UMLS:C0019163 - FACTA Search

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

Human Hepatitis B Virus (HBV) replication is accomplished by its own polymerase. The HBV RNase H domain of HBV polymerase has been expressed in Escherichia coli and purified by affinity column chromatography. The MBP-RNase H fusion protein (43 kDa MBP plus 17 kDa HBV RNase H domain) was proved to be RNase H by in vitro activity assay, inhibitor studies, and mutagenesis. The HBV RNase H domain represented the optimal RNase H activity in the presence of either 8 mM MgCl2 or 16 mM MnCl2. In Tris-Cl buffer, the optimum pH for MBP-RNase H fusion protein is between 7.7 and 8.2. The MBP-RNase H fusion protein required 40 mM monovalent cation for its enzyme activity, whereas it showed lower activity at a salt concentration of more than 100 mM. Ribonucleoside Vanadyl complex (RAV) and 2'-deoxyadenosine 5'-monophosphate (dAMP) inhibited the RNase H activity. Moreover, the mutation of highly conserved amino acids in the HBV RNase H domain diminished the RNase H activity. These results clearly suggest that the RNase H activity is separable from viral HBV polymerase enzymatic activities.
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PMID:RNase H activity of human hepatitis B virus polymerase expressed in Escherichia coli. 914 47

Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.
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PMID:Properties of monoclonal antibodies directed against hepatitis B virus polymerase protein. 1019 15

Genome replication of hepadnavirus proceeds by reverse transcription from a viral pregenomic RNA template by a virally encoded polymerase that possesses protein-priming, reverse transcriptase, DNA polymerase, and RNase H activities. Characterization of this enzyme has been hampered by failure to purify an active enzyme from virions and difficulties in expressing an active polymerase in heterologous systems. In this study, we constructed human hepatitis B virus polymerase cDNA under the control of a phage T7 promoter and expressed it in a rabbit reticulocyte lysate-coupled transcription-translation system. In vitro site-directed mutagenesis confirmed that the recombinant polymerase cDNA produced three products: a full-length protein (approximately 94 kDa), an internally initiated protein (approximately 81 kDa), and an N-terminal protein (approximately 40 kDa). The in vitro expressed polymerase possessed protein priming activity, as demonstrated by 32P-dGTP-labeling of the full size polymerase and the N-terminal portion of the molecule in an in vitro priming assay. The polymerase also exhibited polymerization activity, as detected in an in vitro polymerase assay by incorporation of radionucleotides into acid-precipitable polynucleotides and by synthesis of human hepatitis B virus (HBV) specific DNA with product lengths between 100 and 500 nucleotides. In addition, the polymerase was found to use an RNA sequence bearing HBV DR1/epsilon stem-loop motif as a template for DNA synthesis. Both the protein-priming and the reverse transcription activities of this recombinant polymerase are dependent on the RNA fragment containing the HBV DR1/epsilon stem-loop sequence known to be required for the polymerase activities. The in vitro systems used in this study will be applicable to further functional and biochemical studies of this enzyme.
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PMID:Expression of an enzymatically active polymerase of human hepatitis B virus in an coupled transcription-translation system. 1043 46

Hepatitis B virus (HBV), although a DNA virus, replicates using reverse transcriptase encoded by the HBV polymerase (pol) gene. The biochemical dissection of HBV pol has been hampered by failure to liberate enzymatically active protein from nucleocapsids. Here, we have employed a yeast-based genetic approach to express the HBV reverse transcriptase. In this strategy, the reverse transcriptase of yeast retrotransposon Ty1 element is replaced with the HBV pol gene to produce the hybrid Ty1/HBV element. Additionally, the indicator gene his3AI is combined in an antisense orientation to the transcripts of the hybrid Ty1/HBVRT element. The splicing of his3AI, cDNA synthesis of the Ty1/HBVRT RNA and subsequent integration relies on the reverse transcriptase activity. The production of histidine prototrophs results from the successful reverse transcription of Ty1/HBVRThis3AI transcripts followed by either homologous recombination or integrase-mediated insertion and subsequent expression of HIS3 gene. Using this approach we successfully detected the reverse transcriptase activity of HBV in yeast strains defective in endogenous Ty1 expression. Consistent with the unique priming activity associated with HBV pol, the minus strand DNA synthesis was protein-primed. Deletion of HBV reverse transcriptase (RT) or RNase H domains resulted in a dramatic drop in histidine prototrophs. The addition of HBV encoded HBx protein in virus-like particles during in vitro RT reaction stimulated the RT reaction by severalfold. Furthermore, in the presence of 3TC, a known inhibitor of HBV reverse transcriptase, yeast His(+) growth of His protrophs was not observed. Thus, this approach, which is based on genetic selection in yeast, is safe, economic, and a reliable strategy with a potential for large scale screening of cofactors and inhibitors of HBV polymerase functions.
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PMID:Expression of hepatitis B virus polymerase in Ty1-his3AI retroelement of Saccharomyces cerevisiae. 1053 36

