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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cis-replicating RNA elements in the 5' and 3' nontranslated regions (NTRs) of the hepatitis C virus (HCV) genome have been thoroughly studied before. However, no cis-replicating elements have been identified in the coding sequences of the HCV polyprotein until very recently. The existence of highly conserved and stable stem-loop structures in the RNA polymerase NS5B coding sequence, however, has been previously predicted (A. Tuplin, J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds, RNA 8:824-841, 2002). We have selected for our studies a 249-nt-long RNA segment in the C-terminal NS5B coding region (NS5BCR), which is predicted to form four stable stem-loop structures (SL-IV to SL-VII). By deletion and mutational analyses of the RNA structures, we have determined that two of the stem-loops (SL-V and SL-VI) are essential for replication of the HCV subgenomic replicon in Huh-7 cells. Mutations in the loop and the top of the stem of these RNA elements abolished replicon RNA synthesis but had no effect on translation. In vitro gel shift and filter-binding assays revealed that purified NS5B specifically binds to SL-V. The NS5B-RNA complexes were specifically competed away by unlabeled homologous RNA, to a small extent by 3' NTR RNA, and only poorly by 5' NTR RNA. The other two stem-loops (SL-IV and SL-VII) of the NS5BCR domain were found to be important but not essential for colony formation by the subgenomic replicon. The precise function(s) of these cis-acting RNA elements is not known.
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PMID:cis-acting RNA signals in the NS5B C-terminal coding sequence of the hepatitis C virus genome. 1545 7

Plasmon-waveguide resonance (PWR) spectroscopy has been used to study the interactions between ligands that correspond to inhibitors, activators or substrates and three integral membrane proteins representing potential drug targets; cyclooxygenases 1 and 2 (COX-1 and -2), integrin alphaVbeta3, and hepatitis C virus RNA polymerase. The proteins were incorporated into an egg phosphatidylcholine bilayer deposited onto the surface of the PWR resonator, and changes in the amplitude and position of the PWR spectra due to mass density increases and conformational transitions have been used to characterize the kinetics and binding affinities corresponding to these interactions. Although the partition of COX-2 into the bilayer was not as efficient as was the case with the other two proteins, sufficient protein could be incorporated to allow ligand binding to be observed. It was also possible to incorporate COX-1 into a lipid bilayer by adding a suspension of microsomal membrane fragments containing this enzyme to a preformed bilayer, and to observe binding of an inhibitory ligand. The interactions between integrin alphaVbeta3 and two ligands with different in vivo efficacies could be distinguished by both spectral measurements and binding kinetics. In the case of the RNA polymerase, the kinetics of PWR spectral changes upon adding a substrate solution to an enzyme-template complex were consistent with those obtained from direct measurements of enzymatic turnover. These experiments demonstrate the utility of PWR spectroscopy to provide novel information regarding drug interactions with membrane proteins in a lipid environment and to distinguish conformational changes induced by binding of various drug molecules.
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PMID:Plasmon-waveguide resonance spectroscopy applied to three potential drug targets: cyclooxygenase-2, hepatitis C virus RNA polymerase and integrin alpha V beta 3. 1553 62

A microbead-based affinity chromatography chip (micro-BACC) controlling hundreds of nanoliters of reaction volume was developed to separate and analyze hepatitis C virus (HCV) RNA polymerase protein by immobilization of an RNA aptamer on beads. A photocleavable linker was conjugated in between the beads and the aptamer to elute the bound RNA polymerase from the RNA aptamer in one step by UV irradiation, resulting in an efficient method to elute and identify the target molecule bound on RNA using a mass spectrometer. This linker showed a cleavage activity over 70% upon UV irradiation at 1050 mW/cm2 for more than 5 min. The photoelution method could prevent the target molecule from contaminations in affinity chromatography caused by elution solutions of high salt concentration, extreme pH and detergent, respectively. In this chip, sample reagents up to 800 nL could be metered quantitatively into the bead chamber using a nanoliter dispenser working, based on surface-guided flow control and pneumatic control by external air pressure on the chip. RNA polymerase eluted after UV irradiation was successfully analyzed by trypsin treatment without additional purification. As a result, using the aptamer, we could detect RNA polymerase from 800 nL hepatitis C patient serum containing 96 fmol HCV RNA polymerase. The detection limit of this system was estimated to be 9.6 fmol HCV RNA polymerase.
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PMID:Microbead-based affinity chromatography chip using RNA aptamer modified with photocleavable linker. 1556 96

Clinical data suggest that iron is a negative factor in chronic hepatitis C; however, the molecular mechanisms by which iron modulates the infectious cycle of hepatitis C virus (HCV) remain elusive. To explore this, we utilized cells expressing a HCV replicon as a well-established model for viral replication. We demonstrate that iron administration dramatically inhibits the expression of viral proteins and RNA, without significantly affecting its translation or stability. Experiments with purified recombinant HCV RNA polymerase (NS5B) revealed that iron binds specifically and with high affinity (apparent Kd: 6 and 60 microM for Fe2+ and Fe3+, respectively) to the protein's Mg2+-binding pocket, thereby inhibiting its enzymatic activity. We propose that iron impairs HCV replication by inactivating NS5B and that its negative effects in chronic hepatitis C may be primarily due to attenuation of antiviral immune responses. Our data provide a direct molecular link between iron and HCV replication.
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PMID:Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C Virus. 1563 67

