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)

An in vitro system that supports the efficient growth of hepatitis C virus (HCV) and reflects its complete in vitro replication cycle has not yet been established. The establishment of a minigene RNA of HCV in mammalian cells could facilitate the study of virus-cell interactions and the molecular pathogenesis of this virus. We constructed a replication-deficient recombinant adenovirus expressing bacteriophage T7 RNA polymerase under the control of CAG promoter (AdexCAT7). A high level of T7 RNA polymerase was detectable for at least 11 days after inoculation. Cells infected with AdexCAT7 were then transfected with plasmids carrying the authentic T7 promoter, the 5' untranslated region (UTR) of encephalomyocarditis virus, a luciferase gene, and a T7 terminator (pT7EMCVLuc) or carrying the modified T7 promoter, the 5'UTR of HCV, a luciferase gene, the coding region of C-terminal of NS5B and the 3'UTR of HCV, a ribozyme of hepatitis D virus and a T7 terminator (pT7HCVLuc). Most of the cell lines examined supported a higher expression of luciferase by transfection with pT7EMCVLuc than with pT7HCVLuc. However, one cell line, FLC4, derived from a human hepatocellular carcinoma, exhibited very high reporter gene expression with pT7HCVLuc. In this cell line, transfection with RNA synthesized in vitro from pT7HCVLuc induced a higher level of reporter gene expression than RNA from pT7EMCVLuc. The T7-adenovirus system for the synthesis of HCV minigenes in vivo provides useful information on the molecular mechanisms of HCV translation in human liver cells.
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PMID:A human liver cell line exhibits efficient translation of HCV RNAs produced by a recombinant adenovirus expressing T7 RNA polymerase. 977 Apr 28

Although hepatitis C virus (HCV) mainly affects hepatocytes, infection is widespread and involves immunologically privileged sites. Whether lymphoid cells represent further targets of early HCV infection, or whether other cells in the hematopoietic microenvironment may serve as a potential virus reservoir, is still unclear. We studied whether pluripotent hematopoietic CD34(+) cells support productive HCV infection and can be used to establish an in vitro infection system for HCV. Six patients were selected as part of a cohort of HCV chronic carriers who developed a neoplastic disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA signal amplification assays were used to detect and quantitate HCV RNA in extracted nucleic acids from purified bone marrow and peripheral blood CD34(+) cells. Direct in situ RT-PCR, flow cytometry analysis, and immunocytochemistry were applied to demonstrate specific viral genomic sequences and structural and nonstructural virus-related proteins in intact cells. Results indicated that both positive and negative HCV RNA strands and viral proteins were present in CD34(+) cells from all HCV-positive patients and in none of the controls. Additional experiments showed that a complete viral cycle took place in CD34(+) cells in vitro. Spontaneous increases in viral titers indicated that virions were produced by infected hematopoietic progenitor cells. To further define the cellular tropism, we attempted to infect CD34(+) cells in vitro. We were unable to demonstrate viral uptake by cells. These findings suggest that HCV replication can occur in the early differentiation stages of hematopoietic progenitor cells, and that they may be an important source of virus production.
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PMID:Hepatitis C virus infection involves CD34(+) hematopoietic progenitor cells in hepatitis C virus chronic carriers. 978 70

The outcome of hepatitis C virus (HCV) infection on patient and graft survival after orthotopic liver transplantation (OLT) has been controversial. An earlier experience with a higher dose of tacrolimus (>/=0.1 mg/kg/d intravenously and >/=0.2 mg/kg/d orally) was associated with a worse clinical outcome in patients infected with HCV. The clinical outcome of 183 liver transplant recipients with end-stage liver disease (ESLD) secondary to HCV infection (HCV group) was compared with a contemporary cohort of 556 patients with HCV infection who underwent transplantation for nonviral, nonmalignant ESLD (control group). All patients were prospectively screened for anti-HCV antibodies and HCV RNA by reverse-transcriptase polymerase chain reaction. All OLT patients were receiving low-dose tacrolimus immunosuppression. Cumulative patient survival rates for the HCV group were 80% after 1 year and 75% after 3 years compared with rates of 84% and 78%, respectively, in the control group (P = .452). Primary graft survival rates at the same time intervals for the HCV group and the control group were 72% and 77.5% at 1 year and 67% and 72% at 3 years, respectively (P = .144). The incidence of re-transplantation (re-OLT) in the HCV group and the control group was 12.6% and 10.4%, respectively (P = .42). Chronic HCV infection as an indication for OLT with a lower dose of tacrolimus immunosuppression (</=0.05 mg/kg/d intravenously and </=0.1 mg/kg/d orally) is associated with a similar patient and graft survival as those without HCV infection.
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PMID:Clinical outcome of patients infected with hepatitis C virus infection on survival after primary liver transplantation under tacrolimus. 979 Nov 54

