Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two similar, yet functionally distinct genomic RNAs are transcribed from the DNA genome of the human hepatitis B virus. The pre-C RNAs encode the precore protein which is proteolytically processed to yield e antigen. The pregenomic RNAs encode both the nucleocapsid protein and reverse transcriptase and serve as the templates for viral DNA replication. To determine whether synthesis of these two RNAs is directed from a single or a closely spaced pair of promoters, we introduced point and insertion mutations into the basal elements of the promoter that directs their synthesis. Transcription from these mutants was examined both in cell-free transcription systems derived from hepatoma (HepG2) and nonliver (HeLa) cell lines and by transient transfection of hepatoma cell lines (Huh7 and HepG2). The data from these experiments indicated that synthesis of the pre-C and pregenomic RNAs is directed by two distinct promoters and that the basal elements of these two promoters partially overlap, yet are genetically separable, with each consisting of its own transcriptional initiator and a TATA box-like sequence situated approximately 25 to 30 bp upstream of its sites of initiation. A 15-bp insertion was found to be sufficient to physically separate these two promoters. Furthermore, these two promoters can be differentially regulated, with the transcriptional activator Sp1 specifically activating transcription from the pregenomic promoter and the hepatocyte nuclear factor 4 specifically repressing transcription from the pre-C promoter. Thus, we conclude that the promoters used in synthesis of the pre-C and pregenomic mRNAs are genetically distinct and separately regulated.
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PMID:Promoters for synthesis of the pre-C and pregenomic mRNAs of human hepatitis B virus are genetically distinct and differentially regulated. 897 Sep 99

Assembly of hepadnaviruses depends on the formation of a ribonucleoprotein (RNP) complex comprising the viral polymerase polypeptide and an RNA segment, epsilon, present on pregenomic RNA. This interaction, in turn, activates the reverse transcription reaction, which is primed by a tyrosine residue on the polymerase. We have shown recently that the formation of this RNP complex in an avian hepadnavirus, the duck hepatitis B virus, depends on cellular factors that include the heat shock protein 90 (Hsp90). We now report that RNP formation also requires ATP hydrolysis and the function of p23, a recently identified chaperone partner for Hsp90. Furthermore, we also provide evidence that the chaperone complex is incorporated into the viral nucleocapsids in a polymerase-dependent reaction. Based on these findings, we propose a model for hepadnavirus assembly and priming of viral DNA synthesis where a dynamic, energy-driven process, mediated by a multi-component chaperone complex consisting of Hsp90, p23 and, potentially, additional factors, maintains the reverse transcriptase in a specific conformation that is competent for RNA packaging and protein priming of viral DNA synthesis.
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PMID:Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids. 900 68

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

Infection with hepatitis B virus can result in asymptomatic seroconversion with viral clearance, fulminant hepatic failure and death, or chronic, typically lifelong, transmissible infection. The mechanism(s) of viral persistence are poorly understood but viral clearance and fulminant hepatic failure are generally thought to result from co-ordinated and effective and abnormally vigorous immune responses, respectively, whereas viral persistence results from immunological failure in addition to poorly characterized viral factors promoting persistence. This paper proposes (1) that the predominant viral factor(s) promoting persistence of hepatitis B virus are homeostatic mechanism(s) responsible for modulating its replication and mutation and (2) that chronic hepatitis B results when these mechanisms are successful and other outcomes occur when these homeostatic mechanism(s) fail. Furthermore, it is proposed that seroconversion (e.g. from HBsAg to anti HBsAg positivity), when it occurs, is a consequence facilitated by restricted viral antigenic diversity and reduced viral replication rather than a proximate cause of it. The specific homeostatic mechanisms proposed--negative feedback inhibition of hepatitis B virus DNA polymerase/reverse transcriptase mediated by HBs antigen and a hepatitis B virus DNA polymerase fidelity modulating function of HBeAg--are consistent with the available data and resolve many paradoxical clinical observations. But, more importantly, this model has clear implications for therapy, including the rational design of drugs and therapeutic vaccines.
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PMID:Hepatitis B virus replication and mutation are autoregulated by interactions between surface antigen and HBeAg and the HBV DNA polymerase: a functional model with therapeutic implications. 904 82

Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction that involves the covalent linkage of the first deoxyribonucleotide to the polymerase polypeptide. We recently expressed human hepatitis B virus (HBV) reverse transcriptase (pol) in insect cells by using the recombinant baculovirus system. The purified protein is active in nucleotide priming and reverse transcription reactions. In this report, we demonstrate that the tyrosine residue at amino acid number 63 within the TP (terminal protein) domain of the polymerase is the site of covalent linkage of the first nucleotide of minus-strand DNA. Analysis of pol polypeptides with mutations in the TP and RT (reverse transcriptase) domains indicated that both domains were required for in vitro nucleotide priming activity. Polymerase proteins with mutations in the TP and RT domains were not capable of complementing each other in the nucleotide priming reaction, suggesting that transcomplementation between full-length polypeptides was not possible. However, when the TP and RT domains were expressed as separate polypeptides, they formed a highly stable complex that was active in nucleotide priming and reverse transcription. The presence of an epsilon stem-loop dramatically increased the nucleotide priming activity in transcomplementation assays, even though full-length pol displayed similar activities in the absence and presence of epsilon. These data raise the possibility that in the transcomplementation assay, epsilon may play a role in the formation of a functional complex between TP and RT, rather than being required only as the template for nucleotide priming. The results indicate that using the baculovirus system, it is possible to dissect the protein-protein and protein-RNA interactions required for HBV genome replication.
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PMID:Transcomplementation of nucleotide priming and reverse transcription between independently expressed TP and RT domains of the hepatitis B virus reverse transcriptase. 906 Jun 59

The genomes of both bacteria and eukaryotic organisms are known to encode selenoproteins, using the UGA codon for seleno-cysteine (SeC), and a complex cotranslational mechanism for SeC incorporation into polypeptide chains, involving RNA stem-loop structures. These common features and similar codon usage strongly suggest that this is an ancient evolutionary development. However, the possibility that some viruses might also encode selenoproteins remained unexplored until recently. Based on an analysis of the genomic structure of the human immunodeficiency virus HIV-1, we demonstrated that several regions overlapping known HIV genes have the potential to encode selenoproteins (Taylor et al. [31], J. Med. Chem. 37, 2637-2654 [1994]). This is provocative in the light of overwhelming evidence of a role for oxidative stress in AIDS pathogenesis, and the fact that a number of viral diseases have been linked to selenium (Se) deficiency, either in humans or by in vitro and animal studies. These include HIV-AIDS, hepatitis B linked to liver disease and cancer, Coxsackie virus B3, Keshan disease, and the mouse mammary tumor virus (MMTV), against which Se is a potent chemoprotective agent. There are also established biochemical mechanisms whereby extreme Se deficiency can induce a proclotting or hemorrhagic effect, suggesting that hemorrhagic fever viruses should also be examined for potential virally encoded selenoproteins. In addition to the RNA stem-loop structures required for SeC insertion at UGA codons, genomic structural features that may be required for selenoprotein synthesis can also include ribosomal frameshift sites and RNA pseudoknots if the potential selenoprotein module overlaps with another gene, which may prove to be the rule rather than the exception in viruses. One such pseudoknot that we predicted in HIV-1 has now been verified experimentally; a similar structure can be demonstrated in precisely the same location in the reverse transcriptase coding region of hepatitis B virus. Significant new findings reported here include the existence of highly distinctive glutathione peroxidase (GSH-Px)-related sequences in Coxsackie B viruses, new theoretical data related to a previously proposed potential selenoprotein gene overlapping the HIV protease coding region, and further evidence in support of a novel frameshift site in the HIV nef gene associated with a well-conserved UGA codon in the 1-reading frame.
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PMID:Genomic structures of viral agents in relation to the biosynthesis of selenoproteins. 915 12

