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Query: UMLS:C0019163 (hepatitis B)
 38,309 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatitis B virus (HBV) is the type member of the hepadnaviridae, small enveloped DNA viruses that replicate through reverse transcription of an RNA intermediate, the pregenome. This reaction occurs usually inside the viral nucleocapsid, the assembly of which requires specific interactions between multiple copies of the core protein, the viral replication enzyme (P protein) and the RNA pregenome which also serves as mRNA for both proteins. Deletion studies have established that specific packaging of the RNA is mediated by a short cis-acting sequence, the encapsidation signal epsilon. Using nuclease sensitivity experiments we provide experimental evidence that part of this sequence can adopt a stem-loop structure that is interrupted by a bulge and a single unpaired U residue. The structural consequences of deletions of the unpaired regions and changes in their primary sequences were investigated in vitro, and their influence on the function of the epsilon-signal was tested in animal cells by monitoring encapsidation of RNAs carrying the mutant epsilon-sequences in front of a 2.7 kb foreign RNA fragment, or within the context of a complete HBV genome. The data indicate that the entire stem-loop structure containing the bulge and the loop is critical for encapsidation competence. While gross alterations in the primary sequences of the unpaired regions interfere with encapsidation, data obtained with additional mutants suggest that the bulge region is more tolerant to sequence changes than the loop.
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PMID:The encapsidation signal on the hepatitis B virus RNA pregenome forms a stem-loop structure that is critical for its function. 769 Apr 71

Human hepatitis B virus (HBV) is a small DNA virus that replicates inside the viral nucleocapsid by reverse transcription of an RNA intermediate. Encapsidation of this RNA pregenome is mediated by the interaction of the viral replication enzyme P with the structured 5'-proximal RNA element epsilon; replication was thought to start in the 3'-proximal direct repeat DR1*. However, recent data obtained with the duck hepatitis B virus indicated a novel, discontinuous mechanism of negative-strand DNA synthesis. Here we demonstrate, using DNA transfection of complete HBV genomes, that the 3'-half of a 6-nucleotide bulge in HBV epsilon whose primary sequence is not important for encapsidation serves as template for a short DNA primer that is subsequently transferred to DR1*. Apparently, P protein copies any template sequence that does not interfere with epsilon structure; however, altered primary sequences can induce polymerase stuttering, resulting in extended primers containing more than one equivalent of the template sequence. The importance of the bulged structure is emphasized by the dependence of primer length on bulge size. Transfer specificity is in part controlled by sequence complementarity. The strategy of using the 5' encapsidation signal as the origin of replication for discontinuous negative-strand DNA synthesis, common to mammalian and avian hepadnaviruses, suggests the evolutionary origin of hepatitis B viruses to lie between that of modern retroviruses and primitive retroelements like the Mauriceville retroplasmid.
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PMID:A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis. 862 50

The hepadnavirus P gene contains amino acid sequences which share homology with all known RNases H. In this study, we made four mutants in which single amino acids of the duck hepatitis B virus (DHBV) RNase H region were altered. In two of them, amino acids at locations comprising the putative catalytic site were changed, while the remaining mutants had alterations at amino acids conserved among hepadnaviruses. Transfection of these mutant genomes into permissive cells resulted in synthesis of several discrete viral nucleic acid species, ranging in apparent sizes from approximately 500 to 3,000 bp, numbered I, II, III, IV, and V. While the locations of the species were similar in all mutants, the proportions of the species varied among the mutants. Analysis of the nucleic acid species revealed that they were hybrid molecules of RNA and minus-strand DNA, indicating that the RNase H activity was missing or greatly reduced in these mutants. Primer extension experiments showed that the mutant viruses initiated minus-strand viral DNA synthesis normally. The 3' termini of minus-strand DNA in species II, III, and IV were mapped just downstream of nucleotides 1659, 1220, and 721, respectively. Species V contained essentially full-length minus-strand viral DNA. A parallel amino acid change in the putative catalytic site of the HBV RNase H domain resulted in accumulation of low-molecular-weight hybrid molecules consisting of RNA and minus-strand DNA and similar in size and pattern to those seen with DHBV. These studies demonstrate experimentally the involvement of the C-terminal portion of the P gene in RNase H activity in both DHBV and human hepatitis B virus and indicate that the amino acids essential for RNase H activity of hepadnavirus P protein are also important for the efficient elongation of minus-strand viral DNA.
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PMID:Amino acids essential for RNase H activity of hepadnaviruses are also required for efficient elongation of minus-strand viral DNA. 870 40