The existence of extrahepatic reservoirs of hepatitis C virus (HCV) replication remains highly controversial. We searched for the presence of HCV-RNA negative strand in various tissues from eight HCV-infected patients who died of acquired immunodeficiency syndrome (AIDS)-related complications. Negative-strand RNA was detected by a Tth-based reverse-transcriptase polymerase chain reaction (RT-PCR), which was optimized for sensitivity and strand specificity on synthetic RNA templates. This assay was capable of detecting about 10(2) genomic Eq molecules of the correct strand while unspecifically detecting >/=10(8) genomic Eq molecules of the incorrect strand. Negative-strand viral RNA was detected in all but one liver, in lymph nodes (5 cases), in pancreas (5 cases), in adrenal gland (2 cases), in thyroid (2 cases), in bone marrow (1 case), and in spleen (1 case). These data suggest a possible presence of HCV replication sites outside the liver, at least in AIDS patients. Whether these findings relate to various extrahepatic manifestations of HCV infection remains to be determined.
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PMID:Search for hepatitis C virus extrahepatic replication sites in patients with acquired immunodeficiency syndrome: specific detection of negative-strand viral RNA in various tissues. 979 27

In previous studies employing interferons (IFNs) in the treatment of chronic hepatitis C, there have been few reliable predictors of sustained responses. We retrospectively evaluated the predictive value of hepatitis C virus (HCV)-RNA measurements in the first few months during consensus interferon (CIFN) treatment using a sensitive reverse-transcriptase polymerase chain reaction assay to determine sustained responses. Data from two large treatment trials, one of IFN-naive patients and one of retreated relapsers and nonresponders, were used, including serum samples at 2-week intervals in the naive study and 8-week intervals in the retreatment study. Patients received initial CIFN (9 microgram) treatment for 6 months and were assessed 6 months after treatment. There were 28 sustained viral responders of 232 CIFN-treated patients. Of the sustained responders, 48% had already cleared HCV RNA from serum (<100 copies/mL) by week 2, 78% by week 4, 81% by week 6, and 96% by week 12. Patients with early HCV-RNA clearance were more likely to have sustained responses than those who responded later. Early clearance of HCV from serum was also associated with greater likelihood of a sustained response to 48 weeks of retreatment with 15 microgram CIFN. Ninety-five percent of the sustained responders were HCV-RNA-negative by week 8 of retreatment. Early assessment of HCV RNA may help in the prediction of sustained responses to IFN and allow the value of continued treatment to be determined early in the course of IFN therapy.
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PMID:Early hepatitis C virus-RNA responses predict interferon treatment outcomes in chronic hepatitis C. The Consensus Interferon Study Group. 979 29

The transmission of perinatal hepatitis C virus (HCV) infection was studied retrospectively in 62 infants born to 54 HCV- and human immunodeficiency virus (HIV)-coinfected women enrolled in a prospective natural history study of HIV transmission. Infant HCV infection was assessed by nested RNA polymerase chain reaction. The overall rate of vertical HCV transmission was 16.4% (9/62). Most HCV-infected children did not develop antibodies to HCV. The rate of HCV infection was higher among HIV-infected infants (40%) than among HIV-uninfected infants (7.5%; odds ratio, 8.2; P = .009). This difference in transmission was not related to differences in maternal HCV load, as measured by branched DNA assay, or mode of delivery. Why HIV-infected infants of HCV- and HIV-coinfected women have significantly higher rates of perinatal HCV transmission remains to be elucidated. The rate of HCV transmission in HIV-uninfected infants of HCV- and HIV-coinfected women is similar to that reported for infants born to HIV-seronegative mothers.
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PMID:Increased transmission of vertical hepatitis C virus (HCV) infection to human immunodeficiency virus (HIV)-infected infants of HIV- and HCV-coinfected women. 980 33

Studies aimed at correlating the intrahepatic hepatitis C virus (HCV)-RNA level and anatomo-clinical features have been difficult because of sensitivity and specificity shortcomings of available techniques. We titered the genomic- and minus-strand HCV RNAs by a strand-specific, semiquantitative, genotype-independent reverse-transcriptase polymerase chain reaction (RT-PCR) in the liver tissue of 61 patients with chronic hepatitis C. Findings were correlated with the levels of HCV RNA in the serum, the HCV genotype, the expression of intrahepatic HCV antigens, the histological activity (using separate scores for the lobular and the portal/periportal necroinflammatory activity and for the fibrosis), and the response to interferon alfa (IFN-alpha) treatment. Genomic- and minus-strand HCV RNA were detected in 59 and 57 liver specimens, respectively. The HCV-RNA level in the serum correlated with the genomic-strand, but not with the minus-strand, HCV-RNA titer in the liver. No correlations were found between either strand of the intrahepatic HCV RNA and the level of expression of HCV antigens in the liver, or with the grading/staging of the underlying liver disease. The response to IFN-alpha treatment could be predicted by the serum HCV-RNA level and genotype, but not by the intrahepatic level of genomic- or minus-strand HCV RNA. These results suggest that, although the detection of the minus-strand HCV RNA reliably identifies the presence of replicating HCV in its target organ, the quantitative measurement of viremia remains the clinically meaningful "golden standard" for assessing the level of HCV replication.
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PMID:Detection of genomic- and minus-strand of hepatitis C virus RNA in the liver of chronic hepatitis C patients by strand-specific semiquantitative reverse-transcriptase polymerase chain reaction. 991 32