Hepatitis C virus (HCV) infection is usually treated with the combination of interferon and ribavirin, but only a small fraction of patients develop a sustained remission. There is need for the development of specific molecular approaches for the treatment of chronic HCV infection. We propose that RNA interference is highly effective antiviral strategy that offers great potential for the treatment of HCV infection. Three plasmid constructs expressing small interfering RNAs (siRNAs) targeted to sequences encoding the structural gene (E2) and non-structural genes (NS3, NS5B) of HCV1a genome were prepared. Antiviral properties of siRNAs against the HCV1a strain were studied in a transient replication model that involved the use of a transcription plasmid containing the full-length HCV genome and an adenovirus expressing T7 RNA polymerase. We found that siRNAs targeted to the E2, NS3 and NS5B regions of the HCV genome efficiently inhibited expression of the HCV core and NS5A protein measured by Western blot analysis and immunocytochemical staining. Intracytoplasmic immunization of siRNAs in HCV-transfected cells efficiently degraded genomic positive strand HCV RNA, as shown by ribonuclease protection assay (RPA). All three siRNAs efficiently inhibited synthesis of replicative negative strand HCV RNA in the transfected cells. A control siRNA plasmid against a Epstein--Barr virus latency gene did not inhibit protein expression and negative strand HCV RNA. These results suggest that RNAi is an effective and alternative approach that can be used to inhibit HCV expression and replication.
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PMID:Small interfering RNA effectively inhibits protein expression and negative strand RNA synthesis from a full-length hepatitis C virus clone. 1597 38

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.
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PMID:Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. 1603 84

The hepatitis C virus (HCV) is a major etiological agent causing chronic hepatitis in humans. Since the virus does not grow in a cell culture, the direct measurement of viral replication is impossible. Therefore, the current study presents a surrogate model system using a viral polymerase and RNA template. A plasmid expressing the HCV NS5B polymerase was maintained with a plasmid containing a reporter gene in an Escherichia coli cell. The reporter construct contained the HCV 5' untranslated region (UTR) followed by a luciferase gene with a specific orientation so that a minus-sense transcript containing the luciferase fused to the 5' UTR was produced after the initial transcription. When the HCV NS5B polymerase was expressed in the same cell, the primary transcript was recognized by the polymerase due to the presence of the minus-sense 5' UTR, and a secondary transcript containing a plus-sense luciferase gene was produced. Thus, a simple luciferase assay was able to measure the HCV NS5B polymerase activity. The production of minus- and plus-sense transcripts was confirmed by an RT-PCR, while the production of HCV NS5B and expression of the reporter luciferase in the bacterial cell were confirmed by immunofluorescence microscopy. The polymerization occurred in the absence of any other viral/host factors. Accordingly, this would appear to be the first study to demonstrate that the heterologous expression of an animal viral RNA polymerase and its template in a bacterial cell can partially reconstitute the polymerization reaction.
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PMID:Partial reconstitution of hepatitis C virus RNA polymerization by heterologous expression of NS5B polymerase and template RNA in bacterial cell. 1609 67

The high prevalence of the disease caused by hepatitis C virus (HCV) and the limited efficacy of interferon-based therapies have stimulated the search for safer and more effective drugs. The development of inhibitors of the HCV NS5B RNA polymerase represents a promising strategy for identifying novel anti-HCV therapeutics. However, the high genetic diversity, mutation rate and turnover of HCV are expected to favour the emergence of drug resistance, limiting the clinical usefulness of polymerase inhibitors. Thus, the characterization of the drug-resistance profile of these antiviral agents is considered crucial for identifying the inhibitors with a higher probability of clinical success. In the absence of an efficient in vitro infection system, HCV sub-genomic replicons have been used to study viral resistance to both nucleoside and non-nucleoside NS5B inhibitors. While these studies suggest that drug-resistant viruses are likely to evolve in vivo, they provide a wealth of information that should help in the identification of inhibitors with improved and distinct resistance profiles that might be used for combination therapy.
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PMID:HCV antiviral resistance: the impact of in vitro studies on the development of antiviral agents targeting the viral NS5B polymerase. 1613 May 21

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.
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PMID:Treating hepatitis C: can you teach old dogs new tricks? 1631 65

The hepatitis C virus RNA polymerase (NS5B) is strictly required for viral replication and thus represents an attractive target for antiviral drug development. In this study, stable HeLa cell lines with an integrated NS5B gene were selected by G418 and then confirmed by genome PCR. Subsequently, transcription and expression of the integrated NS5B genes were demonstrated by RT-PCR and Western blot analysis. Further analysis demonstrated enzymatic activity of the expressed NS5B polymerase. The stable HeLa cell lines should be useful for the identification of NS5B inhibitors and for studying the mechanisms of HCV replication.
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PMID:Establishment of stable HeLa cell lines expressing enzymatically active hepatitis C virus RNA polymerase. 1632 44


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