Formation of a functional hepatitis B virus replication initiation complex involves a major structural alteration in the RNA template.

The DNA genome of a hepatitis B virus is generated by reverse transcription of the RNA pregenome. Replication initiation does not involve a nucleic acid primer; instead, the hepadnavirus P protein binds to the structured RNA encapsidation signal epsilon, from which it copies a short DNA primer that becomes covalently linked to the enzyme. Using in vitro-translated duck hepatitis B virus (DHBV) P protein, we probed the secondary structure of the protein-bound DHBV epsilon RNA (Depsilon) and observed a marked conformational change compared to free Depsilon RNA. Several initiation-competent mutant RNAs with a different free-state structure were similarly altered, whereas a binding-competent but initiation-deficient variant was not, indicating the importance of the rearrangement for replication initiation and suggesting a mechanistic coupling to encapsidation.

Evolutionary conservation in the hepatitis B virus core structure: comparison of human and duck cores.

BACKGROUND: Hepatitis B virus is a major human pathogen which has been extensively studied, yet its structure is unknown. Cryo-electron microscopy of the viral cores expressed in Escherichia coli or isolated from infected liver provides a means for determining the structure of the hepatitis B nucleocapsid. RESULTS: Using cryo-electron microscopy and three-dimensional image reconstruction, we have determined the structures of duck and human hepatitis B virus cores and find that they have similar dimer-clustered T = 3 and T = 4 icosahedral organizations. The duck virus core protein sequence differs from the human in both length and amino acid content; however, the only significant structural differences observed are the lobes of density on the lateral edges of the projecting (distal) domain of the core protein dimer. The different cores contain varying amounts of nucleic acid, but exhibit similar contacts between the core protein and the nucleic acid. Immunoelectron microscopy of intact cores has localized two epitopes on the core surface corresponding to residues 76-84 and 129-132. CONCLUSIONS: The bacterial expression system faithfully reproduces the native hepatitis B virus core structure even in the absence of the complete viral genome. This confirms that proper assembly of the core is independent of genome packaging. Difference imaging and antibody binding map three sequence positions in the structure: the C terminus and the regions near amino acids 80 and 130. Finally, we suggest that the genome-core interactions and the base (proximal) domain of the core dimer are evolutionarily conserved whereas the projecting domain, which interacts with the envelope proteins, is more variable.

Hepatitis B virus replication.

Hepatitis B virus (HBV) is the causative agent of B-type hepatitis in humans and the prototypic member of the hepadnaviruses. It is a small enveloped DNA virus that replicates via reverse transcription. Although hepadnaviruses are similar to retroviruses in basic life cycle and genome organization, recent studies have revealed several unique hepadnaviral strategies that optimize exploitation of their extremely small circular DNA genomes.

A carbene-generating photoaffinity probe for beta-adrenergic receptors.

A new radioiodinated (2.2 Ci/mumol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [125I]ICYP-diazirine binds with high affinity (Kd = 60 pM) to beta-receptors from turkey erythrocyte membranes. Upon irradiation, [125I]ICYP-diazirine is covalently incorporated in a Mr 40 000 protein. Stereoselective inhibition of photolabeling by the (-)enantiomers of alprenolol and isoproterenol indicated that the Mr 40 000 protein contains a beta-adrenergic binding site. The yield of specific labeling was up to 8.2% of total beta-receptor binding sites. The Mr 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [125I]ICYP-diazirine resulted in specific labeling of two proteins with Mr 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximately Mr 67 000) was labeled specifically.

Topological analysis of the hepatitis B virus core particle by cysteine-cysteine cross-linking.

