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.

Developments in Cell-Penetrating Peptides as Antiviral Agents and as Vehicles for Delivery of Peptide Nucleic Acid Targeting Hepadnaviral Replication Pathway.

Alternative therapeutic approaches against chronic hepatitis B virus (HBV) infection need to be urgently developed because current therapies are only virostatic. In this context, cell penetration peptides (CPPs) and their Peptide Nucleic Acids (PNAs) cargoes appear as a promising novel class of biologically active compounds. In this review we summarize different in vitro and in vivo studies, exploring the potential of CPPs as vehicles for intracellular delivery of PNAs targeting hepadnaviral replication. Thus, studies conducted in the duck HBV (DHBV) infection model showed that conjugation of (D-Arg)8 CPP to PNA targeting viral epsilon (ε) were able to efficiently inhibit viral replication in vivo following intravenous administration to ducklings. Unexpectedly, some CPPs, (D-Arg)8 and Decanoyl-(D-Arg)8, alone displayed potent antiviral effect, altering late stages of DHBV and HBV morphogenesis. Such antiviral effects of CPPs may affect the sequence-specificity of CPP-PNA conjugates. By contrast, PNA conjugated to (D-Lys)4 inhibited hepadnaviral replication without compromising sequence specificity. Interestingly, Lactose-modified CPP mediated the delivery of anti-HBV PNA to human hepatoma cells HepaRG, thus improving its antiviral activity. In light of these promising data, we believe that future studies will open new perspectives for translation of CPPs and CPP-PNA based technology to therapy of chronic hepatitis B.

Characterization of novel hepadnaviral RNA species accumulated in hepatoma cells treated with viral DNA polymerase inhibitors.

Inhibitors of hepadnaviral DNA polymerases are predicted to inhibit both minus and plus strand of viral DNA synthesis and arrest viral DNA replication at the stage of pregenomic (pg) RNA-containing nucleocapsids. However, analyses of the RNA species of human and duck hepatitis B viruses (HBV and DHBV, respectively) in hepatoma cells treated with viral DNA polymerase inhibitors revealed the genesis of novel RNA species migrating slightly faster than the full-length pgRNA. The DNA polymerase inhibitor-induced accumulation of these RNA species were abolished in the presence of alpha-interferon or HBV nucleocapsid assembly inhibitors. Moreover, they were protected from microccocal nuclease digestion and devoid of a poly-A tail. These characteristics suggest that the novel RNA species are most likely generated from RNase H cleavage of encapsidated pgRNA, after primer translocation and synthesis of the 5' terminal portion of minus strand DNA. In support of this hypothesis, DNA polymerase inhibitor treatment of chicken hepatoma cells transfected with a DHBV genome encoding an RNase H inactive DNA polymerase (E696H) failed to produce such RNA species. Our results thus suggest that the currently available DNA polymerase inhibitors do not efficiently arrest minus strand DNA synthesis at the early stage in hepatocytes. Hence, development of novel antiviral agents that more potently suppress viral DNA synthesis or viral nucleocapsid assembly inhibitors that are mechanistically complementary to the currently available DNA polymerase inhibitors are warranted.

Comparative Transcriptomic Analysis of Primary Duck Hepatocytes Provides Insight into Differential Susceptibility to DHBV Infection.

Primary duck hepatocytes (PDH) displays differential susceptibility to duck hepatitis B virus when maintained in the media supplemented with fetal bovine serum or dimethyl sulfoxide (DMSO) which has been widely used for the maintenance of hepatocytes, and prolonging susceptibility to hepadnavirus. However the mechanism underlying maintenance of susceptibility to hepadnavirus by DMSO treatment remains unclear. In this study, a global transcriptome analysis of PDHs under different culture conditions was conducted for investigating the effects of DMSO on maintenance of susceptibility of PDH to DHBV in vitro. The 384 differential expressed genes (DEGs) were identified by comparisons between each library pair (PDHs cultured with or without DMSO or fresh isolated PDH). We analyzed canonical pathways in which the DEGs were enriched in Hepatic Fibrosis / Hepatic Stellate Cell Activation, Bile Acid Biosynthesis and Tight Junction signaling. After re-annotation against human genome data, the 384 DEGs were pooled together with proteins belonging to hepatitis B pathway to construct a protein-protein interaction network. The combination of decreased expression of liver-specific genes (CYP3A4, CYP1E1, CFI, RELN and GSTA1 et al) with increased expression of hepatocyte-dedifferentiation-associated genes (PLA2G4A and PLCG1) suggested that in vitro culture conditions results in the fading of hepatocyte phenotype in PDHs. The expression of seven DEGs associated with tight junction formation (JAM3, PPP2R2B, PRKAR1B, PPP2R2C, MAGI2, ACTA2 and ACTG2) was up-regulated after short-term culture in vitro, which was attenuated in the presence of DMSO. Those results could shed light on DHBV infection associated molecular events affected by DMSO.