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

Hepatitis B virus (HBV) mutants have recently been identified in patients with acute or fulminant as well as chronic infections. Naturally occurring mutations have been identified in all viral genes and regulatory elements, most notably in the genes coding for the structural envelope and nucleocapsid proteins. Mutations in the gene coding for the hepatitis B surface antigen (HBsAg) may result in infection or viral persistence despite the presence of antibodies against HBsAg (anti-HBs) ("vaccine escape" or "immune escape"). Mutations in the gene encoding the pre-core/ core protein (pre-core stop codon mutant) result in a loss of hepatitis B e antigen (HBeAg) and seroconversion to antibodies to HBeAg (anti-HBe) with persistence of HBV replication (HBeAg minus mutant). Mutations in the core gene may lead among others to an "immune escape" due to a T cell receptor antagonism. Mutations in the gene coding for the polymerase/reverse transcriptase can be associated with viral persistence or resistance to nucleoside analogues. Thus, HBV mutations may affect the natural course of infection, viral clearance and response to antiviral therapy. Apart from the precore/core stop codon mutations, the exact contribution of specific mutations to diagnosis and therapy of HBV infection as well as patient management in clinical practice remain to be established.
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PMID:[Hepatitis B virus mutants--clinical significance]. 926 94

For evaluation of anti-hepatitis B virus (HBV) drugs, we have employed the HBV transgenic mouse in which virion-like particles can be assayed in the serum. Bispivaloyloxymethyl-9-(2-phosphonylmethoxyethyl)-adenine [bis (POM) PMEA] 100 mg/kg/day, 2',3'-dideoxy-3'-thiacytidine [(+-)-BCH189] 200 mg/kg/day and a placebo were orally administered to mice twice a day for 14 days. Anti-viral effects were monitored by checking the levels of serum HBV DNA by the semiquantitative polymerase chain reaction, HBsAg and HBeAg by enzyme immunoassay, and replicative intermediates in the liver by Southern blotting. As expected, decrease from the 10(0.5) to 10(3) copies of HBV DNA per microl of sera detected before the treatment to the undetectable level was evident for all five animals treated with bis(POM) PMEA 100 mg/kg/day. However (+-)-BCH189 200 mg/kg/day, which is known to act as the inhibitor of reverse transcriptase for HBV or HIV in vivo and in vitro, did not suppress HBV DNA levels in the transgenic mouse. Thus, we were able to detect the effects of anti-HBV drugs semi-quantitatively, and confirm differences in drug efficacy.
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PMID:Evaluation of anti-hepatitis B virus (HBV) drugs using the HBV transgenic mouse: application of the semiquantitative polymerase chain reaction (PCR) for serum HBV DNA to monitor the drug efficacy. 940 31

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are known to be associated with hepatocellular carcinoma (HCC). In this study, we investigated the prevalence of the newly described hepatitis G virus (HGV) in patients with HCC. The sera of 85 patients (66 male, 19 female, 61 +/- 11 years) with HCC were studied for the presence of HGV RNA by reverse transcriptase-polymerase chain reaction. Seventeen (20%) of 85 patients with HCC, 10 (16%) of 61 patients with chronic hepatitis B without HCC and 14 (20%) of 68 patients with chronic hepatitis C without HCC were infected with HGV, a significantly higher proportion when compared with two (2%) of 85 healthy controls (P < 0.01). When grouped according to the underlying cause of liver disease, HCC patients with HBV infection (33%), HCV infection (21%), alcoholic liver disease (17%), or cryptogenic cirrhosis (15%) had similar serum levels of HGV RNA. Four of the 17 (24%) HGV-positive patients with HCC were coinfected with HBV and six (35%) with HCV; thus, 59% of HGV-positive patients with HCC were coinfected with other hepatotropic viruses. Seven (41%) HGV-positive patients were infected with HGV only. Patients with HGV infection were more likely to have a history of blood transfusion than patients without HGV infection (P = 0.024). Hence, the prevalence of HGV is significantly higher in patients with HCC in comparison with the healthy population.
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PMID:Prevalence of hepatitis G virus in patients with hepatocellular carcinoma. 943 Mar 61


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