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.

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.

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.

The pregenome/C RNA of duck hepatitis B virus is not used for translation of core protein during the early phase of infection in vitro.

Over the course of duck hepatitis B virus (DHBV) replication, one type of RNA (pregenome/C RNA, 3.5 kb) that corresponds to the whole genome of DHBV is generated from the transcription of viral cccDNA. Previous work has proposed three functions for the pregenome/C RNA: it can serve as the pregenome and be packaged into the core protein during the process of replication, and it encodes the mRNA for both the capsid protein and the viral polymerase. However, little is known about the timing of these functions during the different stages of viral infection. In this study, a reverse transcription quantitative real-time PCR assay was developed to analyze the dynamic transcription process of the pregenome/C RNA. The dynamic expression of the core protein was investigated using an indirect immunofluorescence assay (IFA) and by western blot analysis. The generation of pregenome/C RNA began at 12 h post infection and peaked at 20 h post infection; however, the core protein was not detectable until 24h post infection. These results demonstrate that the core protein appeared approximately 12h later than the pregenome/C RNA. These results suggest that the DHBV pregenome/C RNA is not used for the translation of the viral core protein during the early stages of infection.

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.

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.

Alpha-interferon suppresses hepadnavirus transcription by altering epigenetic modification of cccDNA minichromosomes.

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the transcriptional template for viral mRNA synthesis. Elimination of cccDNA is the prerequisite for either a therapeutic cure or immunological resolution of HBV infection. Although accumulating evidence suggests that inflammatory cytokines-mediated cure of virally infected hepatocytes does occur and plays an essential role in the resolution of an acute HBV infection, the molecular mechanism by which the cytokines eliminate cccDNA and/or suppress its transcription remains elusive. This is largely due to the lack of convenient cell culture systems supporting efficient HBV infection and cccDNA formation to allow detailed molecular analyses. In this study, we took the advantage of a chicken hepatoma cell line that supports tetracycline-inducible duck hepatitis B virus (DHBV) replication and established an experimental condition mimicking the virally infected hepatocytes in which DHBV pregenomic (pg) RNA transcription and DNA replication are solely dependent on cccDNA. This cell culture system allowed us to demonstrate that cccDNA transcription required histone deacetylase activity and IFN-α induced a profound and long-lasting suppression of cccDNA transcription, which required protein synthesis and was associated with the reduction of acetylated histone H3 lysine 9 (H3K9) and 27 (H3K27) in cccDNA minichromosomes. Moreover, IFN-α treatment also induced a delayed response that appeared to accelerate the decay of cccDNA. Our studies have thus shed light on the molecular mechanism by which IFN-α noncytolytically controls hepadnavirus infection.

Concerted action of activation-induced cytidine deaminase and uracil-DNA glycosylase reduces covalently closed circular DNA of duck hepatitis B virus.

Covalently closed circular DNA (cccDNA) forms a template for the replication of hepatitis B virus (HBV) and duck HBV (DHBV). Recent studies suggest that activation-induced cytidine deaminase (AID) functions in innate immunity, although its molecular mechanism of action remains unclear, particularly regarding HBV restriction. Here we demonstrated that overexpression of chicken AID caused hypermutation and reduction of DHBV cccDNA levels. Inhibition of uracil-DNA glycosylase (UNG) by UNG inhibitor protein (UGI) abolished AID-induced cccDNA reduction, suggesting that the AID/UNG pathway triggers the degradation of cccDNA via cytosine deamination and uracil excision.

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.

Spectroscopic characterization and fusogenic properties of preS domains of duck hepatitis B virus.

