Relaxing the restricted structural dynamics in the human hepatitis B virus RNA encapsidation signal enables replication initiation in vitro.

Hepadnaviruses, including hepatitis B virus (HBV) as a major human pathogen, replicate their tiny 3 kb DNA genomes by capsid-internal protein-primed reverse transcription of a pregenomic (pg) RNA. Initiation requires productive binding of the viral polymerase, P protein, to a 5´ proximal bipartite stem-loop, the RNA encapsidation signal ε. Then a residue in the central ε bulge directs the covalent linkage of a complementary dNMP to a Tyr sidechain in P protein´s Terminal Protein (TP) domain. After elongation by two or three nucleotides (nt) the TP-linked DNA oligo is transferred to a 3´ proximal acceptor, enabling full-length minus-strand DNA synthesis. No direct structural data are available on hepadnaviral initiation complexes but their cell-free reconstitution with P protein and ε RNA (Dε) from duck HBV (DHBV) provided crucial mechanistic insights, including on a major conformational rearrangement in the apical Dε part. Analogous cell-free systems for human HBV led at most to P-ε binding but no detectable priming. Here we demonstrate that local relaxation of the highly basepaired ε upper stem, by mutation or via synthetic split RNAs, enables ε-dependent in vitro priming with full-length P protein from eukaryotic translation extract yet also, and without additional macromolecules, with truncated HBV miniP proteins expressed in bacteria. Using selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) we confirm that upper stem destabilization correlates with in vitro priming competence and show that the supposed bulge-closing basepairs are largely unpaired even in wild-type ε. We define the two 3´ proximal nt of this extended bulge as main initiation sites and provide evidence for a Dε-like opening of the apical ε part upon P protein binding. Beyond new HBV-specific basic aspects our novel in vitro priming systems should facilitate the development of high-throughput screens for priming inhibitors targeting this highly virus-specific process.

Establishment of a hydrodynamic delivery system in ducks.

Chronic hepatitis B virus (HBV) poses a significant global health challenge as it can lead to acute or chronic liver disease and hepatocellular carcinoma (HCC). To establish a safety experimental model, a homolog of HBV-duck HBV (DHBV) is often used for HBV research. Hydrodynamic-based gene delivery (HGD) is an efficient method to introduce exogenous genes into the liver, making it suitable for basic research. In this study, a duck HGD system was first constructed by injecting the reporter plasmid pLIVE-SEAP via the ankle vein. The highest expression of SEAP occurred when ducks were injected with 5 µg/mL plasmid pLIVE-SEAP in 10% bodyweight volume of physiological saline for 6 s. To verify the distribution and expression of exogenous genes in multiple tissues, the relative level of foreign gene DNA and ß-galactosidase staining of LacZ were evaluated, which showed the plasmids and their products were located mainly in the liver. Additionally, ß-galactosidase staining and fluorescence imaging indicated the delivered exogenous genes could be expressed in a short time. Further, the application of the duck HGD model on DHBV treatment was investigated by transferring representative anti-HBV genes IFNα and IFNγ into DHBV-infected ducks. Delivery of plasmids expressing IFNα and IFNγ inhibited DHBV infection and we established a novel efficient HGD method in ducks, which could be useful for drug screening of new genes, mRNAs and proteins for anti-HBV treatment.

Identification of avihepadnaviruses and circoviruses in an unexplained death event in farmed ducks.

Research into disease pathogens can greatly benefit from viral metagenomics technology. Using this technique, we investigated potential disease pathogens that resulted in the death of many ducks on a duck farm. Two duck circoviruses (DuCV) and one duck hepatitis B virus (DHBV) were detected and identified, and all three strains were closely related to avian-associated viruses. Two duck circoviruses had 81.64%-97.65% genome-wide sequence identity to some reference strains, and duck hepatitis B virus shared 75.85%-98.92% identity with other strains. Clinical characteristics of the diseased ducks, including ruffled feathers, lethargy, and weight loss, were comparable to those observed in cases of DuCV infection. Further research is needed to determine whether coinfection with DHBV leads to liver damage and exacerbation of the disease.

Activities of endogenous APOBEC3s and uracil-DNA-glycosylase affect the hypermutation frequency of hepatitis B virus cccDNA.

