Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming.

Hepadnaviruses, including human hepatitis B virus (HBV), replicate their tiny DNA genomes by protein-primed reverse transcription of a pregenomic (pg) RNA. Replication initiation as well as pgRNA encapsidation depend on the interaction of the viral polymerase, P protein, with the ε RNA element, featuring a lower and an upper stem, a central bulge, and an apical loop. The bulge, somehow assisted by the loop, acts as template for a P protein-linked DNA oligo that primes full-length minus-strand DNA synthesis. Phylogenetic conservation and earlier mutational studies suggested the highly based-paired ε structure as crucial for productive interaction with P protein. Using the tractable duck HBV (DHBV) model we here interrogated the entire apical DHBV ε (Dε) half for sequence- and structure-dependent determinants of in vitro priming activity, replication, and, in part, in vivo infectivity. This revealed single-strandedness of the bulge, a following G residue plus the loop subsequence GUUGU as the few key determinants for priming and initiation site selection; unexpectedly, they functioned independently of a specific structure context. These data provide new mechanistic insights into avihepadnaviral replication initiation, and they imply a new concept towards a feasible in vitro priming system for human HBV.

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.

Human hepatitis B virus production in avian cells is characterized by enhanced RNA splicing and the presence of capsids containing shortened genomes.

Experimental studies on hepatitis B virus (HBV) replication are commonly done with human hepatoma cells to reflect the natural species and tissue tropism of the virus. However, HBV can also replicate, upon transfection of virus coding plasmids, in cells of other species. In such cross-species transfection experiments with chicken LMH hepatoma cells, we previously observed the formation of HBV genomes with aberrant electrophoretic mobility, in addition to the those DNA species commonly seen in human HepG2 hepatoma cells. Here, we report that these aberrant DNA forms are mainly due to excessive splicing of HBV pregenomic RNA and the abundant synthesis of spliced DNA products, equivalent to those also made in human cells, yet at much lower level. Mutation of the common splice acceptor site abolished splicing and in turn enhanced production of DNA from full-length pgRNA in transfected LMH cells. The absence of splicing made other DNA molecules visible, that were shortened due to the lack of sequences in the core protein coding region. Furthermore, there was nearly full-length DNA in the cytoplasm of LMH cells that was not protected in viral capsids. Remarkably, we have previously observed similar shortened genomes and non-protected viral DNA in human HepG2 cells, yet exclusively in the nucleus where uncoating and final release of viral genomes occurs. Hence, two effects reflecting capsid disassembly in the nucleus in human HepG2 cells are seen in the cytoplasm of chicken LMH cells.

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.

Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner.

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.

Quantitative assessment of the antiviral potencies of 21 shRNA vectors targeting conserved, including structured, hepatitis B virus sites.

BACKGROUND & AIMS: RNA interference (RNAi) may offer new treatment options for chronic hepatitis B. Replicating via an RNA intermediate, hepatitis B virus (HBV) is known to be principally vulnerable to RNAi. However, beyond delivery, the relevant issues of potential off-target effects, target site conservation in circulating HBV strains, and efficacy of RNAi itself have not systematically been addressed, nor can the different existing data be quantitatively compared. The aim of this study was to provide such information. METHODS: To focus on the intracellular RNAi process itself and minimise other variables affecting overall RNAi efficacy, we used a robust co-transfection system to quantitatively assess the relative potencies of 21 small-hairpin (sh) RNA vectors, targeting conserved sites throughout the HBV genome, against viral RNAs, proteins, nucleocapsids, and secreted virions under standardised conditions. RESULTS: The approach enabled a distinct efficacy ranking, with the six most potent shRNAs achieving 95% reductions in virion formation, sequence-specifically and without detectable interferon induction, yet by differentially affecting different steps. Efficacy correlated poorly with predictions and was not principally abolished by target structure. Sequence comparisons suggest that truly conserved, RNAi-targetable sequences comprise less than 500 nucleotides of the circulating HBV genomes. CONCLUSIONS: The HBV genome can harbour only a finite number of optimal target sites, but current predictions are poorly suited to constrain the number of possible candidates. However, the small size of the highly conserved sequence space suggests experimental identification as a viable option.

