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 5' and 3' untranslated regions (UTR) of the hepatitis C virus (HCV) genome contain stem-loop structures, which are important in viral gene expression and replication. In this study, the functional roles of the predicted stem-loop structures of HCV 5' UTR and 3' UTR in viral gene expression were examined using a chimeric clone of full-length HCV genomic cDNA clone and the gene for green fluorescent protein (GFP). High level expression of the HCV-GFP chimera in Huh-7 cells was accomplished by using a replication defective adenovirus that expresses T7 RNA polymerase and transcription plasmid containing full-length HCV-GFP chimera under the control of a T7 promoter. The HCV-GFP clone, with deletion of stem-loop I, expressed proteins in transfected Huh-7 cells at comparable levels to the wild type HCV clone. Other mutations of the 5' UTR, which either deleted or altered the base pairing of stem-loops II to IV, completely abolished the expression of HCV-GFP chimera. In contrast, deletion of 3' UTR sequences had no effect on HCV protein expression. These findings suggest that the stem-loop structures II to IV of HCV 5' UTR are necessary for protein expression, but that stem loop I is dispensable for protein translation. The stem-loop structures of 3' UTR of HCV genome appear to have no direct role in viral gene expression.
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PMID:Stem-loop structures II-IV of the 5' untranslated sequences are required for the expression of the full-length hepatitis C virus genome. 1260 98

Parvovirus B19 has been proposed as the etiological agent of fulminant hepatitis (FH) or hepatitis-associated aplastic anemia (HAA). We studied the prevalence of parvovirus B19 in liver-tissue samples from patients with FH and HAA and from control subjects. In the first study, parvovirus B19 DNA was detected by nested polymerase chain reaction (PCR) in 4 of 15 livers from patients with FH and in 3 of 22 livers from patients with nonviral hepatic disease. In a second confirmatory study, livers were tested for parvovirus B19 and its variant erythroviruses, V9 and A6. Tissues were also tested by reverse-transcriptase PCR for the presence of parvovirus B19 transcripts as a marker of viral replication. There was no significant difference in the prevalence of parvovirus B19 DNA in livers from patients with FH or HAA, compared with liver-tissue samples from patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection; parvovirus B19 transcripts were not detected. There was a significant increase (P<.1) in the prevalence of variant erythrovirus sequences in livers of patients with HBV or HCV hepatitis, the reason for which is currently unknown.
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PMID:Prevalence of parvovirus B19 in liver tissue: no association with fulminant hepatitis or hepatitis-associated aplastic anemia. 1272 38

Nucleoside chain terminators represent one of the most promising classes of antiviral drug for DNA viruses and retroviral infection; however, they have not been fully explored against RNA viral polymerases. In this report, we investigate the notion of employing canonical 3'-deoxyribonucleoside triphosphates (3'-dNTPs) as a chain terminator for hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp). Using a HCV RNA transcript-dependent RNA elongating assay, we found that they inhibit NS5B RdRp with K(i) ranged from 0.7 to 23 microM. Additional structure-activity relationship studies showed that removal of 2'-hydroxyl group, elimination of ribose's 2',3'-carbon-carbon bond, or addition of 5-methyl group to a pyrimidine base is detrimental to 3'-dNTP's potency. Direct evidence was obtained that all four canonical 3'-dNTP are incorporated into elongating RNA chains and the incorporation terminates NS5B RdRp-catalyzed RNA synthesis. The K(i) values for each of 3'-dNTPs were determined in the single nucleotide incorporation experiments. The nucleoside form of 3'-dNTPs was further evaluated in a cell culture-based HCV subgenomic replicon assay. The discrepancy between the potent in vitro activity and the weak cellular activity of these chain terminators was discussed in the context of nucleoside metabolism. This proof of concept study demonstrates that canonical 3'-dNTPs can function as an effective chain terminator for HCV NS5B RdRp with cytidine as the preferred nucleoside scaffold. Our results further sheds light on the potential hurdles that need to be overcome for successful development of active nucleoside chain terminators in vivo for a viral RNA polymerase, especially the HCV NS5B RdRp.
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PMID:Canonical 3'-deoxyribonucleotides as a chain terminator for HCV NS5B RNA-dependent RNA polymerase. 1276 72