Duck Hepatitis B Virus cccDNA Amplification Efficiency in Natural Infection Is Regulated by Virus Secretion Efficiency.

Previous mutation based studies showed that ablating synthesis of viral envelope proteins led to elevated hepadnaviral covalently closed circular DNA (cccDNA) amplification, but it remains unknown how cccDNA amplification is regulated in natural hepadnaviral infection because of a lack of research system. In this study we report a simple procedure to prepare two identical duck hepatitis B virus inocula, but they possess 10-100-fold difference in cccDNA amplification in infected cell culture. We demonstrate that the infected cells with higher cccDNA amplification significantly reduce the virus secretion efficiency that results in higher accumulation of relaxed circular DNA (rcDNA) and DHBsAg in the cells. The infected cells with lower cccDNA amplification significantly increase the virus secretion efficiency that leads to lower intracellular rcDNA and DHBsAg accumulation. In contrast with the findings generated in the mutation based experimental system, the regulation of cccDNA amplification in natural hepadnaviral infection bypasses direct regulation of the cellular envelope proteins concentration, instead it modulates virus secretion efficiency that ultimately impacts the intracellular rcDNA concentration, an important factor determining the destination of the synthesized rcDNA in infected cells.

Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?

Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5' end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5' DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5' DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5' end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo.

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.

Taraxacum mongolicum extract exhibits a protective effect on hepatocytes and an antiviral effect against hepatitis B virus in animal and human cells.

In order to validate the antiviral effect against hepatitis B virus (HBV) of Taraxacum mongolicum (T. mongolicum), the protective effect on hepatocytes, and antiviral properties against duck hepatitis B virus (DHBV) and HBV of T. mongolicum extract (TME) were evaluated in chemically-injured neonatal rat hepatocytes, DHBV-infected duck fetal hepatocytes and HBV-transfected HepG2.2.15 cells, respectively. The results demonstrated that TME at 50-100 µg/ml improved D-galactosamine (D-GalN), thioacetamide (TAA) and tert-butyl hydroperoxide (t-BHP)-injured rat hepatocytes, and produced protection rates of 42.2, 34.6 and 43.8% at 100 µg/ml, respectively. Furthermore, TME at 1-100 µg/ml markedly inhibited DHBV DNA replication. Additionally, TME at 25-100 µg/ml reduced HBsAg and HBeAg levels and produced inhibition rates of 91.39 and 91.72% at 100 µg/ml, respectively. TME markedly inhibited HBV DNA replication at 25-100 µg/ml. The results demonstrate the potent antiviral effect of T. mongolicum against HBV effect. The protective of TME effect on hepatocytes may be achieved by its ability to ameliorate oxidative stress. The antiviral properties of TME may contribute to blocking protein synthesis steps and DNA replication. Furthermore, major components of TME were quantificationally analyzed. These data provide scientific evidence supporting the traditional use of TME in the treatment of hepatitis.

Generation of stable 3'-mRNA cleavage fragments induced by siRNA in cells with high-levels of duck hepatitis B virus replication.