Monoclonal antibodies providing topological information on the duck hepatitis B virus core protein and avihepadnaviral nucleocapsid structure.

The icosahedral capsid of duck hepatitis B virus (DHBV) is formed by a single core protein species (DHBc). DHBc is much larger than HBc from human HBV, and no high-resolution structure is available. In an accompanying study (M. Nassal, I. Leifer, I. Wingert, K. Dallmeier, S. Prinz, and J. Vorreiter, J. Virol. 81:13218-13229, 2007), we used extensive mutagenesis to derive a structural model for DHBc. For independent validation, we here mapped the epitopes of seven anti-DHBc monoclonal antibodies. Using numerous recombinant DHBc proteins and authentic nucleocapsids from different avihepadnaviruses as test antigens, plus a panel of complementary assays, particle-specific and exposed plus buried linear epitopes were revealed. These data fully support key features of the model.

Hepatitis B virus DNA is subject to extensive editing by the human deaminase APOBEC3C.

UNLABELLED: APOBEC3G (A3G) and APOBEC3C (A3C), 2 members of the APOBEC family, are cellular cytidine deaminases displaying broad antiretroviral activity. A3G inhibits hepatitis B virus (HBV) production by interfering with HBV replication without hypermutating the majority of HBV genomes. In contrast, A3C has little effect on HBV DNA synthesis. The aim of this study was to further dissect the mechanisms by which A3G and A3C interfere with the HBV life cycle. Immunoprecipitation experiments demonstrated that both A3G and A3C bind to the HBV core protein. A ribonuclease (RNase) treatment resulted in the nearly complete dissociation of the HBV core protein from A3G, whereas the HBV core-A3C complex was more stable. Interestingly, the majority of the newly synthesized HBV DNA genomes displayed extensive G-to-A mutations in the presence of A3C, whereas no A3C-induced HBV RNA mutations were detected. These findings support a model in which the RNA-dependent entrapment of A3G into the preassembly complex hampers subsequent steps in capsid formation. On the other hand, A3C is readily packaged into replication-competent capsids and efficiently deaminates newly synthesized HBV DNA. CONCLUSION: These findings demonstrate that HBV is highly vulnerable to the editing activity of an endogenous human deaminase and suggest that A3C could contribute to innate anti-HBV host responses.

APOBEC-mediated interference with hepadnavirus production.

APOBEC3G is a cellular cytidine deaminase displaying broad antiretroviral activity. Recently, it was shown that APOBEC3G can also suppress hepatitis B virus (HBV) production in human hepatoma cells. In the present study, we characterized the mechanisms of APOBEC-mediated antiviral activity against HBV and related hepadnaviruses. We show that human APOBEC3G blocks HBV production in mammalian and nonmammalian cells and is active against duck HBV as well. Early steps of viral morphogenesis, including RNA and protein synthesis, binding of pregenomic RNA to core protein, and self-assembly of viral core protein, were unaffected. However, APOBEC3G rendered HBV core protein-associated full-length pregenomic RNA nuclease-sensitive. Ongoing reverse-transcription in capsids that had escaped the block in morphogenesis was not significantly inhibited. The antiviral effect was not modulated by abrogating or enhancing expression of the accessory HBV X protein, suggesting that HBV X protein does not represent a functional homologue to the HIV vif protein. Furthermore, human APOBEC3F but not rat APOBEC1 inhibited HBV DNA production. Viral RNA and low-level DNA produced in the presence of APOBEC3F or rat APOBEC1 occasionally displayed mutations, but the majority of clones were wild-type. In conclusion, APOBEC3G and APOBEC3F but not rat APOBEC1 can downregulate the production of replication-competent hepadnaviral nucleocapsids. In contrast to HIV and other retroviruses, however, APOBEC3G/3F-mediated editing of nucleic acids does not seem to represent an effective innate defense mechanism for hepadnaviruses.