In order to shed light on the hepatitis B virus fusion mechanism and to explore the fusogenic capabilities of preS regions, a recombinant duck hepatitis B virus (DHBV) preS protein (DpreS) containing six histidines at the carboxy-terminal end has been obtained. The DpreS domain, which has an open and mostly nonordered conformation as indicated by fluorescence and circular dichroism spectroscopies, has the ability to interact with negatively charged phospholipid vesicles. The observed interaction differences between neutral and acidic phospholipids can be interpreted in terms of an initial ionic interaction between the phospholipid polar headgroup and the protein followed by the insertion of probably the N-terminal region in the cellular membrane. Fluorescence polarization studies detect a decrease of the transition enthalpy together with a small modification of the transition temperature, typical effects of integral membrane proteins. The interaction of the protein with acidic phospholipid vesicles induces aggregation, lipid mixing, and leakage of internal contents, properties that have been ascribed to membrane destabilizing proteins. The fact that the preS domains of the hepadnaviruses have little similarity but share a very similar hydrophobic profile points to the importance of the overall three-dimensional structure as well as to its conformational flexibility and the distribution of polar and apolar amino acids on the expression of their destabilizing properties rather than to a particular amino acid sequence. The results presented herein argue for the involvement of DpreS in the initial steps of DHBV infection. Taken together with previously reported results, the conclusion that both S and preS regions participate in the fusion process of the hepadnaviridae family may be drawn.

Carbonyl J acid derivatives block protein priming of hepadnaviral P protein and DNA-dependent DNA synthesis activity of hepadnaviral nucleocapsids.

Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploiting in vitro priming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 µM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives.

The unstable part of the apical stem of duck hepatitis B virus epsilon shows enhanced base pair opening but not pico- to nanosecond dynamics and is essential for reverse transcriptase binding.

Hepatitis B virus (HBV) replication starts with binding of reverse transcriptase (RT) to the apical stem-loop region of epsilon, a conserved element of the RNA pregenome. For duck HBV, an in vitro replication system has provided molecular details of this interaction. Further insights can be obtained from the structure and dynamics of the duck and human apical stem-loops. Previously, we reported these for the human apical stem-loop. Here, we present the same for the duck counterpart. Unlike its human counterpart, the duck apical stem is unstable in its middle/upper part and contains noncanonical base pairs. This dynamics study is the first of an unstable RNA-DNA stem. Similar to the human stem, the duck apical stem comprises two helical segments with a bend angle of ca. 10 degrees , separated by a nonpaired mobile U residue. It is capped by a well-structured conserved UGUU loop with two residues mobile on the pico- to nanosecond time scale, one of which is involved in RT binding. Remarkably, the unstable middle/upper part of the stem does not show enhanced pico- to nanosecond time scale dynamics. Instead, adenine dispersion relaxation studies indicate enhanced millisecond time scale dynamics involving base pair opening. It can then be concluded that base pair opening is essential for epsilon-RT binding, because stabilization of the stem abolishes binding. We hypothesize that binding occurs by conformational capture of bases in the base pair open state. The unstable secondary structure of the apical stem-loop makes duck epsilon-RT binding unusual in light of recent classifications of RNA target interactions that assume stable secondary structures.

[Effect of ampelopsis of Ampelopsis grossedentata on duck hepatitis B virus].

OBJECTIVE: We have evaluated the direct effect of ampelopsis (APS) on duck hepatitis B virus (DHBV) replication in ducklings in vivo. METHOD: One-day-old ducklings were infected with DHBV. After infection for 7 days, the animals were treated with APS at dosages of 70, 150, 300 mg x kg(-1) of body weight via the oral route. The drug was given twice per day for 10 days continuously, and normal saline was used as control. The blood was drawn from the posterior tibial vein of all ducks before treatment (T0), after the medication for 5 (T5), 10 (T10) days and withdrawal of the drug for 3 days (P3). DHBV DNA in duck serum was detected by dot blot. RESULT: The duck serum DHBV-DNA levels were reduced in the group of APS (150, 300 mg x kg(-1)) after treated for 5 and 10 days and the levels of DHBV-DNA did not markedly relapse in both groups of APS after withdrawal of the drug for 3 days. We provide the first evidence that APS can efficiently inhibits DHBV replication in ducks in vivo. CONCLUSION: APS therefore warrants further investigation as a potential therapeutic agent for HBV infections.

