Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019163 (hepatitis B)
 38,309 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activities of the hepadnavirus polymerases are known to include those of DNA polymerase, reverse transcriptase and RNase H. To date, it has been difficult or impossible to clone and express the product as an active enzyme. In this study, full length capped RNA encoding Duck Hepatitis B Virus (DHBV) polymerase was produced by in vitro transcription from a T7 promoter. The RNA was translated in a rabbit reticulocyte lysate system and produced an 35S-Methionine labelled 79 Kd band on SDS-polyacrylamide gel electrophoresis. The translation product showed DNA polymerase and reverse transcriptase activities on exogenous templates (respectively) of DNA or RNA with random DNA hexamer primers. The same RNA transcripts were also microinjected into Xenopus oocytes, but appeared to be toxic and gave no detectable translation product. Production of hepadnavirus polymerase by in vitro transcription/translation may provide a useful tool for structure/function and pharmacological studies on this important group of polymerases.
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PMID:Duck hepatitis B virus polymerase produced by in vitro transcription and translation possesses DNA polymerase and reverse transcriptase activities. 128 90

A duck hepatitis B virus (DHBV) genome cloned from a domestic duck from the People's Republic of China has been sequenced and exhibits no variation in sequences known to be important in viral replication or generation of gene products. Intrahepatic transfection of a dimer of this viral genome into ducklings did not result in viremia or any sign of virus infection, indicating that the genome was defective. Functional analysis of this mutant genome, performed by transfecting the DNA into a chicken hepatoma cell line capable of replicating wild-type virus, indicated that viral RNA is not encapsidated. However, virus core protein is made and can assemble into particles in the absence of encapsidation of viral nucleic acid. Using genetic approaches, it was determined that a change of cysteine to tyrosine in position 711 in the polymerase (P) gene C terminus led to this RNA-packaging defect. By site-directed mutagenesis, it was found that while substitution of Cys-711 with tryptophan also abolished packaging, substitution with methionine did not affect packaging or viral replication. Therefore, Cys-711, which is conserved in all published sequences of DHBV, may not be involved in a disulfide bridge structure essential to viral RNA packaging or replication. Our results, showing that a missense mutation in the region of the DHBV polymerase protein thought to be primarily the RNase H domain results in packaging deficiency, support the previous findings that multiple regions of the complex hepadnaviral polymerase protein may be required for viral RNA packaging.
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PMID:Naturally occurring point mutation in the C terminus of the polymerase gene prevents duck hepatitis B virus RNA packaging. 130 4

The polymerase (P) gene of hepadnaviruses encodes a large polypeptide that appears to participate in several steps in the viral life cycle: packaging of viral RNA, providing the primer for synthesis of minus-strand DNA, synthesizing minus-strand DNA from an RNA template and plus-strand DNA from a DNA template, and degrading viral RNA in RNA-DNA hybrids. To assist in the assignment of these functions to domains of the duck hepatitis B virus polymerase protein, we have constructed a series of substitution mutations and a large insertion mutation, based in part on amino acid sequence comparisons with other proteins known to exhibit reverse transcriptase (RT) and RNase H activities. We found that changes in highly conserved sequences in putative RT and RNase H domains in the carboxy-terminal half of the protein dramatically reduced synthesis of both strands of viral DNA without major effects on RNA packaging into subviral cores. Thus we can uncouple RNA packaging and DNA synthesis but cannot separate RT and RNase H activities as has been done with human hepatitis B virus. The viability of a mutant with a large insertion (123 amino acids) upstream of the RT and RNase H domain indicates that a hinge region may separate parts of the polymerase protein implicated in priming and polymerization.
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PMID:Effects of insertional and point mutations on the functions of the duck hepatitis B virus polymerase. 169 97