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) plays a key role in the persistence of viral infection. We have previously shown that overexpression of an antiviral factor APOBEC3G (A3G) induces hypermutation in duck HBV (DHBV) cccDNA, whereas uracil-DNA-glycosylase (UNG) reduces these mutations. In this study, using cell-culture systems, we examined whether endogenous A3s and UNG affect HBV cccDNA mutation frequency. IFNγ stimulation induced a significant increase in endogenous A3G expression and cccDNA hypermutation. UNG inhibition enhanced the IFNγ-mediated hypermutation frequency. Transfection of reconstructed cccDNA revealed that this enhanced hypermutation caused a reduction in viral replication. These results suggest that the balance of endogenous A3s and UNG activities affects HBV cccDNA mutation and replication competency.

Effect of Hepatocyte Targeting Nanopreparation Syringopicroside on Duck Hepatitis B Virus and Evaluation of Its Safety.

Syringopicroside is a kind of iridoid monomer compound isolated from Syringa oblata exhibiting a potent effect against hepatitis B virus (HBV). The therapeutic effect and safety of syringopicroside-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (SYR-NP) were studied on the model of HBV-infected ducklings and on cultured HepG2.2.15 cells. HBV DNA in ducklings was assessed by fluorescence quantitative PCR. In HepG2.2.15 cells, the content of HBsAg and HBeAg were assayed. Acute toxicity of SYR-NP was studied in ICR mice in 12 h and 7 days after SYR-NP administration. The serum levels of HBV DNA in ducklings treated with SYR-NP in a high dose was significantly lower than in the control. In HepG2.2.15 cells treated with different doses of SYR-NP, the concentrations of HBsAg and HBeAg were significantly below the control. Acute toxicity test showed high safety of SYR-NP. Thus, SYR-NP can inhibit replication of HBV DNA and protect the liver tissue.

Glucose Homeostasis Is Dysregulated in Ducks Infected with Duck Hepatitis B Virus.

INTRODUCTION: The association between hepatitis B virus (HBV) infection and the development of diabetes remains controversial. This study examined the effect of HBV infection on glucose homeostasis using a duck HBV (DHBV) model. METHODS: Plasma DHBV DNA was detected by quantitative polymerase chain reaction (PCR). Tissue infection of DHBV was determined by detecting DHBV covalently closed circular DNA (cccDNA) with a method of rolling circle amplification combined with cross-gap PCR, and verified by fluorescence in situ hybridization assay. An intravenous injection glucose tolerance test (GTT) was used to analyze the effect of DHBV infection on glucose tolerance. RESULTS: Of the finally included 97 domestic ducks, 53 (54.6%) were congenitally infected by DHBV. The positive rate of DHBV cccDNA in the liver, kidney, pancreas, and skeletal muscle of the infected ducks was 100, 75.5, 67.9, and 47.2%, respectively. The DHBV-infected ducks had higher blood glucose levels at 15 and 30 min post-load glucose (p < 0.01 and p < 0.001, respectively) in the GTT, much more individuals with greater glucose area under curve (p < 0.01), and a 57% impaired glucose tolerance (IGT) rate, as compared with noninfected controls. In addition, the subgroups of the infected ducks with DHBV cccDNA positive in skeletal muscle maintained the higher blood glucose level up to 2 h post-load glucose during the GTT and had a 76% IGT rate. CONCLUSION: These results suggest that DHBV intrahepatic and extrahepatic infection impairs glucose tolerance, and thus evidence the association of DHBV infection with the dysregulation of glucose metabolism.

Distribution of Hepatitis B Virus Nuclear DNA.