Therapeutic small interfering RNAs (siRNAs) have attracted a lot of interest both in basic biomedical sciences as well as in translational medicine. Apart from their therapeutic efficacy adverse effects of siRNAs must be addressed. The generation of stable mRNA cleavage fragments and the translation of N-truncated proteins induced by antisense oligodeoxynucleotides (ASOs) have been reported. Similar to ASOs, siRNAs are considered to function via an antisense mechanism that promotes the cleavage of the target mRNA. To further investigate whether the stable mRNA cleavage fragments also occur in siRNA we constructed a short hairpin RNA (shRNA) expression plasmid, pshRNA794, containing the same sequence reported in experiments using ASOs which directly targeted the overlapping region of the pre-genomic mRNA (pgmRNA) and sub-genomic mRNA (sgmRNA) of duck hepatitis B virus (DHBV). The shRNA resulted in a 70.9% and 69.9% reduction of the DHBV mRNAs in LMH and HuH-7 cells, respectively. In addition a 70% inhibition of the DHBV DNA level was observed. Interestingly, 3'-mRNA cleavage fragments were detected in LMH but not in HuH-7 cells. Taken together, our findings demonstrate that the ASO sequence was also effective in siRNA. Importantly, our results provide direct evidence that stable 3'-mRNA fragments were generated by siRNA in cells with high levels of DHBV replication. Whether these can cause adverse RNAi effects needs to be explored further.

Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver.

Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10(5)-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis.

Isolation and identification of an anti-hepatitis B virus compound from Hydrocotyle sibthorpioides Lam.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydrocotyle sibthorpioides (Apiaceae) have been used as a folk remedy for the treatment of fever, edema, detoxication, throat pain, psoriasis and hepatitis B virus infections in China. The aim of this study is to isolate and identify an anti-HBV compound from this herb. MATERIALS AND METHODS: A compound (saponin) was isolated from the active ethanol extract using bioassay-guided screening. The structure of the saponin was elucidated by spectroscopic methods and compared with published data. The anti-HBV activity of the saponin was evaluated by detecting the levels of HBV antigens, extracellular HBV DNA, nuclear covalent closed circular DNA (cccDNA) and five HBV promoters in HepG2.2.15 cells. In addition, the levels of serum HBsAg/HBeAg, DHBV DNA, ALT/AST and hepatic pathological changes were analyzed in DHBV-infected ducks. RESULTS: The chemical analysis indicated that the saponin isolated from Hydrocotyle sibthorpioides is asiaticoside. The pharmacodynamics experimental studies showed that asiaticoside effectively suppressed the levels of HBsAg/HBeAg, extracellular HBV DNA and intracellular cccDNA in a dose-dependent manner. Furthermore, experiments demonstrated that asiaticoside markedly reduced viral DNA transcription and replication by inhibiting the activities of core, s1, s2, and X gene promoters. In addition, asiaticoside markedly reduced DHBV replication without any obvious signs of toxicity. The levels of serum DHBV DNA, HBsAg/HBeAg were increased 3 days after drug withdrawal, but the levels rebounded slightly in the asiaticoside treatment groups compared with the 3TC treatment group. Moreover, analysis of the serum ALT/AST levels and the liver pathological changes indicated that asiaticoside could alleviate liver damage. CONCLUSIONS: Our results show that asiaticoside could efficiently inhibit HBV replication both in vitro and in vivo, and asiaticoside may be a major bioactive ingredient in Hydrocotyle sibthorpioides.

Potent inhibition of late stages of hepadnavirus replication by a modified cell penetrating peptide.

Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication of Duck Hepatitis B Virus (DHBV), a reference model for human HBV. Amongst twelve CatLip peptides we identified Deca-(Arg)8 having a particularly potent antiviral activity, leading to a drastic inhibition of viral particle secretion without detectable toxicity. Inhibition of virion secretion was correlated with a dose-dependent increase in intracellular viral DNA. Deca-(Arg)8 peptide did neither interfere with DHBV entry, nor with formation of mature nucleocapsids nor with their travelling to the nucleus. Instead, Deca-(Arg)8 caused envelope protein accumulation in large clusters as revealed by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)8-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis via interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside the mode of inhibition. Deca-(Arg)8 may be a useful tool for elucidating the hepadnaviral secretory pathway, which is not yet fully understood. Moreover we provide the first evidence that a modified CPP displays a novel antiviral mechanism targeting another step of viral life cycle compared to what has been so far described for other enveloped viruses.

C-terminal substitution of HBV core proteins with those from DHBV reveals that arginine-rich 167RRRSQSPRR175 domain is critical for HBV replication.

