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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our research is directed towards enhancing the understanding of the molecular biology of dengue virus replication with the ultimate goal being to develop novel antiviral strategies based on preventing critical inter- or intra-molecular interactions required for the normal virus life cycle. The viral RNA-dependent RNA polymerase (NS5) and the viral helicase (NS3) interaction offers a possible target for inhibitors to bind and prevent replication. In this study the yeast-two hybrid system was used to show that a small region of NS5 interacts with NS3, and also with the cellular nuclear transport receptor importin-beta. Furthermore, intramolecular interaction between the two putative domains of NS5 can also be detected by the yeast two-hybrid assay. We have also modified the colony lift assay for the beta-galactosidase reporter activity in intact yeast cells which reflects the strength of interaction between two proteins to a microtiter plate format. This assay offers a unique opportunity to screen for small molecule compounds that block physiologically important interactions.
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PMID:Characterisation of inter- and intra-molecular interactions of the dengue virus RNA dependent RNA polymerase as potential drug targets. 1134 63

Field male Aedes aegypti (L.) and Aedes albopictus (Skuse) adults caught from fixed monitoring stations weekly for 1 yr were screened for dengue viruses (DEN-1, DEN-2, DEN-3, and DEN-4). The assay was carried out using a single-step reverse transcription (or transcriptase)-polymerase chain reaction (PCR) (RT-PCR) followed by a semi-nested PCR using an upstream consensus primer and four type-specific primers within the nonstructural protein three gene (NS3) of dengue viruses. The diagnostic fragments for DEN-1, DEN-2, DEN-3, and DEN-4 serotypes were of sizes 169, 362, 265, and 426 bp, respectively. Results showed that in Singapore 1.33% and 2.15% of Aedes aegypti and Aedes albopictus adult male mosquitoes, respectively, were positive for dengue viruses. The serotypes detected in male Ae. aegypti was DEN-1 (44%), followed by DEN-2 (22.2%) and DEN-3 (22.2%), and DEN-4 (11.1%). For Aedes albopictus males, the serotype was DEN-4 (38.9%), followed by DEN-2 (33.3%), DEN-3 (16.7%), and DEN-1 (11.1%).
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PMID:Detection of dengue viruses in field caught male Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Singapore by type-specific PCR. 1147 26

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

We recently developed a model for flavivirus infection in mice and hamsters using the Modoc virus (MODV), a flavivirus with no known vector (P. Leyssen, A. Van Lommel, C. Drosten, H. Schmitz, E. De Clercq, and J. Neyts, 2001, Virology 279, 27-37). We now present the coding and noncoding sequence of MODV. The Modoc virus genome was determined to be 10,600 nucleotides in length with a single open reading frame extending from nucleotides 110 to 10,234, encoding 3374 amino acids. The deduced gene order of the single open reading frame is C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, which is exactly the same as that of the mosquito- and tick-borne flaviviruses. It is flanked by a 5'- and 3'-untranslated region (UTR) of 109 and 366 nucleotides, respectively. Alignment of the MODV amino acid sequence with that of 20 other flaviviruses revealed several regions with high sequence similarity corresponding to functionally important domains (e.g., the serine protease/helicase/NTPase of NS3 and the methyltransferase/RNA-dependent RNA polymerase of NS5) and conserved sites for proteolytic cleavage by viral and cellular proteases. Phylogenetic analysis of the entire coding region confirmed the classification of MODV within the flaviviruses with no known vector, which is in agreement with previous findings based on partial NS5 sequences. A detailed comparative analysis of the putative folding patterns of the 5'- and 3'-UTR of MODV and of the tick- and mosquito-borne viruses was carried out. Structural elements in the 5'- and 3' UTR of MODV that are preserved among vector-borne flaviviruses were noted and so were structural elements distinguishing the MODV UTRs from mosquito-borne and tick-borne flaviviruses. Also the putative secondary structure of circularized MODV RNA is presented.
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PMID:Complete genome sequence, taxonomic assignment, and comparative analysis of the untranslated regions of the Modoc virus, a flavivirus with no known vector. 1185 6

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

Tamana bat virus (TABV, isolated from the bat Pteronotus parnellii) is currently classified as a tentative species in the genus FLAVIVIRUS: We report here the determination and analysis of its complete coding sequence. Low but significant similarity scores between TABV and member-viruses of the genus Flavivirus were identified in the amino acid sequences of the structural, NS3 and NS5 genes. A series of cysteines located in the envelope protein and the most important enzymatic domains of the virus helicase/NTPase, methyltransferase and RNA-dependent RNA polymerase were found to be highly conserved. In the serine-protease domain, the catalytic sites were conserved, but variations in sequence were found in the putative substrate-binding sites, implying possible differences in the protease specificity. In accordance with this finding, the putative cleavage sites of the TABV polyprotein by the virus protease are substantially different from those of flaviviruses. The phylogenetic position of TABV could not be determined precisely, probably due to the extremely significant genetic divergence from other member-viruses of the family FLAVIVIRIDAE: However, analysis based on both genetic distances and maximum-likelihood confirmed that TABV is more closely related to the flaviviruses than to the other genera. These findings have implications for the evolutionary history and taxonomic classification of the family as a whole: (i) the possibility that flaviviruses were derived from viruses infecting mammals rather than from mosquito viruses cannot be excluded; (ii) using the current criteria for the definition of genera in the family Flaviviridae, TABV should be assigned to a new genus.
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PMID:Genome sequence analysis of Tamana bat virus and its relationship with the genus Flavivirus. 1223 26

