoded as an N-Terminal Extension of the Duck Hepatitis B Virus Polymerase Gene

The duck hepatitis B virus (DHBV) pregenomic RNA is a bicistronic mRNA encoding the core and polymerase proteins. Thirteen AUGs (C2 to C14) and 10 stop codons (S1 to S10) are located between the C1 AUG for the core protein and the P1 AUG that initiates polymerase translation. We previously found that the translation of the DHBV polymerase is initiated by ribosomal shunting. Here, we assessed the biosynthetic events after shunting. Translation of the polymerase open reading frame was found to initiate at the C13, C14, and P1 AUGs. Initiation at the C13 AUG occurred through ribosomal shunting because translation from this codon was cap dependent but was insensitive to blocking ribosomal scanning internally in the message. C13 and C14 are in frame with P1, and translation from these upstream start codons led to the production of larger isoforms of P. We named these isoforms "pre-P" by analogy to the pre-C and pre-S regions of the core and surface antigen open reading frames. Pre-P was produced in DHBV16 and AusDHBV-infected duck liver and was predicted to exist in 80% of avian hepadnavirus strains. Pre-P was not encapsidated into DHBV core particles, and the viable strain DHBV3 cannot make pre-P, so it is not essential for viral replication. Surprisingly, we found that pre-P is an N-linked glycoprotein that is secreted into the medium of cultured cells. These data indicate that DHBV produces an additional protein that has not been previously reported. Identifying the role of pre-P may improve our understanding of the biology of DHBV infection.

Antiviral therapy with entecavir combined with post-exposure “prime-boost” vaccination eliminates duck hepatitis B virus-infected hepatocytes and prevents the development of persistent infection

Short-term antiviral therapy with the nucleoside analogue entecavir (ETV), given at an early stage of duck hepatitis B virus (DHBV) infection, restricts virus spread and leads to clearance of DHBV-infected hepatocytes in ∼ 50% of ETV-treated ducks, whereas widespread and persistent DHBV infection develops in 100% of untreated ducks. To increase the treatment response rate, ETV treatment was combined in the current study with a post-exposure “prime-boost” vaccination protocol. Four groups of 14-day-old ducks were inoculated intravenously with a dose of DHBV previously shown to induce persistent DHBV infection. One hour post-infection (p.i.), ducks were primed with DNA vaccines that expressed DHBV core (DHBc) and surface (pre-S/S and S) antigens (Groups A, B) or the DNA vector alone (Groups C, D). ETV (Groups A, C) or water (Groups B, D) was simultaneously administered by gavage and continued for 14 days. Ducks were boosted 7 days p.i. with recombinant fowlpoxvirus (rFPV) strains also expressing DHBc and pre-S/S antigens (Groups A, B) or the FPV-M3 vector (Groups C, D). DHBV-infected hepatocytes were observed in the liver of all ducks at day 4 p.i. with reduced numbers in the ETV-treated ducks. Ducks treated with ETV plus the control vectors showed restricted spread of DHBV infection during ETV treatment, but in 60% of cases, infection became widespread after ETV was stopped. In contrast, at 14 and 67 days p.i., 100% of ducks treated with ETV and “prime-boost” vaccination had no detectable DHBV-infected hepatocytes and had cleared the DHBV infection. These findings suggest that ETV treatment combined with post-exposure “prime-boost” vaccination induced immune responses that eliminated DHBV-infected hepatocytes and prevented the development of persistent DHBV infection.

