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)

The (-) enantiomer of 3'-thiacytidine (lamivudine) has been found to be a potent inhibitor of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) replication. Mutation of methionine to valine or isoleucine at the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the HIV reverse transcriptase has been shown to be responsible for lamivudine resistance in HIV. The hepadnaviruses also have the YMDD motif in their DNA polymerase. Therefore, it is possible that hepadnaviruses could develop lamivudine resistance by a similar mutation at this motif. We analyzed the HBV from a liver transplantation patient who developed recurrent HBV viremia during lamivudine treatment. The polymerase gene was amplified by polymerase chain reaction (PCR), and the region coding for the YMDD motif was sequenced. The pretreatment HBV sequence coded for YMDD, while the lamivudine-resistant mutant HBV coded for YIDD (tyrosine, isoleucine, aspartate, aspartate). With the documented changes in the YMDD motif of lamivudine-resistant HIV, it is likely that the methionine-to-isoleucine mutation in the YMDD motif of the HBV polymerase contributes significantly to the lamivudine-resistance of HBV isolated from this patient.
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PMID:Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. 878 48

DNA polymerase activity was assayed in hepatitis B virus (HBV) and core particles isolated from chronic producer lines. The particle-associated DNA polymerase activity, which was found to be limited to incorporation of only a few nucleotides, was inhibited by the 5'-triphosphates of nucleoside analogs. The 1-beta-L (1S,4R) and 1-beta-D (1R,4S) enantiomers of antiviral nucleoside analogs were compared for the ability to inhibit incorporation of natural nucleoside triphosphates into the viral DNA. Previously, both enantiomers of several analogs were found to be substrates for human immunodeficiency virus type 1 reverse transcriptase (HIV RT); the 1-beta-D enantiomers of some pairs were preferred as substrates. In contrast, the 1-beta-L enantiomers of all pairs tested were the more potent inhibitors of labeled substrate incorporation into hepatitis B virus DNA; the concentration required to inhibit the incorporation of the natural substrate by 50% was 6-fold to several hundred-fold lower than the concentration of the 1-beta-D enantiomer required for the same inhibitory effect. This preference for the 1-beta-L enantiomers was observed for both RNA-directed synthesis in core particles and DNA-directed synthesis in viral particles. The observed antiviral effect of the nucleoside analogs in cell culture seemed to be limited chiefly by their phosphorylation in cells.
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PMID:DNA polymerase activity of hepatitis B virus particles: differential inhibition by L-enantiomers of nucleotide analogs. 878 5

The polymerase encoded by human hepatitis B virus, which has reverse transcriptase and RNase H activity, binds to its pregenomic RNA template in a two-step process involving a terminal redundancy. Both first strand and second strand DNA synthesis involve primer translocation and second strand synthesis involves a template jump. Three parts of the genome, including the so-called core promoter, are known to show deletions in strains usually arising after long-standing HBV infection, but also in some patients treated with interferon. A computer-based study of RNA template folding in the core promoter region, accommodating well-known point mutations, has generated a model for the 3' DR1 primer binding site as being part of a superstructure encompassing an already well-established stem-loop. Depending on the identity of nucleotides 1762 and 1764, the DR1 region may assume two alternative secondary structures which stabilize it as a primer binding site to different extents. Remarkably, one of these structures includes a pronounced loop which coincides with at least 12 related deletions seen in HBV DNA from different patients. Thus according to the model, the 5'- and 3'-ends of pregenomic RNA, which share primary sequences but have separate functions, are not structural equivalents. An RNA superstructure near the 3'-end of all HBV transcripts could have far-reaching implications for the modulation of both genome replication and post-transcriptional processing.
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PMID:A revised secondary structure model for the 3'-end of hepatitis B virus pregenomic RNA. 881 Oct 80

Hepatitis B virus replication is very sensitive to lamivudine. A single amino acid change in human immunodeficiency virus reverse transcriptase is responsible for high-level resistance to this compound. Duck hepatitis B virus mutants were created bearing the analogous amino acid change in the duck hepatitis B virus polymerase. Viral DNA production was reduced 92% for the wild-type virus at 2 micrograms of lamivudine per ml, while the mutants required 40 micrograms of lamivudine per ml to inhibit replication by greater than 80%.
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PMID:Generation of duck hepatitis B virus polymerase mutants through site-directed mutagenesis which demonstrate resistance to lamivudine [(--)-beta-L-2', 3'-dideoxy-3'-thiacytidine] in vitro. 884 15

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

The recently identified hepatitis G virus (HGV) is parenterally transmitted; the impact of sexual transmission is unknown. Moreover, it is unclear what proportion of HGV-infected persons may develop persistent viremia. Sera from injecting drug users (IDUs), non-drug-injecting homosexual and bisexual men with high levels of sexual risk behavior, and blood donors were tested for HGV RNA and hepatitis C virus (HCV) RNA by reverse transcriptase-polymerase chain reaction and for antibodies to human immunodeficiency virus, hepatitis B virus, and HCV. HGV RNA was detected in 33% of IDUs (n = 130), 11% of homosexual and bisexual men (n = 101), and 2% of blood donors (n = 90). HGV RNA seroprevalence significantly decreased with increasing time since first drug injection, whereas the seroprevalences of both HCV RNA and anti-HCV antibody increased. Thus, a high proportion of HGV-infected persons may clear the virus and develop protective antibodies. The relatively high HGV RNA prevalence among non-drug-injecting homosexual and bisexual men indicates that sexual contact may be another important route of HGV transmission.
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PMID:Detection of the hepatitis G virus genome among injecting drug users, homosexual and bisexual men, and blood donors. 894 Feb 25