A number of hepatitis C virus (HCV) proteins, including NS5B, the RNA-dependent RNA polymerase, were detected in membrane fractions from Huh7 cells containing autonomously replicating HCV RNA replicons. These membrane fractions were used in a cell-free system for the analysis of HCV RNA replication. Initial characterization revealed a reaction in which the production of replicon RNA increased over time at temperatures ranging from 25 to 40 degrees C. Heparin sensitivity and nucleotide starvation experiments suggested that de novo initiation was occurring in this system. Both Mn2+ and Mg2+ cations could be used in the reaction; however, concentrations of Mn2+ greater than 1 mM were inhibitory. Compounds shown to inhibit recombinant NS3 and NS5B activity in vitro were found to inhibit RNA synthesis in the cell-free system. This system should be useful for biochemical analysis of HCV RNA synthesis by a multisubunit membrane-associated replicase and for evaluating potential antiviral agents identified in biochemical or cell-based screens.
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PMID:Hepatitis C virus RNA synthesis in a cell-free system isolated from replicon-containing hepatoma cells. 1252 37

In vitro RNA-dependent RNA polymerase assays revealed that the JEV replication complex (RC) synthesized viral RNA utilizing a semiconservative and asymmetric mechanism. Peak viral replicase activity and levels of viral RNA observed 15-18 h postinfection (h p.i.) preceded maximum viral titers in the culture medium seen 21 h p.i. Among divalent cations, Mg(2+) was essential and exhibited cooperative binding for its two replicase-binding sites. Mn(2+), despite sixfold higher affinity for the replicase, elicited only 70% of the maximum Mg(2+)-dependent activity, and deficit of either cation led to synthesis of incomplete RNA products. We also determined as a first instance for a flavivirus RC, kinetic parameters using cytoplasmic "virus-induced heavy membranes" after depleting endogenous nucleotides. Exhaustive trypsin treatment, which degraded the bulk of NS3 and NS5, had no effect on replicase activity, suggesting that the active flaviviral RC resides behind a membrane barrier and recruits minuscule proportions of the replicase proteins.
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PMID:Characterization of RNA synthesis, replication mechanism, and in vitro RNA-dependent RNA polymerase activity of Japanese encephalitis virus. 1266 4

Recently, cell-based replicon systems for hepatitis C virus (HCV), in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. To date, one limitation of using these replicon systems to advance drug discovery is the inability of other genotypic derivatives, beyond those of two distinct strains of genotype 1b (HCV-N and Con1), to stably replicate in Huh7 cells. In this report, we evaluated a series of replicon genotype 1a-1b chimeras, as well as a complete genotype 1a replicon clone. A subgenomic replicon construct containing only type 1a sequences failed to generate stable colonies in Huh7 cells even after repeated attempts. Furthermore, addition of an NS5A adaptive mutation (S2204I) which enhances type 1b replicon efficiency was insufficient to confer replication to the wild-type 1a replicon. This subgenomic replicon was subsequently found to be inefficiently translated in Huh7 cells compared to a type 1b replicon, and the attenuation of translation mapped to the N-terminal region of NS3. Therefore, to ensure efficient translation and thereby support replication of the 1a genome, the coding sequence for first 75 residues from type 1a were replaced with the type 1b (strain Con 1) NS3 coding sequence. Although nonstructural proteins were expressed at lower levels with this replicon than with type 1b and although the amount of viral RNA was also severalfold lower (150 copies of positive-strand RNA per cell), the replicon stably replicated in Huh7 cells. Notwithstanding this difference, the ratio of positive- to negative-strand RNA of 26 was similar to that found with the type 1b replicon. Similar results were found for a 1b replicon expressing the type 1a RNA-dependent RNA polymerase. These 1a hybrid replicons maintained sensitivity to alpha interferon (IFN-alpha), albeit with an eightfold-higher 50% inhibitory concentration than type 1b replicons. Evidence is provided herein to confirm that this differential response to IFN-alpha may be attributed directly to the type 1a polymerase.
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PMID:Replication studies using genotype 1a subgenomic hepatitis C virus replicons. 1269 37

A review of the patent literature for inhibitors of hepatitis C virus (HCV) is presented for the period of January 2001 to December 2001. This review focuses on inhibitors of virally encoded enzymes/proteins, especially HCV NS3 serine protease and NS5B RNA-dependent RNA polymerase (RDRP), both of which have emerged as primary anti-HCV targets as a result of detailed understanding of their biological functions and the high quality structural information available. A number of potent small molecule inhibitors for these enzymes have been disclosed during this period of time. Inhibitors of other virally encoded enzymes have also been reported, but in lower numbers. With recent advances in obtaining a stable HCV cell-based assay to facilitate screening and selection of inhibitors, it is highly likely that effective small molecule antiviral therapies for HCV infection should soon emerge.
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PMID:Inhibitors of hepatitis C--a review of the recent patent literature. 1286 76


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