Peptide Aptamer-mediated Inhibition of Target Proteins by Sequestration into Aggresomes

Peptide aptamers (PAs) can be employed to block the intracellular function of target proteins. Little is known about the mechanism of PA-mediated protein inhibition. Here, we generated PAs that specifically bound to the duck hepatitis B virus (HBV) core protein. Among them, PA34 strongly blocked duck HBV replication by inhibiting viral capsid formation. We found that PA34 led to a dramatic intracellular redistribution of its target protein into perinuclear inclusion bodies, which exhibit the typical characteristics of aggresomes. As a result, the core protein is efficiently removed from the viral life cycle. Corresponding findings were obtained for bioactive PAs that bind to the HBV core protein or to the human papillomavirus-16 (HPV16) E6 protein, respectively. The observation that PAs induce the specific sequestration of bound proteins into aggresomes defines a novel mechanism as to how this new class of intracellular inhibitors blocks the function of their target proteins.

Entecavir therapy combined with DNA vaccination for persistent duck hepatitis B virus infection.

This study was designed to test the efficacy of antiviral treatment with entecavir (ETV) in combination with DNA vaccines expressing duck hepatitis B virus (DHBV) antigens as a therapy for persistent DHBV infection in ducks. Ducks were inoculated with 10(9) DHBV genomes at 7 days of age, leading to widespread infection of the liver and viremia within 7 days, and were then treated orally with either ETV (0.1 mg/kg of body weight/day) or distilled water from 21 days posthatch for 244 days. Treatment with ETV caused a 4-log drop in serum DHBV DNA levels within 80 days and a slower 2- to 3-log drop in serum DHBV surface antigen (DHBsAg) levels within 120 days. Following withdrawal of ETV, levels of serum DHBV DNA and DHBsAg rebounded to match those in the water-treated animals within 40 days. Sequential liver biopsy samples collected throughout the study showed that ETV treatment reduced DHBV DNA replicative intermediates 70-fold in the liver, while the level of the stable, template form, covalently closed circular DNA decreased only 4-fold. ETV treatment reduced both the intensity of antigen staining and the percentage of antigen-positive hepatocytes in the liver, but the intensity of antigen staining in bile duct cells appeared not to be effected. Intramuscular administration of five doses of a DNA vaccine expressing the DHBV presurface, surface, precore, and core antigens, both alone and concurrently with ETV treatment, on days 50, 64, 78, 127, and 141 did not result in any significant effect on viral markers.

Nuclear localization of the duck hepatitis B virus capsid protein: detection and functional implications of distinct subnuclear bodies in a compartment associated with RNA synthesis and maturation.

We have reinvestigated the subcellular distribution of the duck hepatitis B virus (DHBV) core protein in infected duck hepatocytes and in transfected cells. By using indirect immunofluorescence, the protein was found to be localized not only in the cytoplasm, as described previously, but also within the cell nucleus, being concentrated in distinct, brightly staining nuclear core bodies (NCBs). In colocalization studies using confocal microscopy, the NCBs were found exclusively in the periphery of nuclear subdomains characterized as splicing factor compartments and distal to other subnuclear domains. Also relevant for their functional significance is that the NCBs formed during the establishment of virus infection, i.e., at very low overall concentrations of newly synthesized core protein, and persisted throughout all stages of infection. Moreover, a subset of NCBs colocalized with foci of pregenomic DHBV RNA present at concentrations detectable by fluorescence in situ hybridization. Taken together, these findings indicate that a minor fraction of the DHBV core protein molecules escapes the major cytoplasmic assembly pathway to accumulate in specific subnuclear domains, and they furthermore suggest that these NCBs serve a role in the synthesis and/or maturation of the DHBV RNA pregenome.

Cis-preferential recruitment of duck hepatitis B virus core protein to the RNA/polymerase preassembly complex