The nucleocapsid, or core particle, of hepatitis B virus is formed by 180 subunits of the core protein, which contains Cys at positions 48, 61, 107 and 183, the latter constituting the C terminus. Upon adventitious oxidation, some or all of these cysteine residues participate in the formation of disulphide bridges, leading to polymerization of the subunits within the particle. To utilize the cysteine residues as topological probes, we reduced the number of possible intersubunit crosslinks by replacing these residues individually, or in all combinations, by serine. A corresponding set of variants was constructed within the context of an assembly-competent core protein variant that lacks the highly basic C-terminal region. Analysis, by polyacrylamide gel electrophoresis under non-reducing conditions, of the oxidative crosslinking products formed by the wild-type and mutant proteins expressed in Escherichia coli, revealed a clear distinction between the three N-proximal, and the C-terminal Cys: N-proximal Cys formed intermolecular disulphide bonds only with other N-proximal cysteine residues, leading to dimerization. Cys48 and Cys61, in contrast to Cys107, could be crosslinked to the homologous cysteine residues in a second subunit, and are therefore located at the dimer interface. Cys 183 predominantly formed disulphide bonds with Cys183 in subunits other than those crosslinked by the N-proximal cysteine residues. Hence, the polymers generated by oxidation of the wild-type protein are S-S-linked dimeric N-terminal domains interconnected via Cys183/Cys183 disulphide bonds. The intermolecular crosslinks between the N-proximal cysteine residues were apparently the same in the C-terminally truncated and in the full-length proteins, corroborating the model in which the N-terminal domain and the C terminus of the HBV core protein form two distinct and structurally independent entities. The strong tendency of the N-terminal domain for dimeric interactions suggests that core protein dimers are the major intermediates in hepatitis B virus nucleocapsid assembly.

Epitopes recognized by antibodies to denatured core protein of hepatitis B virus.

Particulate and denatured core protein as well as e-antigen (HBe) of hepatitis B virus (HBV) differ in part immunologically but this has not been studied in sufficient detail. Therefore, in this study the B-cell immune response to native and denatured HBV core protein which both can exhibit HBe-specific epitopes was examined using a panel of mouse MABs and rabbit polyclonal antibodies to native and denatured core protein and polyclonal anti-HBe/anti-HBc antibodies from sera of infected patients. Epitope mapping was performed using a set of partially overlapping synthetic HBc peptides, carboxy-terminally truncated HBc proteins and various HBc fusion proteins. A major immunogenic region between amino acids 134-140 and two less immunogenic regions, one spanning amino acids 2-10 and one with three partially overlapping epitopes between amino acid positions 138 and 154, were defined by mouse MABs. Polyclonal rabbit antibodies to denatured HBc, woodchuck and ground squirrel hepatitis core proteins (WHc and GSHc) recognized similar epitopes but in addition occasionally region 61-85, and the latter was also recognized on particulate HBc. Two antigenic regions (amino acid positions 2-10 and 138-145) were found to be exposed on HBe from human serum, and were recognized by mouse anti-HBe but not by anti-HBc antibodies from sera of infected patients. This study demonstrates a more complex pattern of HBc and HBe epitopes than detected previously and provides tools to study conformational changes which may take place during HBc/HBe processing, transport and core particle assembly.

Total chemical synthesis of a gene for hepatitis B virus core protein and its functional characterization.

We have chemically synthesized a DNA duplex of 560 nucleotides that codes for the hepatitis B virus (HBV) core protein. The synthetic gene contains 27 unique internal restriction sites. Thereby, it can easily be mutagenized by replacement of rather short restriction fragments. A number of restriction recognition sequences are in common between the synthetic and the authentic gene, thus allowing for the transfer of synthetic segments into the cloned viral genome. Several unexpected mutations in the synthetic gene were readily corrected utilizing the multiple unique restriction sites. In Escherichia coli, the expression level of the synthetic gene product amounts to about 4% of the total soluble protein. It forms particles closely resembling native HBV cores. After transfer of the synthetic gene into the viral genome, transient expression in a hepatoma cell line yields proteins indistinguishable from the native gene products. The synthetic gene thus provides a useful tool for studies on the structure and function of the isolated HBV core protein as well as the gene and its various products in the viral life-cycle.