To investigate the contributions of carboxyl-terminal nucleic acid binding domain of HBV core (C) protein for hepatitis B virus (HBV) replication, chimeric HBV C proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) C protein for the corresponding regions of HBV C protein. All chimeric C proteins formed core particles. A chimeric C protein with 221-262 amino acids of DHBV C protein, in place of 146-185 amino acids of the HBV C protein, supported HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis: 40% amino acid sequence identity or 45% homology in the nucleic-acid binding domain of HBV C protein was sufficient for pgRNA encapsidation and DNA synthesis, although we predominantly detected spliced DNA. A chimeric C protein with 221-241 and 251-262 amino acids of DHBV C, in place of HBV C 146-166 and 176-185 amino acids, respectively, could rescue full-length DNA synthesis. However, a reciprocal C chimera with 242-250 of DHBV C ((242)RAGSPLPRS(250)) introduced in place of 167-175 of HBV C ((167)RRRSQSPRR(175)) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the arginine-rich (167)RRRSQSPRR(175) domain may be critical for efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be important.

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.

Identification of disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in viral infection and persistence and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment. Therefore, there is an urgent need for the development of novel therapeutic agents that directly target cccDNA formation and maintenance. By employing an innovative cell-based cccDNA assay in which secreted HBV e antigen is a cccDNA-dependent surrogate, we screened an in-house small-molecule library consisting of 85,000 drug-like compounds. Two structurally related disubstituted sulfonamides (DSS), termed CCC-0975 and CCC-0346, emerged and were confirmed as inhibitors of cccDNA production, with low micromolar 50% effective concentrations (EC(50)s) in cell culture. Further mechanistic studies demonstrated that DSS compound treatment neither directly inhibited HBV DNA replication in cell culture nor reduced viral polymerase activity in the in vitro endogenous polymerase assay but synchronously reduced the levels of HBV cccDNA and its putative precursor, deproteinized relaxed circular DNA (DP-rcDNA). However, DSS compounds did not promote the intracellular decay of HBV DP-rcDNA and cccDNA, suggesting that the compounds interfere primarily with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck HBV-infected primary duck hepatocytes. This is the first attempt, to our knowledge, to identify small molecules that target cccDNA formation, and DSS compounds thus potentially serve as proof-of-concept drug candidates for development into therapeutics to eliminate cccDNA from chronic HBV infection.

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.

A high level of mutation tolerance in the multifunctional sequence encoding the RNA encapsidation signal of an avian hepatitis B virus and slow evolution rate revealed by in vivo infection.

In all hepadnaviruses, protein-primed reverse transcription of the pregenomic RNA (pgRNA) is initiated by binding of the viral polymerase, P protein, to the ε RNA element. Universally, ε consists of a lower stem and an upper stem, separated by a bulge, and an apical loop. Complex formation triggers pgRNA encapsidation and the ε-templated synthesis of a DNA oligonucleotide (priming) that serves to generate minus-strand DNA. In vitro systems for duck hepatitis B virus (DHBV) yielded important insights into the priming mechanism, yet their relevance in infection is largely unexplored. Moreover, additional functions encoded in the DHBV ε (Dε) sequence could affect in vivo fitness. We therefore assessed the in vivo performances of five recombinant DHBVs bearing multiple mutations in the upper Dε stem. Three variants with only modestly reduced in vitro replication competence established chronic infection in ducks. From one variant but not another, three adapted new variants emerged upon passaging, as demonstrated by increased relative fitness in coinfections with wild-type DHBV. All three showed enhanced priming and replication competence in vitro, and in one, DHBV e antigen (DHBeAg) production was restored. Pronounced impacts on other Dε functions were not detected; however, gradual, synergistic contributions to overall performance are suggested by the fact of none of the variants reaching the in vivo fitness of wild-type virus. These data shed more light on the P-Dε interaction, define important criteria for the design of future in vivo evolution experiments, and suggest that the upper Dε stem sequences provided an evolutionary playground for DHBV to optimize in vivo fitness.

Goose haemorrhagic hepatitis caused by a new subtype duck hepatitis type 1 virus.