Previous mutagenesis studies with hepatitis B virus (HBV) suggest that continued interactions with core are required for several steps in genomic replication. To examine core-polymerase (Pol) interactions, insect cells were coinfected with baculovirus constructs that independently expressed core and Pol. The results demonstrated several features with implications that core plays an interactive role with HBV Pol: (i) core coprecipitated with constructs expressing full-length Pol as well as the terminal protein (TP), reverse transcriptase (RT) and RNase H domains of Pol, independently; (ii) coprecipitation of core was not dependent on the presence of an epsilon stem-loop sequence; and (iii) core-Pol complexes migrated as intact capsid particles, as detected by sucrose gradient analysis. To analyze the structural and sequence requirements of core in recognition of Pol, a series of core mutants with two- to four-amino-acid insertions or carboxy-terminal deletions were assessed for Pol interaction. The results indicated that capsid formation is required but not sufficient for interaction with Pol and that the TP and RT domains of Pol have different requirements for interaction with core. To map the core binding sites on Pol, a panel of amino- and carboxy-terminal deletion mutants of the TP and RT domains of Pol were analyzed for interaction with core. At least three separate core binding sites on Pol were detected. This analysis begins to define basic requirements for core-Pol interactions, but further study is necessary to delineate the effects of these interactions on encapsidation and genome replication.
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PMID:Interaction between hepatitis B virus core protein and reverse transcriptase. 1109 Jan 44

Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (epsilon) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA primer that is templated by epsilon and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in Escherichia coli, followed by complex reconstitution in RRL. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could trans complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of epsilon-dependent DNA primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism.
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PMID:Reconstitution of a functional duck hepatitis B virus replication initiation complex from separate reverse transcriptase domains expressed in Escherichia coli. 1146 13

Ribonuclease H (RNaseH) recognizes and efficiently cleaves the RNA strand of DNA-RNA hybrids, but has no inherent sequence selectivity. However, the formation of DNA-RNA hybrids does require specific sequence recognition. On the basis of this concept, we wondered whether antisense oligonucleotides complementary to target RNA covalently linked to RNase H could be used to direct specific cleavage events mediated by RNase H. The aim of this research was to couple a DNA oligonucleotide to RNase H to confer specificity of ribonuclease activity toward hepatitis B viral (HBV) mRNA. A modified 13-base oligonucleotide that is specific for the DR1 region of HBV mRNA was conjugated to modified E. coli RNase H using a water soluble cross-linker. A 1200 base fragment of HBV RNA including the DR1 region was synthesized as a substrate using T7 RNA polymerase. Incubation of the RNase H-oligonucleotide conjugate with the RNA transcript resulted in cleavage of the HBV mRNA transcript in a concentration dependent manner. Eighty-five percent of substrate was cleaved under optimal conditions. Controls consisting of RNase H alone, oligonucleotide alone, and incubation of the conjugate with an unrelated mRNA substrate resulted in no cleavage activity. RNase H coupled to an HBV antisense oligonucleotide can specifically cleave target HBV transcripts.
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PMID:A ribonuclease H-oligo DNA conjugate that specifically cleaves hepatitis B viral messenger RNA. 1156 95