The correlation between 3 assays for hepatitis C virus (HCV) RNA quantification and their respective accuracy in predicting the response to interferon and interferon/ribavirin therapy was evaluated by analysing pre-treatment sera from 100 patients. A total of 97%, 100%, and 98% of the patients tested positive by the branched DNA 2.0 assay (Quantiplex), a multi-cycle reversed transcriptase polymerase chain reaction quantitative assay (Superquant) and the Roche Amplicor Monitor assay, respectively. The correlations between the assays, in all patients and in the major genotypes 1, 2, and 3, were significant, although the levels detected by the Amplicor Monitor assay were more than 1 log lower than by the other assays. Sustained virological responders to interferon therapy, but not to combination therapy, had lower baseline viral levels than long-term non-responders (p = 0.002 by Quantiplex 2.0; p = 0.008 by Superquant; p = 0.06 by Roche Amplicor Monitor). Pre-treatment viral load greater than 3 x 10(6) Eq or copies/ml by the Quantiplex 2.0 and Superquant assays and greater than 100,000 copies/ml by the Amplicor Monitor assay predicted long-term non-response in 94%, 93% and 91% of the interferon treated patients, respectively. In conclusion, acceptable correlations between available commercial quantitative assays were found. High baseline viral load predicted long-term non-response to interferon monotherapy, whereas it did not to interferon/ribavirin combination therapy.
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PMID:Comparison of 3 quantitative HCV RNA assays--accuracy of baseline viral load to predict treatment outcome in chronic hepatitis C. 1006 40

NS5B of the hepatitis C virus is an RNA template-dependent RNA polymerase and therefore the key player of the viral replicase complex. Using a highly purified enzyme expressed with recombinant baculoviruses in insect cells, we demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or GMP. Enhancement of RNA synthesis was found with various heteropolymeric RNA templates, with poly(C)-oligo(G)12 but not with poly(A)-oligo(U)12. Several amino acid substitutions in polymerase motifs B, C, and D previously shown to be crucial for RdRp activity were tested for GTP stimulation of RNA synthesis. Most of these mutations, in particular those affecting the GDD motif (motif C) strongly reduced or completely abolished activation by GTP, suggesting that the same NTP-binding site is used for stimulation and RNA synthesis. Since GTP did not affect the overall RNA binding properties or the elongation rate, high concentrations of GTP appear to accelerate a rate-limiting step at the level of initiation of RNA synthesis. Finally, enhancement of RNA synthesis by high GTP concentrations was also found with NS5B of the pestivirus classical swine fever virus, but not with the 3D polymerase of poliovirus. Thus, stimulation of RdRp activity by GTP is evolutionarily conserved between the closely related hepaciviruses and pestiviruses but not between these and the more distantly related picornaviruses.
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PMID:Selective stimulation of hepatitis C virus and pestivirus NS5B RNA polymerase activity by GTP. 1019 56

Numerous investigators have postulated that one mechanism by which hepatitis C virus (HCV) may evade the immune system is through the formation of escape mutants. This hypothesis is based largely on the observed mutability of the viral genome resulting in evolution of diverse quasispecies over the course of infection. That such diversification is a product of viral RNA polymerase infidelity, immune-driven selection or a combination of the two processes has not been addressed. We have examined sequence variability in a specific segment of HCV RNA encoding a known immunodominant region of the viral helicase, amino acids 358-375 of the non-structural 3 protein. Using sequence-specific oligonucleotide probe hybridization and automated DNA sequencing, we report a high frequency of mutations, essentially all of which result in amino acid replacements. To assess the biological impact of such mutations, corresponding chemically synthesized peptides were compared to wild-type peptide in T cell proliferation assays. We observed that a sizeable fraction of such peptides stimulated attenuated or negligible levels of proliferation by peripheral T cells from a chronically infected patient. This observation is consistent with expectations for immune-mediated selection of escape variants at the epitope level. We postulate that such a mechanism may be important in the immunopathogenesis of HCV infections.
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PMID:Identification of antigenic escape variants in an immunodominant epitope of hepatitis C virus. 1032 11


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