Constructs expressing the core, surface, X, or polymerase proteins of hepatitis B virus were transfected into human cells. In transient assays, only the polymerase inhibited the responses to interferons alpha and gamma (IFN-alpha and -gamma). Stable expression of the polymerase was achieved in the cell line 2fTGH, which carries an IFN-inducible marker gene, by growth under conditions that select for inhibition of the response to IFN-alpha, but the clones grew poorly. When expressed alone, the terminal protein domain of the polymerase gene inhibited the response to IFN-alpha and the reverse transcriptase plus RNase H domains appeared to be toxic. Clones of cells expressing terminal protein alone, selected for the loss of response to IFN-alpha, grew normally and had no detectable response to IFN-alpha, IFN-gamma, or double-stranded RNA. Binding of IFN-alpha to these cells was not impaired but did not lead to activation of the E alpha subunit of the IFN-induced transcription factor E. These observations are of potential importance in relation to the pathogenesis of chronic hepatitis B virus infection and the resistance of such infection to IFN-alpha therapy.
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PMID:Expression of the terminal protein region of hepatitis B virus inhibits cellular responses to interferons alpha and gamma and double-stranded RNA. 170 74

We characterized 11 DNA polymerase mutants of human hepatitis B virus (HBV) which contain single missense or nonsense mutations in the various domains within this gene. Except for mutant 738, a tight association between DNA replication and RNA packaging of these missense pol mutants was observed. Further analysis of HBV core particle-associated RNA indicated that only the 3.5-kb core-specific RNA, but not the precore-specific RNA, is selectively packaged in this tissue culture system. Previously, we have demonstrated that only the 3.5-kb core-specific RNA can serve as an efficient template for pol translation. Taken together, our results suggest that selectivity of HBV RNA packaging occurs as a result of selective translation of pol-containing mRNAs. Furthermore, our data suggest that the RNA encapsidation domain of pol overlaps with all of the domains of pol involved in the synthesis of terminal protein, as well as DNA replication. Finally, on the basis of gradient centrifugation analysis, a pol defect appeared to have no negative effect on the assembly or stability of core particles. A new method to assay RNA encapsidation, as well as potential RNase H activity, is reported.
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PMID:Pregenomic RNA encapsidation analysis of eleven missense and nonsense polymerase mutants of human hepatitis B virus. 171 Feb 85

Reverse transcription of RNA into duplex DNA requires accurate initiation of both minus and plus strand DNA synthesis; this in turn requires the generation of specific primer molecules. We have examined plus strand primer generation in the hepatitis B viruses, small DNA viruses that replicate via reverse transcription. The plus strand primer in these viruses is a short capped RNA derived from the 5' end of the RNA template by cleavage at a specific set of sites. To elucidate the cleavage mechanism we constructed a series of viral mutants bearing alterations in and around the cleavage sites. Our results reveal that the cleavage reaction is sequence-independent and indicate that the cleavage sites are positioned by measurement of the distance from the 5' end of the RNA. Comparison of these findings with what is known about RNase H-mediated primer generation in retroviruses and other retroid elements suggests that, despite many divergent features, some common molecular features are preserved.
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PMID:Sequence-independent RNA cleavages generate the primers for plus strand DNA synthesis in hepatitis B viruses: implications for other reverse transcribing elements. 191 7

To correlate the hepatitis B virus P gene with the enzymatic activities predicted to participate in hepadnavirus reverse transcription, a series of P gene mutants containing missense mutations, in-phase insertions, and in-phase deletions was constructed by site-directed mutagenesis. These mutants were tested in the context of otherwise intact hepatitis B virus genomes for the ability to produce core particles containing the virus-associated polymerase activity. The results obtained suggest that the P protein consists of three functional domains and a nonessential spacer arranged in the following order: terminal protein, spacer, reverse transcriptase/DNA polymerase, and RNase H. The first two domains are separated by a spacer region which could be deleted to a large extent without significant loss of endogenous polymerase activity. In cotransfection experiments, all P gene mutants could be complemented in trans by constructs expressing the wild-type gene product but not by a second P gene mutant. This indicates that the multifunctional P gene is expressed as a single translational unit and independent of the core gene and furthermore that the gene product is freely diffusible and not processed before core assembly.
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PMID:Mutational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity. 215 28