Packaging of up to 240 subunits of a 17 kDa nuclease into the interior of recombinant hepatitis B virus capsids.

The icosahedral nucleocapsid of hepatitis B virus (HBV) consists of multiple subunits of a single 183 amino acids (aa) core protein encasing the viral genome. However, recombinant core protein alone also forms capsid-like particles. We have recently shown that a 238 aa protein centrally inserted into the core protein can be displayed on the particle surface. Here we demonstrate that replacement of the C-terminal basic domain by the 17 kDa Staphylococcus aureus nuclease also yields particles but that in these the foreign domains are located in the interior. The packaged nuclease is enzymatically active, and the chimeric protein forms mosaic particles with the wild-type core protein. Hence the HBV capsid is useful as a molecular platform which, dependent on the fusion site, allows foreign protein domains to either be packaged into or be exposed on the exterior of the particle. These results are of relevance for the use of the HBV capsid as a vaccine carrier, and as a target for antiviral therapy.

Expression of hepatitis B virus core gene products with specific immunoreactivity for e antigen (HBeAg) in Saccharomyces cerevisiae.

The e antigen of the hepatitis B virus (HBeAg) was expressed in S. cerevisiae. Yeast-derived HBeAg exhibits high HBe antigenicity while lacking any HBc antigenicity. The production yield of HBeAg expressed in yeast is dependent on the host strains and the nature of the leader sequences used in the plasmid constructions. The recombinant antigen is not secreted into the medium, independent from the leader sequences which are used. A simple extraction procedure was developed, enabling the isolation of HBeAg from the cells without killing them. Recombinant HBeAg derived from yeast can replace plasma-derived antigen in ELISA for determining antibodies to HBeAg.

Hepatitis B virus replication: novel roles for virus-host interactions.

Chronic hepatitis B continues to be one of the most widespread and serious viral infections in humans worldwide. Several fundamental aspects of the molecular biology of its causative agent, hepatitis B virus, are meanwhile understood in some detail. However, recent research has emphasized that the dependence of the viral infectious cycle on cellular factors is far greater than previously anticipated. More and more intracellular interactions between viral and cellular components are discovered, and probably each individual step of genome replication will turn out to involve several host factors. Prominent examples are the activation of the viral reverse transcriptase, P protein, by chaperones, and the nucleocytoplasmic trafficking of viral nucleic acids by as yet unidentified components of the host machinery. Some of these new developments will be described here but many more can be expected to follow. Identifying these host factors and characterizing their interactions with the viral components will certainly reveal novel targets for specific antiviral strategies.

Novel molecular approaches toward therapy of chronic hepatitis B.

More than 300 million people worldwide are chronically infected with hepatitis B virus (HBV), and are at greatly increased risk of developing liver cirrhosis and eventually primary liver carcinoma. While infection can, with relative success, be prevented by vaccination, no generally effective therapy for chronic hepatitis B is available. Hence there is an urgent need for novel antiviral strategies. Recent advances in our understanding of the mechanisms underlying virus replication and assembly provide opportunities for the rational design of molecules that could specifically interfere with these processes; some of these possibilities are discussed in this review.

Conserved cysteines of the hepatitis B virus core protein are not required for assembly of replication-competent core particles nor for their envelopment.