Crotty et al. recently proposed the primary antiviral action of ribavirin to be that of a potent RNA mutagen [Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nat. Med. 6, 1375-1379]. Here we investigate the effect of ribavirin triphosphate (RTP) on RNA synthesis catalyzed by a full-length hepatitis C virus (HCV) RNA polymerase in vitro. HCV polymerase can use RTP as a nucleotide substrate in a template-dependent manner, incorporating it opposite a pyrimidine (C or U) template residue, but not a purine (A or G). Kinetic analysis revealed that incorporation of ribavirin monophosphate (RMP) across from C is 3 times more efficient catalytically than that across from U, as determined by the k(cat)/K(m) parameter. The efficiency of RMP incorporation, however, is 50-100 fold lower than that of the natural NMP. RMP incorporation does not lead to termination of RNA chain synthesis, as evidenced by the ability of the polymerase to extend its RNA product many nucleotides beyond the site of RMP incorporation. However, multiple-RMP incorporation at low GTP concentrations induced the formation of stalled elongation complexes, particularly at the template region containing consecutive C residues. Most, but not all, such elongation blocks can be relieved by the re-addition of GTP. When ribavirin is present in the RNA template, pyrimidine (but neither purine nor ribavirin) monophosphate is incorporated opposite ribavirin, but at an exceedingly low catalytic efficiency (200-3000-fold lower) compared to the efficiencies of those templated by A or G. Consequently, the level of RNA synthesis on a ribavirin-containing template is significantly reduced. These findings suggest that ribavirin not only is mutagenic but also interferes with HCV polymerase-mediated RNA synthesis.
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PMID:Mutagenic and inhibitory effects of ribavirin on hepatitis C virus RNA polymerase. 1295 Jan 73

Although the hepatitis C virus (HCV) genome is synthesized by the virus-encoded RNA-dependent RNA polymerase NS5B, other viral and cellular factors are assumed to be required for template-specific initiation and regulation of RNA-synthesis. The cellular protein La, which normally associates with RNA polymerase III transcripts, also interacts with the 5'- and 3'-untranslated regions of several RNA viruses, including HCV. To investigate whether other viral gene products may be involved in this interaction, we constructed an HCV cDNA expression library in bacteriophage T7 allowing portions of the HCV polyprotein to be displayed on the phage surface. Screening of the phage library against La resulted in selection of clones displaying the N-terminal region of HCV NS5A. Co-precipitation of full-length and truncated forms of recombinant NS5A with La revealed that the N-terminal region of NS5A was both necessary and sufficient for binding to La. Although this region of NS5A is essential for HCV replication, the role of the NS5A-La interaction in the infected cell remains to be established.
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PMID:Interaction of hepatitis C virus NS5A with La protein revealed by T7 phage display. 1296 47

Ribavirin (RBV), a guanosine analogue, has been suggested to exert an antiviral action against hepatitis C virus (HCV) by causing lethal mutations and suppressing RNA polymerase in vitro, but the mechanism of its clinical therapeutic effects is currently unknown. To test the hypothesis that RBV could act both as an RNA mutagen and inhibit viral RNA synthesis in vivo, we studied the evolution of the nucleotide sequences of HCV RNA at the nonstructural (NS) 5B region in patients receiving RBV, placebo, or interferon alfa (IFN-alpha) monotherapy. The RBV group showed a slightly more accelerated evolution rate of HCV RNA quasispecies than either the IFN-alpha or placebo group. RBV caused preferentially A-to-G and U-to-A mutations. Interestingly, an NS5B amino acid 415 Phe-to-Tyr (F415Y) mutation emerged in all (5 of 5) patients infected with HCV genotype 1a during the RBV treatment. Subsequently, the parental 415F strain reemerged in some patients after the treatment was discontinued. The effect of the amino acid substitution at NS5B415 on HCV RNA replication was then investigated using an HCV subgenomic replicon in Huh7 cells. We showed that treatment of replicon cells with RBV reduced the HCV RNA level of NS5B415F replicon, but not NS5B415Y, in a dose-dependent manner. Thus, NS5B F415Y mutation represents an RBV-resistant variant. The 3-dimensional modeling and structure analysis of NS5B protein revealed that the 415th amino acid is located at the P helix region of the thumb subdomain, which may interact with the minor groove of the template-primer duplex in the putative RNA-binding cleft. In conclusion, RBV could work as a weak mutagen for HCV RNA in HCV-infected patients. Furthermore, the selection of an RBV-resistant variant with a single amino acid substitution in NS5B suggested that RBV may directly interact with HCV RNA polymerase, thus interfering with its enzymatic activity.
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PMID:Identification of a ribavirin-resistant NS5B mutation of hepatitis C virus during ribavirin monotherapy. 1451 67

