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)

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

Hepatitis B virus (HBV) is a small DNA virus that replicates by reverse transcription of a terminally redundant RNA, the pregenome. Specific packaging of this transcript into viral capsids is mediated by interaction of the reverse transcriptase, P protein, with the 5'-proximal encapsidation signal epsilon, epsilon-function is correlated with the formation of a hairpin structure containing a bulge and a loop, each consisting of 6 nt. To analyse the importance of primary sequence in these regions, we have combined selection of encapsidation competent individuals from pools of randomized epsilon-sequences in transfected cells with in vitro amplification, thus bypassing the current experimental limitations of the HBV system. While no alterations of the authentic loop sequence were detectable, many different sequences were tolerated in the 3'-part of the bulge. However, at the two 5'-proximal bulge positions the wt sequence was strongly selected for, indicating that for RNA packaging close contacts between protein and the 5'- but not the 3'-part of the bulge are important. Such a bipartite organisation provides a structural basis for the recently demonstrated special role of the 3'-part of the bulge as template for the first nucleotides of (-)-strand DNA in HBV reverse transcription.
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PMID:Distinct requirements for primary sequence in the 5'- and 3'-part of a bulge in the hepatitis B virus RNA encapsidation signal revealed by a combined in vivo selection/in vitro amplification system. 747 35

The replication of hepatitis B virus DNA proceeds through reverse transcription of a pregenomic RNA intermediate, a reaction that takes place within viral nucleocapsids and is catalyzed by the viral P protein. P protein is involved in all phases of the reaction, serving as (a) a recognition factor for the selective encapsidation of the pregenomic RNA template; (b) the protein primer for the initiation of minus strand DNA synthesis; (c) the reverse transcriptase and DNA polymerase involved in strand elongation; and (d) the RNaseH activity required to remove RNA template prior to plus strand synthesis. P protein is capable of site-specific RNA recognition, specifically binding to a stem-loop structure at the 5' end of pregenomic RNA. This interaction is required for both RNA encapsidation and reverse transcription.
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PMID:Hepatitis B virus reverse transcriptase and its many roles in hepadnaviral genomic replication. 752 20

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

CD151 (PETA-3/SFA-1) is a member of the Transmembrane 4 Superfamily (TM4SF) of cell-surface proteins and, like other TM4SF members CD9 and CD63, is expressed by platelets, megakaryocytes and endothelial cells. The precise function of CD151 is unknown however complexes containing CD151 and beta1 integrins have been isolated from a number of cell systems and studies using anti-CD151 monoclonal antibodies have suggested a role in transmembrane signalling and cell adhesion. To further investigate the function of CD151 we have determined the genomic organisation of mouse CD151 (Cd151). Cd151 spans 4 kb and contains six coding region exons. Using 5' RACE and reverse transcriptase-polymerase chain reaction (RT-PCR) we have identified three 5' UTR splice variants which arise through alternate splicing of three exons. Splice variants were detected in a number of mouse tissues by RT-PCR. Analysis of the Cd151 genomic structure reveals a high degree of structural conservation with other TM4SF molecules supporting the theory that family members have arisen from gene duplication of a common ancestral gene. Cd151 maps to chromosome 7, in close linkage to the p gene (OCA2 in humans), and helps define a boundary in the human/mouse homology relationships.
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PMID:Characterisation of the mouse homologue of CD151 (PETA-3/SFA-1); genomic structure, chromosomal localisation and identification of 2 novel splice forms. 960 68

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, 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


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