An interdomain RNA binding site on the hepadnaviral polymerase that is essential for reverse transcription.

The T3 motif on the duck hepatitis B virus reverse transcriptase (P) is proposed to be a binding site essential for viral replication, but its ligand and roles in DNA synthesis are unknown. Here, we found that T3 is needed for P to bind the viral RNA, the first step in DNA synthesis. A second motif, RT-1, was predicted to assist T3. T3 and RT-1 appear to form a composite RNA binding site because mutating T3 and RT-1 had similar effects on RNA binding, exposure of antibody epitopes on P, and DNA synthesis. The T3 and RT-1 motifs bound RNA non-specifically, yet they were essential for specific interactions between P and the viral RNA. This implies that specificity for the viral RNA is provided by a post-binding step. The T3:RT-1 motifs are conserved with the human hepatitis B virus and may be an attractive target for novel antiviral drug development.

Anti-hepatitis B virus activity and mechanisms of recombinant human serum albumin-interferon-alpha-2b fusion protein in vitro and in vivo.

AIMS: To evaluate the anti-hepatitis B virus (anti-HBV) effects and mechanisms of recombinant human serum albumin-interferon-alpha-2b fusion protein (rHSA-IFNalpha-2b) in vitro and in vivo. METHODS: The inhibiting effects on HBV replication were examined in the HepG2 2.2.15 cell line and in ducks, and the expressions of signal transducers and transactivator 1 (STAT1), IFN-stimulated gene factor 3 (ISGF3) and 2',5'-oligoadenylate synthetase 1 (OAS1) were investigated by the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. RESULTS: In vitro,at concentrations from 0.075 to 1.2 nmol/l, rHSA-IFNalpha-2b inhibited the releases of extracellular hepatitis B surface antigen, hepatitis B e antigen and HBV DNA in a dose-dependent manner; rHSA- IFNalpha-2b also increased the levels of STAT1, ISGF3 and OAS1. In vivo, rHSA-IFNalpha-2b reduced the levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin and duck hepatitis B virus (DHBV) DNA in the sera of DHBV-infected ducks. CONCLUSIONS: We provide the first evidence that rHSA-IFNalpha-2b significantly inhibits HBV replication in HepG2 2.2.15 cells and in ducks, and that the antiviral effect of rHSA-IFNalpha-2b in vivo is more potent than that of IFNalpha-2b. The anti-HBV mechanism probably operates by triggering the JAK-STAT signaling pathway and increasing the expression of OAS1.

Zinc finger proteins designed to specifically target duck hepatitis B virus covalently closed circular DNA inhibit viral transcription in tissue culture.

Duck hepatitis B virus (DHBV) is a model virus for human hepatitis B virus (HBV), which infects approximately 360 million individuals worldwide. Nucleoside analogs can decrease virus production by inhibiting the viral polymerase; however, complete clearance by these drugs is not common because of the persistence of the HBV episome. HBV DNA is present in the nucleus as a covalently closed circular (cccDNA) form, where it drives viral transcription and progeny virus production. cccDNA is not the direct target of antiviral nucleoside analogs and is the source of HBV reemergence when antiviral therapy is stopped. To target cccDNA, six different zinc finger proteins (ZFP) were designed to bind DNA sequences in the DHBV enhancer region. After the binding kinetics were assessed by using electrophoretic mobility shift assays and surface plasmon resonance, two candidates with dissociation constants of 12.3 and 40.2 nM were focused on for further study. The ZFPs were cloned into a eukaryotic expression vector and cotransfected into longhorn male hepatoma cells with the plasmid pDHBV1.3, which replicates the DHBV life cycle. In the presence of each ZFP, viral RNA was significantly reduced, and protein levels were dramatically decreased. As a result, intracellular viral particle production was also significantly decreased. In summary, designed ZFPs are able to bind to the DHBV enhancer and interfere with viral transcription, resulting in decreased production of viral products and progeny virus genomes.