Duck hepatitis type 1 virus (DHV-1) causes a fatal disease in ducklings but there is no report of DHV-1 isolation from goose. Recently, cases of a new disease in overfeeding geese were reported from China. The cases were characterized by haemorrhagic hepatitis lesions on liver after post mortem examinations. The flocks showed about 20-40% morbidity and less than 5% mortality. The histopathological lesions showed destroyed structure of hepatocytic tissue, severe vacuolation and necrosis of hepatocytes. Viral antigen could be detected by monoclonal antibody against duck hepatitis type 1 virus (DHV-1) in the cytoplasm of positive hepatocytes. PCR amplified viral sequences with primers specific for recent Korean-like duck hepatitis type 1 virus (DHV-1C). Alignment of the complete sequence demonstrated that the isolated JT strain from goose exhibiting 95.9% identity to DHV-1C AP-03337 strain, and only 75.3% to classical DHV-1 virus. 80% goslings developed haemorrhagic hepatitis after infection with JT strain. This is the first report on the involvement of a DHV-1 virus in goose.

RNA elements directing translation of the duck hepatitis B Virus polymerase via ribosomal shunting.

The duck hepatitis B virus (DHBV) reverse transcriptase (P) is translated from the downstream position on a bicistronic mRNA, called the pregenomic RNA, through a poorly characterized ribosomal shunt. Here, the positions of the discontinuous ribosomal transfer during shunting were mapped, and RNA elements important for shunting were identified as a prelude to dissecting the shunting mechanism. Mutations were introduced into the DHBV genome, genomic expression vectors were transfected into cells which support reverse transcription, and P translation efficiency was defined as the ratio of P/mRNA. Five observations were made. First, ribosomes departed from sequences that comprise the RNA stem-loop called ε that is key to viral replication, but the known elements of ε were not needed for shunting. Second, at least two landing sites for ribosomes were found on the mRNA. Third, all sequences upstream of ε, most sequences between the cap and the P AUG, and sequences within the P-coding region were dispensable for shunting. Fourth, elements on the mRNA involved in reverse transcription or predicted to be involved in shunting on the basis of mechanisms documented in other viruses, including short open reading frames near the departure site, were not essential for shunting. Finally, two RNA elements in the 5' portion of the mRNA were found to assist shunting. These observations are most consistent with shunting being directed by signals that act through an uncharacterized RNA secondary structure. Together, these data indicate that DHBV employs either a novel shunting mechanism or a major variation on one of the characterized mechanisms.

Helioxanthin analogue 8-1 inhibits duck hepatitis B virus replication in cell culture.

BACKGROUND: Current approved anti-HBV treatment cannot completely eliminate HBV infection, and emergence of resistant virus is an important treatment issue. Effective anti-HBV agents with different mechanisms of action on novel target sites are needed for the treatment of HBV infection and for combating the resistant virus, alone or in combination with current anti-HBV strategies. Helioxanthin analogue 8-1 displayed potent anti-HBV activity in human HBV in vitro and in animal models, with a unique antiviral mechanism. Its antiviral activity in other HBV system needs further study. METHODS: The anti-duck hepatitis B virus (DHBV) activity of 8-1, an analogue of a natural product, helioxanthin, was studied in the DHBV inducible cell line, dstet5, in comparison to and in combination with the nucleoside analogue, lamivudine (3TC). RESULTS: Helioxanthin analogue 8-1 exhibited anti-DHBV activity as demonstrated by quantification of viral DNA, RNA, covalently closed circular DNA and protein synthesis. Analogue 8-1 did not affect the stability of cellular macromolecules and did not have a sustained antiviral effect after drug removal. When DHBV replication was induced, virus-harbouring cells were more susceptible to the cytotoxicity of 8-1 than non-induced cells. CONCLUSIONS: 8-1 exhibited effective inhibition on DHBV replication. The combination of 8-1 with 3TC resulted in additional anti-DHBV activity. Viral induced cells displayed higher susceptibility to 8-1 treatment than non-induced cells. HBV X protein might not be an essential factor in the initiation of the biological activity of 8-1, as demonstrated by its absence in DHBV. These findings warrant further development of 8-1 for the treatment of chronic hepatitis B and its associated diseases.