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 rapidly emerging and sometimes complicated field of HCV diagnostics can be simplified by classification of tests into two general categories: serologic tests which screen for anti-HCV antibodies, and molecular tests which are used to assess HCV viremia and characterize viral infection at the genetic level. Antibody tests include the highly sensitive screening enzyme immunoassays (current versions: EIA-2 and EIA-3), and supplemental tests such as the recombinant immunoblot assay (RIBA-2). Molecular assays such as HCV RNA polymerase chain reaction (PCR) may play an important role in confirming HCV infection in several clinical situations, such as immunosuppressed patients with chronic hepatitis C, patients with acute hepatitis who might be in the diagnostic "window" period prior to seroconversion, and seropositive patients with normal ALT values. Quantitative HCV-RNA tests, such as quantitative PCR (Q-PCR) and branched DNA (bDNA), provide valuable tools for assessing the level of HCV viremia prior to and during therapy. Genotype tests allow classification of HCV infection in one of six distinct HCV genotypes, although the clinical relevance of HCV genotype tests has not been established.
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PMID:Use and interpretation of HCV diagnostic tests in the clinical setting. 1556 57

It is unclear whether the current antiviral treatment for chronic hepatitis C virus (HCV) infection results in complete elimination of the virus, or whether small quantities of virus persist. Our study group comprised 17 patients with chronic HCV who had sustained virological response (SVR) after interferon/ribavirin treatment. Serum and peripheral blood mononudear cells were collected 2 to 3 times at 3- to 6-month intervals starting 40 to 109 months (mean, 64.2 +/- 18.5 months) after the end of therapy. In addition, lymphocyte and macrophage cultures were established at each point. In 11 patients, frozen liver tissue samples were available from follow-up biopsies performed 41 to 98 months (mean, 63.6 +/- 16.7 months) after therapy. Presence of HCV RNA was determined by sensitive reverse-transcriptase polymerase chain reaction, and concentration of positive and negative strands was determined by a novel quantitative real-time reverse transcriptase polymerase chain reaction. Only 2 of 17 patients remained consistently HCV RNA negative in all analyzed compartments. HCV RNA was detected in macrophages from 11 patients (65%) and in lymphocytes from 7 patients (41%). Viral sequences were also detected in 3 of 11 livers and in sera from 4 patients. Viral replicative forms were found in lymphocytes from 2 and in macrophages from 4 patients. In conclusion, our results suggest that in patients with SVR after therapy, small quantities of HCV RNA may persist in liver or macrophages and lymphocytes for up to 9 years. This continuous viral presence could result in persistence of humoral and cellular immunity for many years after therapy and could present a potential risk for infection reactivation.
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PMID:Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C. 1594 Jun 51

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

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 hepatitis B virus (HBV), as a major cause of acute and chronic hepatitis in humans, contains a partial double-stranded circular DNA genome of 3.2kb that is transcribed into the 3.5-, 2.4-, 2.1-, and 0.7-kb viral transcripts by the host RNA polymerase II. The HBV X (HBx) gene is consistently expressed in all four HBV viral mRNAs and thus an ideal target for developing viral inhibitors via a gene therapeutic approach. In this study, we show that two HBx-specific small interfering RNAs (siRNA), HBx1 and HBx3, significantly decrease both viral RNA and protein levels, and completely block replication in cultured cells co-transfected with a siRNA expression plasmid and an HBV replication-competent vector. To further confirm these antiviral activities of selected siRNAs in small animals, we established acute and chronic HBV mouse models by hydrodynamic injection of this plasmid containing the full-length HBV genome. Selected HBx-specific siRNAs also induced a significant anti-viral effect in living animals. Our findings should facilitate the development of an alternative therapeutic agent against HBV infection, particularly HBV-derived hepatocellular carcinoma (HCC) in which HBx has been known as one of the major pathological factors.
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PMID:Efficient inhibition of hepatitis B virus replication by small interfering RNAs targeted to the viral X gene in mice. 1644 3

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis. A substantial proportion of patients with chronic hepatitis C eventually develop hepatocellular carcinoma (HCC), which is one of the leading causes of death worldwide. Therefore, efficient antiviral treatments for HCV have long been needed. A recently developed combination therapy of pegylated interferon and ribavirin has dramatically improved the outcome of antiviral therapy for HCV infection. In genotype 1b HCV infection, 48 weeks of the combination therapy achieved eradication of the virus in 50% of patients, and in genotype 2 HCV infection, 24 weeks of the therapy resulted in viral eradication in 80%-90% of patients. By this eradication, an improvement in the hepatic fibrosis, an inhibition of HCC development, and an improvement in life expectancy were attained. Patients who did not respond to the combination therapy may be treated with long-term interferon monotherapy, which is not intended to eradicate HCV, but will lower the serum alanine aminotransferase (ALT) level. Thus, the treatment for HCV infection has progressed significantly, but therapies with new modalities, such as inhibitors of viral protease or RNA polymerase, are still being awaited.
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PMID:Antiviral treatment of hepatitis C: present status and future prospects. 1710 84