Using the structured RNA encapsidation signal (D(epsilon)) and the reverse transcriptase (P protein) of duck hepatitis B virus (DHBV) as an example, we devised a sensitive mapping procedure that yields accurate information on the minimal RNA sequence required for interaction with a few nanograms of an RNA-binding protein. RNAs from pools of end-labeled, partially hydrolyzed transcripts that bound to in vitro translated His-tagged P protein were isolated using immobilized Ni2+-ions. Size analysis by PAGE is consistent with a gradual gain in binding-competence from a minimum of 5 to a maximum of 8 base pairs in the basal stem of D(epsilon). The procedure should be generally applicable to the convenient and precise fine mapping of RNA-protein interactions.
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PMID:A sensitive procedure for mapping the boundaries of RNA elements binding in vitro translated proteins defines a minimal hepatitis B virus encapsidation signal. 893 98

Hepatitis B viruses replicate via reverse transcription of an RNA intermediate. This RNA pregenome serves as mRNA and is packaged into capsids and reverse transcribed. Both processes require the interaction of the viral reverse transcriptase, P protein, with the 5'-proximal epsilon-signals on the pregenome. For epsilon of human hepatitis B virus (HBV), the presence of a functionally important stem-loop structure with a central bulge, part of which acts as template for a short primer of first-strand DNA synthesis, has been experimentally confirmed. Based on phylogeny and its functional similarities to epsilon, the D epsilon-signal of duck hepatitis B virus (DHBV) had been proposed to have a similar structure which does not, however, correspond to the most stable computer prediction. We have therefore experimentally determined the secondary structures of D epsilon and of the H epsilon-signal of heron hepatitis B virus which differs considerably from D epsilon in primary sequence yet interacts productively with DHBV P protein. Our data support an HBV epsilon-like structure for both D epsilon and H epsilon; in particular the bulge is highly conserved, in accord with its special function in replication. However, the apical loop in H epsilon is much enlarged suggesting that, by an induced-fit mechanism, both RNAs may adopt a new, probably similar conformation in the complex with P protein.
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PMID:Experimental confirmation of a hepatitis B virus (HBV) epsilon-like bulge-and-loop structure in avian HBV RNA encapsidation signals. 901 50

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.
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PMID:Sequence- and structure-specific determinants in the interaction between the RNA encapsidation signal and reverse transcriptase of avian hepatitis B viruses. 918 60

Human foamy virus (HFV) is the prototype of the Spumavirus genus of retroviruses. These viruses have a genomic organization close to that of other complex retroviruses but have similarities to hepadnaviruses such as human hepatitis B virus (HBV). Both HFV and HBV express their Pol protein independently of their structural proteins. Retroviruses and hepadnaviruses differ in their requirements for particle assembly and genome packaging. Assembly of retroviral particles containing RNA genomes requires only the Gag structural protein. The Pol protein is not required for capsid assembly, and the Env surface glycoprotein is not required for release of virions from the cell. In contrast, assembly of extracellular HBV particles containing DNA requires core structural protein and polymerase (P protein) for assembly of nucleocapsids and requires surface glycoproteins for release from the cell. We investigated the requirements for synthesis of extracellular HFV particles by constructing mutants with either the pol or env gene deleted. We found that the Pol protein is dispensable for production of extracellular particles containing viral nucleic acid. In the absence of Env, intracellular particles are synthesized but few or no extracellular particles could be detected. Thus, foamy virus assembly is distinct from that of other reverse transcriptase-encoding mammalian viruses.
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PMID:The roles of Pol and Env in the assembly pathway of human foamy virus. 1052 50

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.
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PMID:Formation of a functional hepatitis B virus replication initiation complex involves a major structural alteration in the RNA template. 977 43

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.
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PMID:Hepatitis B virus replication: novel roles for virus-host interactions. 1051 65

Hepatitis B viruses replicate through reverse transcription of an RNA intermediate. In contrast to retroviral reverse transcriptases, their replication enzyme, P protein, does not use a nucleic acid primer but initiates DNA synthesis de novo from within an RNA stem-loop structure called epsilon. A short DNA oligonucleotide is copied from epsilon and covalently attached to P protein, and then synthesis is arrested. The information for initiation site selection and synthesis arrest must be contained in the structure of the P protein/epsilon complex. Because P protein activity depends on cellular chaperones this complex can as yet only be generated by in vitro translation of duck hepatitis B virus P protein in rabbit reticulocyte lysate; functional interaction with its cognate RNA element Depsilon can be monitored by the covalent labeling of P protein during primer synthesis. Combining this in vitro priming reaction and a set of chimeric RNA-DNA Depsilon analogues, we found that only five ribose residues in the 57-nucleotide stem-loop were sufficient to provide a functional template; these are a single residue in the template region and the two base pairs at the tip of the lower stem. The base identities in the very same region are essential as well. The presence of this 2'-OH- and base-dependent determinant shortly downstream of the initiation site suggests a mechanism that can account for both initiation site selection and programmed primer synthesis arrest.
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PMID:A small 2'-OH- and base-dependent recognition element downstream of the initiation site in the RNA encapsidation signal is essential for hepatitis B virus replication initiation. 1060 40


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