Chronic hepatitis B affects over 300 million people who are at risk of developing liver cancer. The basis for the persistence of hepatitis B virus (HBV) in hepatocytes, even in the presence of available antiviral therapies, lies in the accumulation of covalently closed circular DNA (cccDNA) in nuclei of infected cells. While methods for cccDNA quantification from liver biopsy specimens and cell lines expressing the virus are known, information about cccDNA formation, stability, and turnover is lacking. In particular, little is known about the fate of cccDNA during cell division. To fill the gaps in knowledge concerning cccDNA biology, we have developed a fluorescence imaging in situ hybridization (FISH)-based assay for the detection of duck hepatitis B virus (DHBV) cccDNA and HBV nuclear DNA in established cell lines. Using FISH, we determined the distribution of cccDNA under conditions mimicking chronic infections with and without antiviral therapy, which prevents de novo viral replication. Our results showed that the copy numbers of viral nuclear DNA can vary by as much as 1.8 orders of magnitude among individual cells and that antiviral therapy leads to a reduction in nuclear DNA in a manner consistent with symmetrical distribution of viral DNA to daughter cells.IMPORTANCE A mechanistic understanding of the stability of HBV cccDNA in the presence of antiviral therapy and during cell division induced by immune-mediated lysis of infected hepatocytes will be critical for the future design of curative antiviral therapies against chronic hepatitis B. Current knowledge about cccDNA stability was largely derived from quantitative analyses of cccDNA levels present in liver samples, and little was known about the fate of cccDNA in individual cells. The development of a FISH-based assay for cccDNA tracking provided the first insights into the fate of DHBV cccDNA and nuclear HBV DNA under conditions mimicking antiviral therapy.

Capsid Phosphorylation State and Hepadnavirus Virion Secretion.

The C-terminal domain (CTD) of hepadnavirus core protein is involved in multiple steps of viral replication. In particular, the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging into immature nucleocapsids (NCs) and the early stage of viral DNA synthesis. For the avian hepadnavirus duck hepatitis B virus (DHBV), CTD is dephosphorylated subsequently to facilitate the late stage of viral DNA synthesis and to stabilize NCs containing mature viral DNA. The role of CTD phosphorylation in virion secretion, if any, has remained unclear. Here, the CTD from the human hepatitis B virus (HBV) was found to be dephosphorylated in association with NC maturation and secretion of DNA-containing virions, as in DHBV. In contrast, the CTD in empty HBV virions (i.e., enveloped capsids with no RNA or DNA) was found to be phosphorylated. The potential role of CTD dephosphorylation in virion secretion was analyzed through mutagenesis. For secretion of empty HBV virions, which is independent of either viral RNA packaging or DNA synthesis, multiple substitutions in the CTD to mimic either phosphorylation or dephosphorylation showed little detrimental effect. Similarly, phospho-mimetic substitutions in the DHBV CTD did not block the secretion of DNA-containing virions. These results indicate that CTD dephosphorylation, though associated with NC maturation in both HBV and DHBV, is not essential for the subsequent NC-envelope interaction to secrete DNA-containing virions, and the CTD state of phosphorylation also does not play an essential role in the interaction between empty capsids and the envelope for secretion of empty virions.IMPORTANCE The phosphorylation state of the C-terminal domain (CTD) of hepatitis B virus (HBV) core or capsid protein is highly dynamic and plays multiple roles in the viral life cycle. To study the potential role of the state of phosphorylation of CTD in virion secretion, we have analyzed the CTD phosphorylation state in complete (containing the genomic DNA) versus empty (genome-free) HBV virions. Whereas CTD is unphosphorylated in complete virions, it is phosphorylated in empty virions. Mutational analyses indicate that neither phosphorylation nor dephosphorylation of CTD is required for virion secretion. These results demonstrate that while CTD dephosphorylation is associated with HBV DNA synthesis, the CTD state of phosphorylation may not regulate virion secretion.

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses.

Hepatitis B virus (HBV), the causative agent of chronic hepatitis B and prototypic hepadnavirus, is a small DNA virus that replicates by protein-primed reverse transcription. The product is a 3-kb relaxed circular DNA (RC-DNA) in which one strand is linked to the viral polymerase (P protein) through a tyrosyl-DNA phosphodiester bond. Upon infection, the incoming RC-DNA is converted into covalently closed circular (ccc) DNA, which serves as a viral persistence reservoir that is refractory to current anti-HBV treatments. The mechanism of cccDNA formation is unknown, but the release of P protein is one mandatory step. Structural similarities between RC-DNA and cellular topoisomerase-DNA adducts and their known repair by tyrosyl-DNA-phosphodiesterase (TDP) 1 or TDP2 suggested that HBV may usurp these enzymes for its own purpose. Here we demonstrate that human and chicken TDP2, but only the yeast ortholog of TDP1, can specifically cleave the Tyr-DNA bond in virus-adapted model substrates and release P protein from authentic HBV and duck HBV (DHBV) RC-DNA in vitro, without prior proteolysis of the large P proteins. Consistent with TPD2's having a physiological role in cccDNA formation, RNAi-mediated TDP2 depletion in human cells significantly slowed the conversion of RC-DNA to cccDNA. Ectopic TDP2 expression in the same cells restored faster conversion kinetics. These data strongly suggest that TDP2 is a first, although likely not the only, host DNA-repair factor involved in HBV cccDNA biogenesis. In addition to establishing a functional link between hepadnaviruses and DNA repair, our results open new prospects for directly targeting HBV persistence.