Hepadnaviral replication requires the concerted action of the polymerase and core proteins to ensure selective packaging of the RNA pregenome into nucleocapsids. Virus assembly is initiated by cis-preferential binding of polymerase to the encapsidation signal ϵ, present on pregenomic RNA. Using the duck hepatitis B virus (DHBV) model, we analyzed how core protein is recruited to the RNA/polymerase preassembly complex. Two sets of trans-complementation assays were performed in cotransfected hepatoma cells. First, a replication-competent DHBV construct was tested for its ability to rescue replication of genomes bearing mutations within the core region. Self-packaging of wild-type pregenomes was more efficient than cross-packaging of core-deficient pregenomes, and this bias was strongly enhanced if mutant pregenomes coded for self-assembly–competent, but packaging-deficient, core proteins. Second, the site of wild-type core protein translation, i.e., pregenomic RNA ( cis) or separate messenger RNA ( trans), was analyzed for its effect on the phenotype of a previously described dominant-negative (DN) DHBV core protein mutant. This mutant forms chimeric nucleocapsids with wild-type core proteins and blocks reverse transcription within most, but not all, mixed particles. Strikingly, suppression of viral DNA synthesis by the mutant increased 100-fold when wild-type core protein was provided in trans. Our results suggest that recruitment of core protein to the DHBV preassembly complex occurs in a cis-preferential manner. This mechanism may account for the leakiness of DN DHBV core protein mutants targeting reverse transcription. (H EPATOLOGY 2002;35:209-216.)

Duck hepatitis B virus replication in primary bile duct epithelial cells.

Primary cultures of intrahepatic bile duct epithelial (IBDE) cells isolated from duckling livers were successfully grown for studies of duck hepatitis B virus (DHBV). The primary IBDE cells were characterized by immunohistochemistry using CAM 5.2, a cytokeratin marker which was shown to react specifically to IBDE cells in duck liver tissue sections and in primary cultures of total duck liver cells. Immunofluorescence assay using anti-duck albumin, a marker for hepatocytes, revealed that these IBDE cultures did not appear to contain hepatocytes. A striking feature of these cultures was the duct-like structures present within each cell colony of multilayered IBDE cells. Normal duck serum in the growth medium was found to be essential for the development of these cells into duct-like structures. When the primary cultures of duck IBDE cells were acutely infected with DHBV, dual-labeled confocal microscopy using a combination of anti-DHBV core proteins and CAM 5.2 or a combination of anti-pre-S1 proteins and CAM 5.2 revealed that the IBDE cell colonies contained DHBV proteins. Immunoblot analysis of these cells showed that the DHBV pre-S1 and core proteins were similar to their counterparts in infected primary duck hepatocyte cultures. Southern blot analysis of infected IBDE preparations using a digoxigenin-labeled positive-sense DHBV riboprobe revealed the presence of hepadnavirus covalently closed circular (CCC) DNA, minus-sense single-stranded (SS) DNA, double-stranded linear DNA, and relaxed circular DNA. The presence of minus-sense SS DNA in the acutely infected IBDE cultures is indicative of DHBV reverse transcriptase activity, while the establishment of a pool of viral CCC DNA reveals the ability of these cells to maintain persistent infection. Taken collectively, the results from this study demonstrated that primary duck IBDE cells supported hepadnavirus replication as shown by the de novo synthesis of DHBV proteins and DNA replicative intermediates.

Signals for bidirectional nucleocytoplasmic transport in the duck hepatitis B virus capsid protein.

Hepadnavirus genome replication involves cytoplasmic and nuclear stages, requiring balanced targeting of cytoplasmic nucleocapsids to the nuclear compartment. In this study, we analyze the signals determining capsid compartmentalization in the duck hepatitis B virus (DHBV) animal model, as this system also allows us to study hepadnavirus infection of cultured primary hepatocytes. Using fusions to the green fluorescent protein as a functional assay, we have identified a nuclear localization signal (NLS) that mediates nuclear pore association of the DHBV nucleocapsid and nuclear import of DHBV core protein (DHBc)-derived polypeptides. The DHBc NLS mapped is unique. It bears homology to repetitive NLS elements previously identified near the carboxy terminus of the capsid protein of hepatitis B virus, the human prototype of the hepadnavirus family, but it maps to a more internal position. In further contrast to the hepatitis B virus core protein NLS, the DHBc NLS is not positioned near phosphorylation target sites that are generally assumed to modulate nucleocytoplasmic transport. In functional assays with a knockout mutant, the DHBc NLS was found to be essential for nuclear pore association of the nucleocapsid. The NLS was found to be also essential for virus production from the full-length DHBV genome in transfected cells and from hepatocytes infected with transcomplemented mutant virus. Finally, the DHBc additionally displayed activity indicative of a nuclear export signal, presumably counterbalancing NLS function in the productive state of the infected cell and thereby preventing nucleoplasmic accumulation of nucleocapsids.

