Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Hepatitis C virus is a positive-stranded RNA virus which is the causal agent for a chronic liver infection afflicting more than 170,000,000 people world-wide. The HCV genome is approximately 9.6 kb in length and the proteome encoded is a polyprotein of a little more than 3000 amino acid residues. This polyprotein is processed by a combination of host and viral proteases into structural and non-structural proteins. The functions of most of these proteins have been established by analogy to other viruses and by sequence homology to known proteins, as well as subsequent biochemical analysis. Two of the non-structural proteins, NS4b and NS5a, are still of unknown function. The development of antivirals for this infectious agent has been hampered by the lack of robust and economical cell culture and animal infection systems. Recent progress in the molecular virology of HCV has come about due to the definition of molecular clones, which are infectious in the chimpanzee, the development of a subgenomic replicon system in Huh7 cells, and the description of a transgenic mouse model for HCV infection. Recent progress in the structural biology of the virus has led to the determination of high resolution three-dimensional structures of a number of the key virally encoded enzymes, including the NS3 protease, NS3 helicase, and NS5b RNA-dependent RNA polymerase. In some cases these structures have been determined in complex with substrates, co-factors (NS4a), and inhibitors. Finally, a variety of techniques have been used to define host factors, which may be required for HCV replication, although this work is just beginning.
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PMID:Recent advances in the molecular biology of hepatitis C virus. 1167 30

The structure of the RNA-dependent RNA polymerase (RdRP) from the rabbit hemorrhagic disease virus has been determined by x-ray crystallography to a 2.5-A resolution. The overall structure resembles a "right hand," as seen before in other polymerases, including the RdRPs of polio virus and hepatitis C virus. Two copies of the polymerase are present in the asymmetric unit of the crystal, revealing active and inactive conformations within the same crystal form. The fingers and palm domains form a relatively rigid unit, but the thumb domain can adopt either "closed" or "open" conformations differing by a rigid body rotation of approximately 8 degrees. Metal ions bind at different positions in the two conformations and suggest how structural changes may be important to enzymatic function in RdRPs. Comparisons between the structures of the alternate conformational states of rabbit hemorrhagic disease virus RdRP and the structures of RdRPs from hepatitis C virus and polio virus suggest novel structure-function relationships in this medically important class of enzymes.
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PMID:Crystal structures of active and inactive conformations of a caliciviral RNA-dependent RNA polymerase. 1167 45

Hepatitis C virus (HCV) NS5B is RNA-dependent RNA polymerase (RdRP), the essential catalytic enzyme for HCV replication. Recently, NS5A has been reported to be important for the establishment of HCV replication in vitro by the adaptive mutations, although its role in viral replication remains uncertain. Here we report that purified bacterial recombinant NS5A and NS5B directly interact with each other in vitro, detected by glutathione S-transferase (GST) pull-down assay. Furthermore, complex formation of these proteins transiently coexpressed in mammalian cells was detected by coprecipitation. Using terminally and internally truncated NS5A, two discontinuous regions of NS5A (amino acids 105-162 and 277-334) outside of the adaptive mutations were identified to be independently essential for the binding both in vivo and in vitro (Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K., and Mkyrakami, S. (1998) J. Biol. Chem. 273, 15479-15486). We previously examined the effect of His-NS5A on RdRP activity of the soluble recombinant NS5Bt in vitro (see Yamashita et al. above). Wild NS5A weakly stimulated at first (when less than 0.1 molar ratio to NS5B) and then inhibited the NS5Bt RdRP activity in a dose-dependent manner. The internal deletion mutants defective in NS5B binding exhibited no inhibitory effect, indicating that the NS5B binding is necessary for the inhibition. Taken together, our results support the idea that NS5A modulates HCV replication as a component of replication complex.
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PMID:Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity. 1180 99

The biochemical properties of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) truncated with C-terminal 21 amino acids and expressed in insect cells were analyzed. The enzyme carried copy-back and de novo RNA synthesis activity but not terminal nucleotidyl transferase activity. k(pol) and K(m) for de novo RNA synthesis were calculated as 10.0 pmol/microg/h and 2.5 microM under 0.5 mM GTP and 2.0 pmol/microg/h and 3.5 microM under 50 microM GTP, respectively. Those for copy-back RNA synthesis were similar under both conditions (k(pol), 1.8 pmol/microg/h; K(m), 3.0 microM). De novo RNA synthesis was activated by 0.5 mM GTP. However, the ratio of GTP to three other NTPs was important for activation. Our HCV RdRp showed high activity for the complementary sequence of the HCV internal ribosomal entry site and a synergistic effect of Mg(2+) to Mn(2+).
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PMID:Kinetic analysis of C-terminally truncated RNA-dependent RNA polymerase of hepatitis C virus. 1181 88