Regulation of multiple stages of hepadnavirus replication by the carboxyl-terminal domain of viral core protein in trans.

UNLABELLED: 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. IMPORTANCE: 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.

Cloning, expression and purification of duck hepatitis B virus (DHBV) core protein and its use in the development of an indirect ELISA for serologic detection of DHBV infection.

Infecting ducks with duck hepatitis B virus (DHBV) is widely accepted as a relevant model for studying aspects of human HBV infection. However, efficient and sensitive diagnostic methods for the various infection models are limited. In order to provide a more simple and convenient method for serologic diagnosis, we improved the production of recombinant DHBV viral capsid protein (core protein) and then used it to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detecting anti-DHBc antibodies (DHBcAg ELISA) in DHBV-infected ducks. Given the positive/negative cut-off value, the maximum dilution of duck sera in which anti-DHBc antibodies could be detected was 1:12,800. In addition, the DHBcAg ELISA displayed no cross reactivity with duck antisera against duck circovirus (DuCV), duck plague virus (DPV), duck hepatitis virus (DHV), duck swollen head septicemia virus (DSHSV), avian influenza virus (AIV), Riemerella anatipestifer, Salmonella anatum, or Escherichia coli. Furthermore, the coefficients of variation (CVs) of inter-assay and intra-assay experiments were both below than 10 %. When compared to PCR for accuracy on clinical samples from cases of suspected DHBV infection, the DHBcAg showed 95.45 % coincidence with PCR. In conclusion, recombinant DHBc was readily produced and used to establish a simple DHBcAg ELISA that provided a highly specific and sensitive method for analysis of clinical samples.

Anti-hepatitis B virus activity of α-DDB-FNCG, a novel nucleoside-biphenyldicarboxylate compound in vitro and in vivo.

A novel codrug, α-DDB-FNCG, was synthesized through coupling of α-biphenyl dimethyl dicarboxylate (α-DDB) and the nucleoside analogue FNCG, via an ester bond. The anti-HBV activity and hepatoprotective effects of this compound were investigated both in vitro and in vivo. In HBV-transfected HepG2.2.15 cell line, the secretion of HBsAg and HBeAg as well as the levels of extracellular and intracellular viral DNA were determined by ELISA and real-time fluorescent quantitative Polymerase Chain Reaction (FQ-PCR), respectively. In DHBV-infected ducks, the viral DNA levels in serum and liver were determined by FQ-PCR. In addition, the levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) in both serum and liver were also examined. The improvement of ducks' livers was evaluated by histopathological analysis. It has been demonstrated that α-DDB-FNCG could suppress the levels of HBV antigens and viral DNA in a time- and dose-dependent manner in the HepG2.2.15 cell line. Furthermore, this codrug could also significantly inhibit the viral DNA replication and reduce the ALT and AST levels in both serum and liver of DHBV-infected ducks, with improved hepatocellular architecture in drug-treated ducks. In short, these results suggest that α-DDB-FNCG could be a promising candidate for further development of new anti-HBV agents with hepatoprotective effects.

Isothiafludine, a novel non-nucleoside compound, inhibits hepatitis B virus replication through blocking pregenomic RNA encapsidation.

AIM: To investigate the action of isothiafludine (NZ-4), a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus (HBV) in vitro and in vivo. METHODS: HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA (pgRNA) and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 (25, 50 or 100 mg·kg⁻¹·d⁻¹) for 15 d. RESULTS: NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC50 value of 1.33 µmol/L, whereas the compound inhibited the cell viability with an IC50 value of 50.4 µmol/L. Furthermore, NZ-4 was active against the replication of various drug-resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 (5, 10, 20 µmol/L) concentration-dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks. CONCLUSION: NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process, thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat HBV infection.

Evaluation of hepatocyteprotective and anti-hepatitis B virus properties of Cichoric acid from Cichorium intybus leaves in cell culture.