Dominant negative mutants of the duck hepatitis B virus core protein interfere with RNA pregenome packaging and viral DNA synthesis.

Dominant negative (DN) mutants of the hepadnaviral core protein are potent inhibitors of viral replication. We have previously shown that fusion of sequences derived from the duck hepatitis B virus (DHBV) polymerase (Pol), DHBV small surface protein (S), bacterial beta-galactosidase (lacZ), or green fluorescent protein (GFP) to the carboxy terminus of the DHBV core protein yields DN mutants that inhibit viral replication at the posttranslational level. To elucidate the mechanism(s) of their antiviral action, we analyzed the effect of the DN mutants on RNA pregenome packaging and nucleocapsid assembly. Core-Pol and core-S, but not core-lacZ or core-GFP, markedly interfered with RNA pregenome packaging. Nucleocapsid formation was not affected by any of the mutants. The DN core-GFP fusion protein formed mixed particles with wild-type core protein in the cytoplasm of cotransfected cells and interfered with reverse transcription of the viral pregenome. A subpopulation of chimeric nucleocapsids, however, was shown to overcome the block in DNA synthesis and produce mature viral DNA. Thus, at least 2 steps within the viral life cycle can be targeted by DN DHBV core proteins: 1) packaging of the viral pregenome; and 2) reverse transcription within mixed particles. The fact that some mixed particles retain replication competence demonstrates a high structural flexibility of nucleocapsids and indicates a possible mechanism of viral escape.

Cellular immune response of ducks to duck hepatitis B virus infection

Duck hepatitis B virus (DHBV) has been a useful model for hepadnavirus infection. There have been few studies on immunity to DHBV and none describing the cell-mediated immune response by acute and chronically infected ducks. A duck hepatitis B antigen-specific blastogenesis assay was used to measure DHBV antigen-specific responses of duck peripheral blood (PBMC) and splenic mononuclear cells (SMCs) from uninfected control ducks, ducks acutely or chronically infected with DHBV, and ducks immune to DHBV. A comparison of the group mean responses by PBMC to DHBV surface antigen (DHBsAg) found that the immune group was significantly different to the other three groups (controls or unexposed, P < 0.0001; acutely infected, P< 0.01; chronically infected, P < 0.01). The responses to DHBsAg by PBMC of the acute group (P< 0.01) were significantly different also to that of the unexposed group. For DHBV core antigen (DHBcAg), significant differences in the responses were found between immune ducks and unexposed (P < 0.0005) and acutely infected (P < 0.05) groups. The SMC showed a significant difference between unexposed ducks and immune ducks (P< 0.05) in the group mean responses to DHBsAg. The responses to DHBcAg were significantly different between the immune group and the acute (P < 0.01) and unexposed (P < 0.01) groups. The group mean of unexposed ducks was also significantly different to that of acutely infected ducks (P < 0.01). This study indicates that the cellular immune response in immune animals differs from acutely and chronically infected ducks. Further studies of these differences may provide some explanations for the differing outcomes of DHBV infection.