To explore functional domains in the hepatitis B virus (HBV) polymerase, two naturally occurring HBV isolates (56 and 2-18) with 98.7% nucleic acid sequence homology but different replication efficiencies were studied. After transfection into HepG2 cells, HBV DNA isolated from intracellular virus core particles was much higher in 56-transfected cells than in cells transfected with 2-18. The structural basis for the difference in replication efficiency between these two isolates was studied by functional domain gene substitution. The complete polymerase (P) gene and its gene segments coding for the terminal protein (TP), spacer (SP), reverse transcriptase (RT), and RNase H in 2-18 were separately replaced with their counterparts from 56 to construct full-length chimeric genomes. Cell transfection analysis revealed that substitution of the complete P gene of 2-18 with the P gene from 56 slightly enhanced viral replication. The only chimeric genome that regained the high replication efficiency of the original 56 isolate was the one with substitution of the RT gene of 2-18 with that from 56. Within the RT region, amino acid differences between isolates 2-18 and 56 were located at positions 617 (methionine versus leucine), 652 (serine versus proline), and 682 (valine versus leucine). Point mutation identified amino acid 652 as being responsible for the difference in replication efficiency. Homologous modeling studies of the HBV RT domain suggest that the mutation of residue 652 from proline to serine might affect the conformation of HBV RT which interacts with the template-primer, leading to impaired polymerase activity.
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PMID:A single amino acid in the reverse transcriptase domain of hepatitis B virus affects virus replication efficiency. 1168 64

Hepadnaviruses and retroviruses are evolutionarily related families because they both require a process of reverse transcription for genome replication. However, hepadnaviruses produce polymerase (pol) and core proteins separately, while retroviruses synthesize a gag-pol fusion protein that is subsequently cleaved by a virally encoded protease to release a functional polymerase. To test whether an additional sequence at the N-terminus of pol in hepatitis B virus (HBV) interferes with its function, we created two plasmids expressing core-pol fusion proteins, core144-pol and core31-pol. Secreted particles obtained from HuH-7 cells, which were cotransfected with a core-pol fusion protein-expressing plasmid and a core-expressing plasmid, showed a positive signal of HBV DNA by the endogenous polymerase assay, indicating that the core-pol fusion proteins retain DNA priming, polymerization and RNase H activities. The fusion protein was detected in the cytoplasm of transfected cells and in secreted virions by immunoprecipitation. Furthermore, we found by immunofluorescence staining that the HBV core-pol fusion protein colocalized with the hepatitis C virus (HCV) core protein in cytoplasm and in lipid droplets. Immunoprecipitation studies showed that the anti-HCV core complex contained the HBV core-pol fusion protein while the anti-HBV pol complex contained the HCV core protein, which supports the hypothesis that the HCV core protein can form a complex with the HBV core-pol fusion protein.
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PMID:Hepatitis B viral polymerase fusion proteins are biologically active and can interact with the hepatitis C virus core protein in vivo. 1170 13

There are two mutually exclusive pathways for plus-strand DNA synthesis in hepadnavirus reverse transcription. The predominant pathway gives rise to relaxed circular DNA, while the other pathway yields duplex linear DNA. Both pathways use the same RNA primer, which is capped and 18 or 19 nucleotides in length. At the completion of minus-strand DNA synthesis, the final RNase H cleavage generates the plus-strand primer. To make relaxed circular DNA, primer translocation must occur, resulting in the transfer of the primer generated at DR1 to the acceptor site (DR2) near the opposite end of the minus-strand DNA. A small fraction of viruses instead make duplex linear DNA after initiating plus-strand DNA synthesis from DR1, a process called in situ priming. We are interested in understanding the mechanism of discrimination between these two pathways. Some variants of duck hepatitis B virus exhibit high levels of in situ priming due to cis-acting mutations. The mechanism by which these mutations act has been obscure. Sequence inspection predicted formation of a small DNA hairpin in the region overlapping these mutations. We have shown that substitutions disrupting base pairing potential in this hairpin led to increased levels of in situ priming. The introduction of compensatory changes to restore base pairing potential led to reduced levels of in situ priming. Thus, formation of the small DNA hairpin overlapping the 5' end of DR1 in the minus strand contributes to the regulation of primer translocation, at least, through inhibition of in situ priming by making the 3' end of the minus-strand DNA a poor template for initiation.
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PMID:Small DNA hairpin negatively regulates in situ priming during duck hepatitis B virus reverse transcription. 1177 73

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