Hepatitis B is the most common serious liver infection in the world. To date, there is still no complete cure for chronic hepatitis B. Natural caffeic acid analogues possess prominent antiviral activity, especially anti-hepatitis B virus (HBV) and anti-human immunodeficiency virus effects. Cichoric acid is a caffeic acid derivative from Cichorium intybus. In the study, the anti-hepatitis B property of cichoric acid was evaluated by the D-galactosamine (D-GalN)-induced normal human HL-7702 hepatocyte injury model, the duck hepatitis B virus (DHBV)-infected duck fetal hepatocytes and the HBV-transfected cell line HepG2.2.15 cells, respectively. The results showed that cichoric acid attenuated significantly D-GalN-induced HL-7702 hepatocyte injury at 10-100 µg/mL and produced a maximum protection rate of 56.26%. Moreover, cichoric acid at 1-100 µg/mL inhibited markedly DHBV DNA replication in infected duck fetal hepatocytes. Also, cichoric acid at 10-100 µg/mL reduced significantly the hepatitis B surface and envelope antigen levels in HepG2.2.15 cells and produced the maximum inhibition rates of 79.94% and 76.41%, respectively. Meanwhile, test compound at 50-100 µg/mL inhibited markedly HBV DNA replication. In conclusion, this study verifies the anti-hepatitis B effect of cichoric acid from Cichorium intybus leaves. In addition, cichoric acid could be used to design the antiviral agents.

An in vivo duck hepatitis B virus model recapitulates key aspects of nucleic acid polymer treatment outcomes in chronic hepatitis B patients.

Nucleic acid polymers (NAPs) are an attractive treatment modality for chronic hepatitis B (CHB), with REP2139 and REP2165 having shown efficacy in CHB patients. A subset of patients achieve functional cure, whereas the others exhibit a moderate response or are non-responders. NAP efficacy has been difficult to recapitulate in animal models, with the duck hepatitis B virus (DHBV) model showing some promise but remaining underexplored for NAP efficacy testing. Here we report on an optimized in vivo DHBV duck model and explore several characteristics of NAP treatment. REP2139 was efficacious in reducing DHBV DNA and DHBsAg levels in approximately half of the treated ducks, whether administered intraperitoneally or subcutaneously. Intrahepatic or serum NAP concentrations did not correlate with efficacy, nor did the appearance of anti-DHBsAg antibodies. Furthermore, NAP efficacy was only observed in experimentally infected ducks, not in endogenously infected ducks (vertical transmission). REP2139 add-on to entecavir treatment induced a deeper and more sustained virological response compared to entecavir monotherapy. Destabilized REP2165 showed a different activity profile with a more homogenous antiviral response followed by a faster rebound. In conclusion, subcutaneous administration of NAPs in the DHBV duck model provides a useful tool for in vivo evaluation of NAPs. It recapitulates many aspects of this class of compound's efficacy in CHB patients, most notably the clear division between responders and non-responders.

TP-RT domain interactions of duck hepatitis B virus reverse transcriptase in cis and in trans during protein-primed initiation of DNA synthesis in vitro.

The hepadnavirus reverse transcriptase (RT) has the unique ability to initiate viral DNA synthesis using RT itself as a protein primer. Protein priming requires complex interactions between the N-terminal TP (terminal protein) domain, where the primer (a specific Y residue) resides, and the central RT domain, which harbors the polymerase active site. While it normally utilizes the cis-linked TP to prime DNA synthesis (cis-priming), we found that the duck hepatitis B virus (DHBV) RT domain, in the context of the full-length RT protein or a mini-RT construct containing only truncated TP and RT domains, could additionally use a separate TP or RT domain in trans as a primer (trans-priming). trans interaction could also be demonstrated by the inhibitory effect (trans-inhibition) on cis-priming by TP and RT domain sequences provided in trans. Protein priming was further shown to induce RT conformational changes that resulted in TP-RT domain dissociation, altered priming site selection, and a gain of sensitivity to a pyrophosphate analog inhibitor. trans-priming, trans-inhibition, and trans-complementation, which requires separate TP and RT domains to reconstitute a functional RT protein, were employed to define the sequences in the TP and RT domains that could mediate physical or functional inter- and intradomain interactions. These results provide new insights into TP-RT domain interactions and conformational dynamics during protein priming and suggest novel means to inhibit protein priming by targeting these interactions and the associated conformational transitions.