Antibodies against the RNase H domain of human hepatitis B virus P protein(s) are frequent markers of acute and chronic virus infection (T. Weimer, K. Weimer, Z.-X. Tu, M.-C. Jung, G. R. Pape, and H. Will, J. Immunol. 143:3750-3756, 1989). In the present study, these antibodies were determined in serial serum samples of experimentally infected chimpanzees and naturally infected human patients with acute and chronic hepatitis B virus infection. Anti-P antibodies were found in the sera of both chimpanzees and humans early in infection shortly after the immunoglobulin M anti-HBc response; they persisted in chronic carriers with ongoing viral replication but declined and disappeared at the time of virus clearance from the sera. These data demonstrate that antibodies to the RNase H domain of the hepatitis B virus P protein are early markers of infection and a signal of ongoing virus replication. Falling titers indicate the decline or end of active virus production and may therefore be a prognostic sign of virus elimination in natural infection and after antiviral therapy.
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PMID:Antibodies to the RNase H domain of hepatitis B virus P protein are associated with ongoing viral replication. 217 Jun 93

The nucleotide sequence of the human spumaretrovirus (HSRV) genome was determined. The 5' long terminal repeat region was analyzed by strong stop cDNA synthesis and S1 nuclease mapping. The length of the RU5 region was determined and found to be 346 nucleotides long. The 5' long terminal repeat is 1,123 base pairs long and is bound by an 18-base-pair primer-binding site complementary to the 3' end of mammalian lysine-1,2-specific tRNA. Open reading frames for gag and pol genes were identified. Surprisingly, the HSRV gag protein does not contain the cysteine motif of the nucleic acid-binding proteins found in and typical of all other retroviral gag proteins; instead the HSRV gag gene encodes a strongly basic protein reminiscent of those of hepatitis B virus and retrotransposons. The carboxy-terminal part of the HSRV gag gene products encodes a protease domain. The pol gene overlaps the gag gene and is postulated to be synthesized as a gag/pol precursor via translational frameshifting analogous to that of Rous sarcoma virus, with 7 nucleotides immediately upstream of the termination codons of gag conserved between the two viral genomes. The HSRV pol gene is 2,730 nucleotides long, and its deduced protein sequence is readily subdivided into three well-conserved domains, the reverse transcriptase, the RNase H, and the integrase. Although the degree of homology of the HSRV reverse transcriptase domain is highest to that of murine leukemia virus, the HSRV genomic organization is more similar to that of human and simian immunodeficiency viruses. The data justify classifying the spumaretroviruses as a third subfamily of Retroviridae.
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PMID:Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus. 245 55

A series of antisera directed against amino acid sequences from different segments of the duck hepatitis B virus (DHBV) P-gene were shown to immunoprecipitate DHBV DNA molecules that were covalently linked to the DHBV DNA terminal protein. Restriction analysis and sizing after protease treatment demonstrated that the P-gene proteins were bound to the 5'-end of the DHBV DNA minus-strand which was mapped to a G-residue in the centre of the repeat sequence DR1. Resistance to alkali treatment indicated a phosphodiester linkage to tyrosine between protein and DNA. Limited protease treatment prior to immunoprecipitation cleaved C-terminal P-proteins from the viral DNA, indicating that the terminal protein forms a separate domain encoded in the N-terminal part of the P-gene. Functional analysis of a deletion mutant confirmed the notion that a non-essential spacer separates the terminal protein from the polymerase domain residing in the C-terminal half of the P-gene. Thus, the major proteins required for hepadnaviral reverse transcription, namely the primer, DNA polymerase, and possibly also RNase H, appear to be synthesized as a polyprotein precursor which is at least initially linked as such to its first DNA product.
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PMID:The amino-terminal domain of the hepadnaviral P-gene encodes the terminal protein (genome-linked protein) believed to prime reverse transcription. 285 56


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