Vascular endothelial growth factor (VEGF) is involved in both development and progression of several epithelial tumours, but its role in hepatocellular carcinoma (HCC) is unclear. Assessment of liver and blood levels of VEGF may provide further insights on angiogenesis in HCC. Tissue mRNA of VEGF-165, VEGF-189 and their receptor KDR was assessed by a semi-quantitative retro-transcriptase polymerase chain reaction, and expressed as target transcript/beta-actin ratio, in 29 patients with HCC, 26 with cirrhosis and 15 with chronic hepatitis. VEGF-165 was also measured by ELISA in plasma samples obtained from both hepatic and femoral veins in additional 58 patients, including 15 with HCC. The liver expression of mRNA of VEGF-165, VEGF-189 and KDR was higher in HCC than in chronic liver diseases (1.54 +/- 0.89 vs 0.62 +/- 0.47, P < 0.0001; 1.09 +/- 0.65 vs 0.64 +/- 0.54, P = 0.003; 1.30 +/- 1.09 vs 0.69 +/- 0.72, P = 0.014). VEGF-165 was higher in HCC tissue than in extra-tumoural tissues (1.44 +/- 0.31 vs 1.03 +/- 0.21, P = 0.0009) and in the cirrhotic tissue of HCC patients than in HCC-free cirrhosis (1.03 +/- 0.23 vs 0.45 +/- 0.45, P = 0.0002). Tissue VEGF-189 mRNA inversely correlated with tumour size and degree of tumour cell proliferation. The hepatic and femoral vein levels of VEGF-165 protein were significantly higher in HCC patients than in cirrhotic patients (66.7 +/- 57.1 vs 24.2 +/- 16.4 pg/mL, P = 0.0001 and 37.1 +/- 42.2 vs 13.5 +/- 9.6 pg/mL, P = 0.001). There was a gradient of VEGF-165 between hepatic and femoral veins in both HCC and cirrhosis. In conclusion, VEGF appears to be involved in the development of HCC and it could be a predictor of HCC development in patients with cirrhosis.
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PMID:Increased expression of vascular endothelial growth factor in small hepatocellular carcinoma. 1724 53

Hepatitis C virus (HCV) is a major causative agent of liver diseases such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Because the current standard therapy, interferon (IFN) or pegylated-IFN alone or in combination with ribavirin, is ineffective on approximately half of the HCV-infected patients, alternative therapeutics are greatly needed. The chemical genetics method is a useful strategy to elucidate molecular mechanisms of the viral life cycle and screen for anti-viral agents. This review focuses on the use of chemical genetics approach to virology, which could be called 'chemical virology', and introduces an example of such analysis. From a cell culture-based screening, an immunosuppressant cyclosporin A (CsA) was identified as an anti-HCV compound. Analysis using CsA as a bioprobe showed that cyclophilin (CyP) B, a cellular target of CsA, regulates the function of HCV RNA polymerase NS5B, which is essential for efficient viral genome replication. By targeting CyP, HCV genome replication was drastically suppressed. Thus, chemical genetics analysis identified CyPB as a cellular cofactor of HCV genome replication and a target for novel anti-HCV agents.
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PMID:Chemical genetics approach to hepatitis C virus replication: cyclophilin as a target for anti-hepatitis C virus strategy. 1729 3

Development of therapeutics for chronic hepatitis C has been hampered by the lack of an efficient cell culture system and a small animal model for the hepatitis C virus (HCV). An RNA replicon system, in which the HCV genome replicates autonomously in cells, and replication competent viruses derived from an HCV genotype 2a JFH1 strain efficiently propagating in Huh7 cells have been developed, and these systems have contributed to the evaluation of anti-HCV drugs targeted to viral and host proteins involved in the replication of HCV. Several compounds counteracting the viral enzymes, such as RNA polymerase and proteases, and host proteins involved in the lipid synthesis and protein folding are reported to have anti-HCV activities based on assessments using these in vitro systems. Furthermore, a mouse model transplanted with human liver fragments was shown to be capable of replicating HCV and has been used to evaluate the efficacy of antiviral drugs in vivo. In this review, we summarize information regarding systems for studying the HCV life cycle and potential new targets for therapeutic intervention for chronic hepatitis C.
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PMID:Evaluation systems for anti-HCV drugs. 1772 Feb 75

Treatment of chronic hepatitis B virus (HBV) infection is aimed at suppressing viral replication to the lowest possible level, and thereby to halt the progression of liver disease and prevent the onset of complications. Two categories of drugs are used in HBV therapy: the interferons, including standard interferon alfa or pegylated interferon alfa, and specific nucleoside or nucleotide HBV inhibitors that target the reverse-transcriptase function of HBV-DNA polymerase. The reported results of clinical trials have used varying definitions of efficacy, failure, and resistance based on different measures of virologic responses. This article discusses HBV virologic markers and tests, and their optimal use both for planning and reporting clinical trials and in clinical practice.
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PMID:Virologic monitoring of hepatitis B virus therapy in clinical trials and practice: recommendations for a standardized approach. 1824 9


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