AIM:To explore the status of extrahepatic hepatitis C virus (HCV) infection and replication in hepatitis C patients,and its potential implication in HCV infection and pathogenicity.METHODS:By reverse-transcriptase poly-merase chain reaction (RT-PCR),in situ hybridization (ISH) and immunohis-tochemistry, HCV RNA, HCV replicative intermediate (minus-strand of HCV RNA), and HCV antigens were detected in 38 autopsy extrahepatic tissue specimens (including 9 kidneys, 9 hearts, 9 pancreas, 5 intestines, 2 adrenal glands, 2 spleens, 1 lymph node, and 1 gallbladder) from 9 hepatitis C patients, respectively; and the status of HCV replication in extrahepatic tissues was studied.RESULTS:By RT-PCR, all 9 patients were positive for HCV RNA in kidney, heart, pancreas, and intestine, but only 6(66.7%) patients were positive for HCV replicative intermediate. HCV RNA and HCV antigens were detected in kidney, heart, pancreas, intestine, adrenal gland, lymph node, and gallbladder in 5(55.6%) and 6(66.7%) patients by ISH and immuno-histochemistry, respectively. HCV RNA and HCV antigens were not detected in these extrahepatic organs in 3(33.3%) patients, although their livers were positive for HCV.HCV replicative intermediate detected by RT-PCR was consistent with HCV RNA and HCV antigens detected by ISH and immunohistochemistry (Kappa =0.42-0.75). HCV RNA and HCV antigens were detected in myocardial cells, epithelial cells of intestinal gladular, interstitial cells of kidney, epithelial cells of tubules and glomerulus, pancreas acinar cells and epithelial cells of pancreatic duct, epithelial cells of mucous membrane sinus of gallbladder, cortex and medulla cells in adrenal gland,and mononuclear cells in lymph node. HCV RNA was also detected in bile duct epithelial cells, sinusoidal cells, and mononuclear cells in liver tissues by ISH.CONCLUSION:HCV can infect extrahepatic tissues, and many various tissue cells may support HCV replication; extrahepatic HCV infection and replication may be of concomitant state in most of patients with hepatitis C. The infected extrahepatic tissues might act as a reservoir for HCV, and play a role in both HCV persistence and reactivation of infection. HCV as an etiologic agent replicating and expressing viral proteins in extrahepatic tissues itself contributes to extrahepatic syndrome associated-HCV infection in a few patients with chronic HCV infection.
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PMID:Hepatitis C virus may infect extrahepatic tissues in patients with hepatitis C. 1181

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is responsible for replication of genomic RNA. A novel nonisotopic assay method is described for detecting its enzymatic activity. The 5' end of the in vitro-transcribed template RNA was attached covalently to the surface of a Covalink module using carbodiimide condensation. The RNA strand containing the 3' untranslated region (3' UTR) of HCV at its 3' end was free in the solution. A purified NS5B polymerase and NTPs along with biotin-labeled UTP were added to this module and the polymerization activity could be detected colorimetrically with streptavidin-conjugated alkaline phosphatase.
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PMID:A nonisotopic assay method for hepatitis C virus NS5B polymerase. 1184

The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K(d), of approximately 22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 A. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.
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PMID:Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase. 1190 26

Hepatitis C virus (HCV) NS5B protein has been shown to have RNA-dependent RNA polymerase (RdRp) activity by itself and is a key enzyme involved in viral replication. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that human eukaryotic initiation factor 4AII (heIF4AII), which is a component of the eIF4F complex and RNA-dependent ATPase/helicase, interacted with NS5B protein. These two proteins were shown to be partially colocalized in the perinuclear region. The binding site in HCV NS5B protein was localized within amino acid residues 495 to 537 near the C terminus. Since eIF4A has a helicase activity and functions in a bidirectional manner, the binding of HCV NS5B protein to heIF4AII raises the possibility that heIF4AII facilitates the genomic RNA synthesis of NS5B protein by unwinding the secondary structure of the HCV genome and is a host component of viral replication complex.
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PMID:Human eukaryotic initiation factor 4AII associates with hepatitis C virus NS5B protein in vitro. 1192 17

Hepatitis C virus (HCV) NS5B has been shown to exhibit RNA-dependent RNA polymerase activity for its viral RNA replication. In this study, we demonstrated the formation of a complex between NS5B and the core protein (NS5B-core protein complex) in mammalian cells, as determined by indirect immunofluorescence and immunoprecipitation analyses. The localization of the core protein was observed to change to the same locus in ER as NS5B locates by its coexpression with NS5B, indicating that the localization of the core protein is determined by NS5B. The truncated NS5B molecule lacking the C-terminal region did not form a complex with the core protein, suggesting that the C-terminal region of NS5B is essential for its interaction with the core protein. Moreover, the change in NS5B localization because of C-terminal deletion indicates that this region includes a certain signal for NS5B retention in ER.
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PMID:Hepatitis C virus core protein binds to a C-terminal region of NS5B RNA polymerase. 1192 15

Continuous efforts are vital to develop new treatment strategies to improve sustained response rates, especially for difficult to treat patients infected with the hepatitis C virus. Despite the introduction of ribavirin, more than 50% of the patients do not eliminate the virus with the current standard therapy of interferon-a (IFN) and ribavirin. Options to further enhance response rates include modification of the IFN-dosing schedule with daily dosing of IFN, new IFN such as consensus interferon or modified IFN with longer half-life and more favourable pharmacokinetics such as pegylated IFN (PEG-IFN). Clinical trials with new IFN showed that consensus IFN may improve response rates in unsuccessfully pre-treated patients and patients with HCV-genotype-1. Treatments with PEG-IFN will double response rates achieved with standard IFN monotherapy. The combination of PEG-IFN and ribavirin improves the virological response to more than 50% and even to more than 80% in patients with genotype 2 or 3. By now, standard therapy of chronic hepatitis C has been changed to the combination of PEG-IFN plus ribavirin. Future anti-viral drugs may comprise molecules that directly inhibit HCV proteins and interfere with viral replication. NS3/4A serine protease, ribonucleic acid (RNA) helicase, RNA-dependent RNA polymerase may be potential targets for new drugs. Furthermore antisense oligonucleotides or ribozymes may become new treatment options to inhibit HCV replication. Finally, immunotherapies to enhance HCV-specific immune responses are also attractive strategies to control HCV infection and to prevent chronic liver disease.
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PMID:Hepatitis C: therapeutic perspectives. 1194 60


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