Replication of hepatitis B virus (HBV) proceeds by reverse transcription of an RNA intermediate inside the viral nucleocapsid formed by the core protein. This protein contains four Cys residues which occur at equivalent positions in the core proteins of all known mammalian hepadnaviruses, suggesting that they might be of structural and/or functional importance. The four His residues of the core protein are located strikingly close to the three N-proximal cysteines. This arrangement is likewise conserved and might indicate the presence of an unconventional Cys-His box element similar to that required for nucleic acid binding in all retroviral NC proteins. In order to test the potential involvement of the core protein cysteines in virus assembly, we transiently expressed in HuH7 cells a mutant HBV genome encoding a core protein in which all cysteines are replaced by serine residues and analyzed the formation of replication-competent cores using the endogenous polymerase reaction. The mutant genome yielded products that were nearly indistinguishable from those produced by a corresponding wild-type genome, virtually ruling out the presence of a functional Cys-His box element in the hepadnaviral core protein. Density gradient analysis showed that the mutant cores were enveloped, though the efficiency of envelopment and/or the stability of the mutant enveloped particles was lowered compared to the wild-type. These data indicate that none of the steps in the viral life cycle from reverse transcription to envelopment was principally impaired. The conservedness of the cysteines might then be related to virus infectivity rather than replication; alternatively, the Cys residues might not be important for the core protein itself, but for the alternative C gene product HBeAg.

The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly.

Assembly of replication-competent hepatitis B virus (HBV) nucleocapsids requires the interaction of the core protein, the P protein, and the RNA pregenome. The core protein contains an arginine-rich C-terminal domain which is dispensable for particle formation in heterologous expression systems. Using transient expression in HuH7 cells of a series of C-terminally truncated core proteins, I examined the functional role of this basic region in the context of a complete HBV genome. All variants containing at least the 144 N-terminal amino acids were assembly competent, but efficient pregenome encapsidation was observed only with variants consisting of 164 or more amino acids. These data indicate that one function of the arginine-rich region is to provide the interactions between core protein and RNA pregenome. However, in cores from the variant ending with amino acid 164, the production of complete positive-strand DNA was drastically reduced. Moreover, almost all positive-strand DNA originated from in situ priming, whereas in wild-type particles, this type of priming not supporting the formation of relaxed circular DNA (RC-DNA) accounted for about one half of the positive strands. Further C-terminal residues to position 173 restored RC-DNA formation, and the corresponding variant did not differ from the full-length core protein in all assays used. The observation that RNA encapsidation and formation of RC-DNA can be genetically separated suggests that the core protein, via its basic C-terminal region, also acts as an essential auxiliary component in HBV replication, possibly like a histone, or like a single-stranded-DNA-binding protein. In contrast to their importance for HBV replication, sequences beyond amino acid 164 were not required for the formation of enveloped virions. Since particles from variant 164 did not contain mature DNA genomes, a genome maturation signal is apparently not required for HBV nucleocapsid envelopment.

Efficient hammerhead ribozyme-mediated cleavage of the structured hepatitis B virus encapsidation signal in vitro and in cell extracts, but not in intact cells.

Hepatitis B virus (HBV), the causative agent of B-type hepatitis in man, is a small enveloped DNA virus that replicates through reverse transcription of an RNA intermediate, the terminally redundant RNA pregenome. An essential highly conserved cis-element present twice on this RNA is the encapsidation signal epsilon, a stem-loop structure that is critical for pregenome packaging and reverse transcription. Epsilon is hence an attractive target for antiviral therapy. Its structure, however, is a potential obstacle to antivirals whose action depends on hybridization, e.g. ribozymes. Here we demonstrate effective in vitro cleavage inside epsilon by hammerhead ribozymes containing flanking sequences complementary to an adjacent less structured region. Upon co-transfection with a HBV expression construct corresponding ribozymes embedded in a U6 snRNA context led to a significant, though modest, reduction in the steady-state level of HBV pregenomes. Inactive ribozyme mutants revealed that antisense effects contributed substantially to this reduction, however, efficient epsilon cleavage by the intracellularly expressed ribozymes was observed in Mg(2+)-supplemented cell lysates. Artificial HBV pregenomes carrying the ribozymes in cis and model RNAs lacking all HBV sequences except epsilon exhibited essentially the same behaviour. Hence, neither the absence of co-localization of ribozyme and target nor a viral component, but rather a cellular factor(s), is responsible for the strikingly different ribozyme activities inside cells and in cellular extracts.