Functional and structural dynamics of hepadnavirus reverse transcriptase during protein-primed initiation of reverse transcription: effects of metal ions.

Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the interaction between the RT and a specific viral RNA template termed epsilon. Protein priming is resistant to a number of RT inhibitors that can block subsequent viral DNA elongation and likely requires a distinct "priming" conformation. Furthermore, protein priming may consist of two distinct stages, i.e., the attachment of the first deoxynucleotide to RT (initiation) and the subsequent addition of 2 or 3 deoxynucleotides (polymerization). In particular, a truncated duck hepatitis B virus RT (MiniRT2) is competent in initiation but defective in polymerization when tested in the presence of Mg(2+). Given the known effects of metal ions on the activities of various DNA and RNA polymerases, we tested if metal ions could affect hepadnavirus RT priming. We report here that Mn(2+), in comparison with Mg(2+), showed dramatic effects on the priming activity of MiniRT2 as well as the full-length RT. First and foremost, MiniRT2 exhibited full polymerization activity in the presence of Mn(2+), indicating that MiniRT2 contains all sequences essential for polymerization but is unable to transition from initiation to polymerization with Mg(2+). Second, the initiation activities of MiniRT2 and the full-length RT were much stronger with Mn(2+). Third, the nucleotide and template specificities during protein priming were decreased in the presence of Mn(2+). Fourth, polymerization was sensitive to inhibition by a pyrophosphate analog in the presence of Mn(2+) but not in the presence of Mg(2+). Finally, limited proteolysis provided direct evidence that the priming active MiniRT2 adopted distinct conformations depending on the presence of Mn(2+) versus that of Mg(2+) and that the transition from initiation to polymerization was accompanied by RT conformational change.

1H, 13C and 15N NMR assignments of Duck HBV primer loop of the encapsidation signal epsilon.

The replication of the hepatitis B virus is initiated by binding of the viral reverse transcriptase protein complex to the apical stem loop of the epsilon element to place it next to the primer loop, from which a four nucleotide DNA primer is subsequently synthesized. Here, we present the (1)H/(13)C/(15)N NMR assignments of the bases and sugars of the 37 residues primer loop of Duck HBV epsilon (BMRB-entry 15786).

1H, 13C and 15N NMR assignments of Duck HBV apical stem loop of the epsilon encapsidation signal.

The replication of Hepatitis B virus is initiated by binding of its reverse transcriptase to the apical stem loop and primer loop of epsilon. Here, we present the (1)H/(13)C/(15)N NMR assignments of the bases and sugars of the 29 residues apical stem loop of Duck HBV epsilon.

Chaperones activate hepadnavirus reverse transcriptase by transiently exposing a C-proximal region in the terminal protein domain that contributes to epsilon RNA binding.

All hepatitis B viruses replicate by protein-primed reverse transcription, employing a specialized reverse transcriptase, P protein, that carries a unique terminal protein (TP) domain. To initiate reverse transcription, P protein must bind to a stem-loop, epsilon, on the pregenomic RNA template. TP then provides a Y residue for covalent attachment of the first nucleotide of an epsilon-templated DNA oligonucleotide (priming reaction) that serves to initiate full-length minus-strand DNA synthesis. epsilon binding requires the chaperone-dependent conversion of inactive P protein into an activated, metastable form designated P*. However, how P* differs structurally from P protein is not known. Here we used an in vitro reconstitution system for active duck hepatitis B virus P combined with limited proteolysis, site-specific antibodies, and defined P mutants to structurally compare nonactivated versus chaperone-activated versus primed P protein. The data show that Hsp70 action, under conditions identical to those required for functional activation, transiently exposes the C proximal TP region which is, probably directly, involved in epsilon RNA binding. Notably, after priming and epsilon RNA removal, a very similar new conformation appears stable without further chaperone activity; hence, the activation of P protein is triggered by energy-consuming chaperone action but may be completed by template RNA binding.