Ribavirin is a broad spectrum antiviral nucleoside that displays activity against a variety of RNA and DNA viruses. Ribavirin is currently used in combination with interferon-alpha for the treatment of hepatitis C virus (HCV) infection and was recently shown to be directly incorporated by the HCV RNA polymerase into RNA products. This capacity ultimately leads to increased mutation rates and drastically reduces the viral fitness. As a first step toward elucidating the nature of the specific interaction between ribavirin and the HCV polymerase, we have utilized fluorescence spectroscopy to monitor precisely the binding of ribavirin triphosphate (RTP) to the viral polymerase. This spectroscopic approach allowed us to clearly separate the RTP binding activity from the concomitant catalytic steps. We report here the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between RTP and an RNA polymerase. We demonstrate that RTP binds to the same active site as nucleotides. Furthermore, we provide evidence that the HCV polymerase cannot only bind to RTP but also to nonphosphorylated ribavirin, albeit with less affinity. By using various combinations of template-primers, we also demonstrate that base pairing is not involved in the initial binding of RTP to the HCV polymerase. Based on the results of circular dichroism and denaturation studies, we show that the RNA polymerase undergoes subtle conformational changes upon the binding of RTP, although the interaction does not significantly modify the stability of the protein. Finally, although metal ions are required for catalytic activity, they are not required for the initial binding of RTP to the polymerase. Such quantitative analyses are of primary importance for the rational design of new ribavirin analogues of potential therapeutic value and provide crucial insights on the interaction between RTP and the HCV RNA polymerase.
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PMID:Initial binding of the broad spectrum antiviral nucleoside ribavirin to the hepatitis C virus RNA polymerase. 1456 44

It has been suggested that cellular proteins are involved in hepatitis C virus (HCV) RNA replication. By using the yeast two-hybrid system, we isolated seven cDNA clones encoding proteins interacting with HCV RNA polymerase (NS5B) from a human liver cDNA library. For one of these, alpha-actinin, we confirmed the interaction by coimmunoprecipitation, immunofluorescent staining and confocal microscopic analysis. Experiments with deletion mutants showed that domains NS5B(84-95), NS5B(466-478), and alpha-actinin(621-733) are responsible for the interaction. Studies of the HCV subgenomic replicon system with small interference RNA indicate that alpha-actinin is essential for HCV RNA replication. Our results suggest alpha-actinin may be a component of the HCV replication complex.
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PMID:Direct interaction between alpha-actinin and hepatitis C virus NS5B. 1462 81

Recently, a benzo-1,2,4-thiadiazine was shown to be a potent, specific inhibitor of the hepatitis C virus (HCV) RNA polymerase [J. Biol. Chem. 277 (2002) 32327]. Herein, we present several lines of evidence to demonstrate that thiadiazine compound 4 (C(21)H(21)N(3)O(4)S) is highly synergistic with interferon-alpha (IFN-alpha) and disrupts HCV replicon RNA synthesis with a distinct kinetic profile. A time course analysis after a single treatment with 5 microM compound 4 showed a loss of viral RNA consistent with replicon RNA half-life, suggesting inhibition of 90% of ongoing or newly initiated replicative intermediates. This finding is consistent with the mechanism of action recently reported for compound 4, an RNA synthesis initiation inhibitor [J. Biol. Chem. 278 (2003) 16602]. Further, unlike IFN-alpha, an immediate reduction of HCV replicon RNA synthesis was apparent upon addition of compound 4. Treatment with IFN-alpha showed a delay of approximately 4h prior to inhibition of viral RNA replication, consistent with its signaling kinetics.
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PMID:Kinetic profile of a heterocyclic HCV replicon RNA synthesis inhibitor. 1462 24

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.
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PMID:Characterization of the inhibition of hepatitis C virus RNA replication by nonnucleosides. 1469 25


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