Cellular immune response of ducks to duck hepatitis B virus infection

Duck hepatitis B virus (DHBV) has been a useful model for hepadnavirus infection. There have been few studies on immunity to DHBV and none describing the cell-mediated immune response by acute and chronically infected ducks. A duck hepatitis B antigen-specific blastogenesis assay was used to measure DHBV antigen-specific responses of duck peripheral blood (PBMC) and splenic mononuclear cells (SMCs) from uninfected control ducks, ducks acutely or chronically infected with DHBV, and ducks immune to DHBV. A comparison of the group mean responses by PBMC to DHBV surface antigen (DHBsAg) found that the immune group was significantly different to the other three groups (controls or unexposed, P < 0.0001; acutely infected, P < 0.01; chronically infected, P < 0.01). The responses to DHBsAg by PBMC of the acute group (P < 0.01) were significantly different also to that of the unexposed group. For DHBV core antigen (DHBcAg), significant differences in the responses were found between immune ducks and unexposed (P < 0.0005) and acutely infected (P < 0.05) groups. The SMC showed a significant difference between unexposed ducks and immune ducks (P < 0.05) in the group mean responses to DHBsAg. The responses to DHBcAg were significantly different between the immune group and the acute (P < 0.01) and unexposed (P < 0.01) groups. The group mean of unexposed ducks was also significantly different to that of acutely infected ducks (P < 0.01). This study indicates that the cellular immune response in immune animals differs from acutely and chronically infected ducks. Further studies of these differences may provide some explanations for the differing outcomes of DHBV infection. J. Med. Virol. 58:19–25, 1999. © 1999 Wiley-Liss, Inc.

Duck hepatitis B virus nucleocapsids formed by N-terminally extended or C-terminally truncated core proteins disintegrate during viral DNA maturation.

Hepadnaviruses are DNA viruses that replicate through reverse transcription of an RNA pregenome. Viral DNA synthesis takes place inside viral nucleocapsids, formed by core protein dimers. Previous studies have identified carboxy-terminal truncations of the core protein that affect viral DNA maturation. Here, we describe the effect of small amino-terminal insertions into the duck hepatitis B virus (DHBV) core protein on viral DNA replication. All insertion mutants formed replication-competent nucleocapsids. Elongation of viral DNA, however, appeared to be incomplete. Increasing the number of additional amino acids and introducing negatively charged residues further reduced the observed size of mature viral DNA species. Mutant core proteins did not inhibit the viral polymerase. Instead, viral DNA synthesis destabilized mutant nucleocapsids, rendering mature viral DNA selectively sensitive to nuclease action. Interestingly, the phenotype of two previously described carboxy-terminal DHBV core protein deletion mutants was found to be based on the same mechanism. These data suggest that (i) the amino- as well as the carboxy-terminal portion of the DHBV core protein plays a critical role in nucleocapsid stabilization, and (ii) the hepadnavirus polymerase can perform partial second-strand DNA synthesis in the absence of intact viral nucleocapsids.

Preparations of Duck Hepatitis B Virions Contain Multiple DNA Polymerase Activities

The hepadnaviral DNA genome is synthesized by a viral-encoded reverse transcriptase, but the nature of this protein(s)in vivoremains obscure. We have previously described studies in which activity gel assays identified multiple DNA polymerase (DNAp) activities associated with highly purified duck hepatitis B virus (DHBV) core particles. We now report that virions isolated from viremic sera are associated with DNA-dependent DNAp activities which are nearly identical to major DNAp activities detected with highly purified DHBV core particles. These results suggest that the virion-associated polymerases are the same as those which are detected with core particles and are likely to represent DHBVpolgene products involved in replication of the genome.