In this study we amplified virtually the entire genomes of hepatitis A virus (a member of the Picornaviridae family), hepatitis B virus (a member of the Hepadnaviridae family), and hepatitis C virus (a member of the Flaviviridae family) by using the recently described technique of long PCR. In order to do this, we first demonstrated, using the lambda phage, that long PCR can be made highly sensitive and the sensitivity can be further enhanced by nested long PCR. We also showed, using tobacco mosaic virus as a model, that a reverse transcriptase reaction can be linked to a long PCR, enabling the nearly full-length amplification of the genomes of RNA viruses. We then applied these techniques to serial dilutions of titrated stocks of well-characterized strains of hepatitis A, B, and C viruses. We amplified the nearly full-length sequence of each of these viruses from a small number of viral genomes, demonstrating the sensitivity of the process.
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PMID:Long PCR and its application to hepatitis viruses: amplification of hepatitis A, hepatitis B, and hepatitis C virus genomes. 894 Apr 52

The hepadnaviral DNA genome is synthesized by a viral-encoded reverse transcriptase, but the nature of this protein(s) in vivo remains obscure. We have previously described studies in which activity gel assays identified multiple DNA polymerase (DNAp) activities associated with highly purified duck hepatitis B virus (DHBV) core particles. We now report that virions isolated from viremic sera are associated with DNA-dependent DNAp activities which are nearly identical to major DNAp activities detected with highly purified DHBV core particles. These results suggest that the virion-associated polymerases are the same as those which are detected with core particles and are likely to represent DHBV pol gene products involved in replication of the genome.
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PMID:Preparations of duck hepatitis B virions contain multiple DNA polymerase activities. 894 31

We tested the sera of 67 consecutive patients for hepatitis G virus (HGV) RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). These patients (42 males and 25 females, median age 35 years, range 13-64 years) had liver disease of unknown aetiology and were without markers of hepatitis (A-E) viruses or signs of genetically determined, autoimmune, alcoholic or drug-induced liver disease. The controls in this study were 110 patients (50 females and 60 males, median age 45 years, range 9-65 years) with chronic hepatitis B virus (HBV) infection (19 patients) or hepatitis C virus (HCV) infection (91 patients). Ten of 67 (14.9%) patients with cryptogenic disease were positive for HGV RNA by at least three separate tests; HGV RNA was also detected in one of 19 (5.3%) hepatitis B surface antigen (HBsAg) carriers and in nine of 91 (16.6%) patients with antibody to HCV. These data suggest that HGV occurs as frequently in HCV-infected patients as in those with cryptogenic disease. Elevated serum gamma glutamyl transpeptidase (gamma-GT) (higher than twice the normal value) and alkaline phosphatase levels were found in eight of 10 (80%) HGV RNA positive patients and in six of 57 (10.5%) HGV RNA negative patients (P < 0.0001). Five (50%) HGV RNA positive patients had non-specific inflammatory bile duct lesions. A statistically significant difference was observed between HGV RNA positive and negative patients with chronic HBV or HCV infections (P < 0.029). Therefore, the spectrum of liver disease associated with HGV is wide, but a characteristic lesion of the bile duct leading to elevation of cholestatic enzymes might be specific for this virus.
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PMID:Hepatitis G virus RNA in the serum of patients with elevated gamma glutamyl transpeptidase and alkaline phosphatase: a specific liver disease? [corrected]. 894 81

The replication of the hepatitis B viral DNA genome proceeds through a pregenomic RNA intermediate. This pregenomic RNA subsequently serves as the template for the formation of the viral DNA by the reverse transcriptase activity of the viral P gene product. The P gene product is believed to be a multifunctional enzyme with DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, and RNase H activities. Detailed biochemical studies of this protein have not been performed because of the inability to obtain sufficient amounts of the enzyme from the virus and by the inability to produce the enzyme in heterologous expression systems. The RNase H activity is essential for viral replication and is believed to be responsible for the degradation of the RNA pregenomic intermediate as well as for generating the short RNA primer that is required for DNA second strand synthesis. We have assembled an expression vector which directs the synthesis of a protein that corresponds to the putative RNase H domain of the P gene product and having a carboxyl-terminal polyhistidine tag to facilitate purification. The protein has been expressed in Escherichia coli and purified to yield 1-2 mg of protein/liter of culture. This protein has RNase H activity as defined by its ability to degrade the RNA component of RNA-DNA hybrids but not the DNA component. The RNase H has a basic optimum pH, is active only in the presence of reducing agents, and is dependent on the presence of divalent cations, with magnesium being preferred over manganese.
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PMID:Expression, purification, and characterization of an active RNase H domain of the hepatitis B viral polymerase. 895 90


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