Extensive mutagenesis of the conserved box E motif in duck hepatitis B virus P protein reveals multiple functions in replication and a common structure with the primer grip in HIV-1 reverse transcriptase.

Hepadnaviruses, including the pathogenic hepatitis B virus (HBV), replicate their small DNA genomes through protein-primed reverse transcription, mediated by the terminal protein (TP) domain in their P proteins and an RNA stem-loop, ε, on the pregenomic RNA (pgRNA). No direct structural data are available for P proteins, but their reverse transcriptase (RT) domains contain motifs that are conserved in all RTs (box A to box G), implying a similar architecture; however, experimental support for this notion is limited. Exploiting assays available for duck HBV (DHBV) but not the HBV P protein, we assessed the functional consequences of numerous mutations in box E, which forms the DNA primer grip in human immunodeficiency virus type 1 (HIV-1) RT. This substructure coordinates primer 3'-end positioning and RT subdomain movements during the polymerization cycle and is a prime target for nonnucleosidic RT inhibitors (NNRTIs) of HIV-1 RT. Box E was indeed critical for DHBV replication, with the mutations affecting the folding, ε RNA interactions, and polymerase activity of the P protein in a position- and amino acid side chain-dependent fashion similar to that of HIV-1 RT. Structural similarity to HIV-1 RT was underlined by molecular modeling and was confirmed by the replication activity of chimeric P proteins carrying box E, or even box C to box E, from HIV-1 RT. Hence, box E in the DHBV P protein and likely the HBV P protein forms a primer grip-like structure that may provide a new target for anti-HBV NNRTIs.

Characterization of nucleosome positioning in hepadnaviral covalently closed circular DNA minichromosomes.

Hepadnaviral covalently closed circular DNA (cccDNA) exists as an episomal minichromosome in the nucleus of virus-infected hepatocytes, and serves as the transcriptional template for the synthesis of viral mRNAs. To obtain insight on the structure of hepadnaviral cccDNA minichromosomes, we utilized ducks infected with the duck hepatitis B virus (DHBV) as a model and determined the in vivo nucleosome distribution pattern on viral cccDNA by the micrococcal nuclease (MNase) mapping and genome-wide PCR amplification of isolated mononucleosomal DHBV DNA. Several nucleosome-protected sites in a region of the DHBV genome [nucleotides (nt) 2000 to 2700], known to harbor various cis transcription regulatory elements, were consistently identified in all DHBV-positive liver samples. In addition, we observed other nucleosome protection sites in DHBV minichromosomes that may vary among individual ducks, but the pattern of MNase mapping in those regions is transmittable from the adult ducks to the newly infected ducklings. These results imply that the nucleosomes along viral cccDNA in the minichromosomes are not random but sequence-specifically positioned. Furthermore, we showed in ducklings that a significant portion of cccDNA possesses a few negative superhelical turns, suggesting the presence of intermediates of viral minichromosomes assembled in the liver, where dynamic hepatocyte growth and cccDNA formation occur. This study supplies the initial framework for the understanding of the overall complete structure of hepadnaviral cccDNA minichromosomes.

In vivo infectivity 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.

Genetic characterization of duck hepatitis B viruses from Anhui Province, China.

Duck hepatitis B virus (DHBV) infection model was frequently used as the experimental model for human hepatitis B virus (HBV) research. In order to decipher the genetic characteristics of DHBVs from Anhui province of China, 120 duck liver tissue samples were collected and subjected to PCR screening, and 28 samples were detected as DHBV positive. Subsequently, five DHBV-positive samples were selected for genome-wide amplification and a comprehensive analysis. Comparative analysis of complete genome sequences using the MegAlign program showed that five strains of DHBVs shared 94.5-96.3% with each other and 93.2-98.7% with other reference strains in GenBank. The phylogenetic analysis showed that all five DHBV strains belonged to the evolutionary branch of "Chinese DHBV" isolates or DHBV-2. Importantly, three potential intra-genotypic recombination events, between strains AAU-6 and Guilin, strains AAU-1 and GD3, and strains AAU-6 and AAU-1, were respectively found using the RDP and SimPlot softwares and considered the first report in avihepadnaviruses. These results not only improve our understanding for molecular prevalence status of DHBV among ducks, but also provide a reference for recombination mechanism of HBV.