Inhibition of viral replication by genetically engineered mutants of the duck hepatitis B virus core protein

The hepatitis B virus (HBV) nucleocapsid consists of 240 viral core proteins that are arranged in a highly symmetrical structure, HBV replication can only take place inside intact nucleocapsids. In the present study, we investigated whether genetically engineered core mutants can inhibit viral replication by interfering with the formation of intact nucleocapsids. Using the duck hepatitis B virus (DHBV) model, a series of core protein mutants was generated. Polymerase chain reaction-amplified fragments from the bacterial lacZ gene expressing up to 282 amino acids were added either to the amino- or carboxy-terminus of the DHBV core protein. In addition, carboxy-terminal extensions were generated by fusing the DHBV core protein with the DHBV small surface protein or various fragments of the viral polymerase. Finally, the green fluorescent protein (GFP) was fused in-frame to the carboxy-terminus of the DHBV core protein. In this chimeric protein, GFP is still functional and can act as a reporter molecule. The various core protein mutants were tested for their potential antiviral activity by cotransfection with a replication-competent DHBV construct into the avian hepatoma cell line LMH. Carboxy-terminal, but not amino-terminal, DHBV core mutants inhibited DHBV replication by up to 90% at an effector-to-target ratio of 1:10, thus displaying a dominant negative phenotype. Antiviral activity was species-specific and caused by posttranslational interference with viral replication. The DHBV core-GFP fusion protein should be an ideal tool to assess the antiviral potential of dominant negative core proteins in vivo. (Hepatology 1996 Aug;24(2):294-9)

Inhibition of viral replication by genetically engineered mutants of the duck hepatitis B virus core protein.

The hepatitis B virus (HBV) nucleocapsid consists of 240 viral core proteins that are arranged in a highly symmetrical structure, HBV replication can only take place inside intact nucleocapsids. In the present study, we investigated whether genetically engineered core mutants can inhibit viral replication by interfering with the formation of intact nucleocapsids. Using the duck hepatitis B virus (DHBV) model, a series of core protein mutants was generated. Polymerase chain reaction-amplified fragments from the bacterial lacZ gene expressing up to 282 amino acids were added either to the amino- or carboxy-terminus of the DHBV core protein. In addition, carboxy-terminal extensions were generated by fusing the DHBV core protein with the DHBV small surface protein or various fragments of the viral polymerase. Finally, the green fluorescent protein (GFP) was fused in-frame to the carboxy-terminus of the DHBV core protein. In this chimeric protein, GFP is still functional and can act as a reporter molecule. The various core protein mutants were tested for their potential antiviral activity by cotransfection with a replication-competent DHBV construct into the avian hepatoma cell line LMH. Carboxy-terminal, but not amino-terminal, DHBV core mutants inhibited DHBV replication by up to 90% at an effector-to-target ratio of 1:10, thus displaying a dominant negative phenotype. Antiviral activity was species-specific and caused by posttranslational interference with viral replication. The DHBV core-GFP fusion protein should be an ideal tool to assess the antiviral potential of dominant negative core proteins in vivo.

Detection of an RNase H activity associated with hepadnaviruses

Replication of the hepadnavirus DNA genome is accomplished via reverse transcription of an intermediate, pregenomic RNA molecule. This process is likely to be carried out by a virally encoded, multifunctional polymerase which possesses DNA- and RNA-dependent DNA polymerase and RNase H activities. However, the nature of the product(s) of the polymerase gene predicted to mediate these functions is unclear. Biochemical studies of the polymerase protein(s) have been limited by its apparent low abundance in virus particles and, until recently, the inability to express active polymerase protein(s) heterologously. We have used activity gel assays to detect DNA- and RNA-dependent DNA polymerase activities associated with highly purified duck hepatitis B virus (DHBV) core particles (S. M. Oberhaus and J. E. Newbold, J. Virol. 67:6558-6566, 1993). Now we report that the same approach identifies a 35-kDa RNase H activity in association with highly purified DHBV core particles and crude preparations of virions from DHBV-infected ducks and woodchuck hepatitis virus-infected woodchucks. This is the first report of the detection of an hepadnavirus-associated RNase H activity. Its apparent size is smaller than any of the DNA polymerase activities that we detected previously and significantly smaller than the full-length protein predicted from the polymerase open reading frame (p85 for DHBV). These data suggest that the viral polymerase and RNase H activities are separable and that these enzymes may coordinate their activities in vivo by forming a complex.