Distinct requirements for primary sequence in the 5'- and 3'-part of a bulge in the hepatitis B virus RNA encapsidation signal revealed by a combined in vivo selection/in vitro amplification system.

Hepatitis B virus (HBV) is a small DNA virus that replicates by reverse transcription of a terminally redundant RNA, the pregenome. Specific packaging of this transcript into viral capsids is mediated by interaction of the reverse transcriptase, P protein, with the 5'-proximal encapsidation signal epsilon, epsilon-function is correlated with the formation of a hairpin structure containing a bulge and a loop, each consisting of 6 nt. To analyse the importance of primary sequence in these regions, we have combined selection of encapsidation competent individuals from pools of randomized epsilon-sequences in transfected cells with in vitro amplification, thus bypassing the current experimental limitations of the HBV system. While no alterations of the authentic loop sequence were detectable, many different sequences were tolerated in the 3'-part of the bulge. However, at the two 5'-proximal bulge positions the wt sequence was strongly selected for, indicating that for RNA packaging close contacts between protein and the 5'- but not the 3'-part of the bulge are important. Such a bipartite organisation provides a structural basis for the recently demonstrated special role of the 3'-part of the bulge as template for the first nucleotides of (-)-strand DNA in HBV reverse transcription.

Specific hepatitis B virus minus-strand DNA synthesis requires only the 5' encapsidation signal and the 3'-proximal direct repeat DR1.

Human hepatitis B virus (HBV) is a small DNA virus that replicates inside the viral nucleocapsid by reverse transcription of an RNA intermediate, the pregenome. The sequences encompassing the encapsidation signal epsilon and the direct repeat DR1 are present in two copies of this terminally redundant transcript. We have recently shown that HBV minus-strand DNA synthesis involves transfer of a short DNA primer copied from 5'-epsilon to 3'-DR1 (DR1*). Using transfection of HBV genomes with lesions in 3'-epsilon, and 5'-DR1 and its preceding sequence, we tested whether these additional elements contribute to the specificity of the transfer reaction. However, while some mutations affected proper plus-strand DNA formation, 5'-epsilon and DR1* were completely sufficient for correct minus-strand DNA production.

Sequence- and structure-specific determinants in the interaction between the RNA encapsidation signal and reverse transcriptase of avian hepatitis B viruses.

Hepatitis B viruses (HBVs) replicate by reverse transcription of an RNA intermediate. Packaging of this RNA pregenome into nucleocapsids and replication initiation depend crucially on the interaction of the reverse transcriptase, P protein, with the cis-acting, 5' end-proximal encapsidation signal epsilon. The overall secondary structure is similar in all of the hepadnaviral epsilon signals, with a lower and an upper stem, separated by a bulge, and an apical loop. However, while epsilon is almost perfectly conserved in all mammalian viruses, the epsilon signals of duck HBV (DHBV) and heron HBV (D epsilon and H epsilon, respectively) differ substantially in their upper stem regions, both in primary sequence and in secondary structure; nonetheless, H epsilon interacts productively with DHBV P protein, as shown by its ability to stimulate priming, i.e., the covalent attachment of a deoxynucleoside monophosphate to the protein. In this study, we extensively mutated the variable and the conserved positions in the upper stem of D epsilon and correlated the functional activities of the variant RNAs in a priming assay with secondary structure and physical P protein binding. These data revealed a proper overall structure, with the bulge and certain key residues, e.g., in the loop, being important constraints in protein binding. Many mutations at the evolutionarily variable positions complied with these criteria and yielded priming-competent RNAs. However, most mutants at the conserved positions outside the loop were defective in priming even though they had epsilon-like structures and bound to P protein; conversely, one point mutant in the loop with an apical structure different from those of D epsilon and H epsilon was priming competent. These results suggest that P protein binding can induce differently structured epsilon RNAs to adopt a new, common conformation, and they support an induced-fit model of the epsilon-P interaction in which both components undergo extensive structural alterations during formation of a priming-competent ribonucleoprotein complex.