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)

Innate immunity is part of the antiviral response. Interferon (IFN)-beta plays a leading role in this system. To investigate the influence of hepatitis C virus (HCV) on innate immunity, we examined the effect of viral proteins on IFN-beta induction. HepG2 cells were co-transfected with plasmids for seven HCV proteins (core protein, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) and the IFN-beta promoter luciferase. Toll-like receptor (TLR) 3 and Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF) play key roles in dsRNA-mediated activation of interferon regulatory factor (IRF)-3 and IFN-beta; therefore, the participation of TLR3/TRIF in NS5B-mediated IFN induction was examined. Among seven HCV proteins, only NS5B, a viral RNA-dependent RNA polymerase (RdRp), activated the IFN-beta promoter. However, mutant NS5B without RdRp activity or template/primer association did not activate the IFN-beta promoter. Activation of the IFN-beta promoter by NS5B required the positive regulatory domain III, a binding sequence for IRF-3. Moreover, IRF-3 was phosphorylated by NS5B. Both inhibition of TLR3 expression by small interfering RNA and expression of the dominant negative form of TRIF significantly reduced NS5B-induced activation of IFN-beta. Of the six other HCV proteins, NS4A, NS4B, and NS5A efficiently inhibited this activation. HCV NS5B is a potent activator of the host innate immune system, possibly through TLR3/TRIF and synthesis of dsRNA. Meanwhile, NS4A, NS4B, and NS5A block IFN-beta induction by NS5B, which may contribute toward the persistence of this virus.
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PMID:Interferon-beta is activated by hepatitis C virus NS5B and inhibited by NS4A, NS4B, and NS5A. 1966 54

NS3 of pestiviruses contains a protease domain and a RNA helicase/NTPase domain. Contradictory results have been reported regarding NS3 in RNA synthesis. To investigate the effect of NS3 on classical swine fever virus (CSFV) NS5B RNA-dependent RNA polymerase activity (RdRp) activity and NS3-NS5B interaction, RdRp reactions, GST-pull-down assays and co-immunoprecipitation analyses containing NS5B and either of NS3 protein and the different truncated NS3 mutants were performed, respectively. We found that NS3 stimulated NS5B RdRp activity in a dose-dependent manner by binding to NS5 through a NS3 protease domain. Furthermore, mapping important regions of the NS3 protease domain was carried out by deletion mutagenesis, associated with RdRp reactions, GST-pull-down assays and co-immunoprecipitation analyses. Results showed that stimulation of CSFV NS5B RdRp activity was obtained by NS3 binding to NS5B through a 31-amino acid fragment at the N-terminal end of NS3 protease domain, which mediated a specific NS3-NS5B interaction.
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PMID:Classical swine fever virus NS3 enhances RNA-dependent RNA polymerase activity by binding to NS5B. 1995 25

The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people. Current therapy relies upon a combination of pegylated interferon-alpha and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC(50)) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log(10) reduction in mean viral load measured 24 h post-dose that was sustained for an additional 120 h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144 h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors.
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PMID:Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. 2139 52

The nucleotide sequence diversity present among hepatitis C virus (HCV) isolates allows rapid adjustment to exterior forces including host immunity and drug therapy. This viral response reflects a combination of a high rate of replication together with an error-prone RNA-dependent RNA polymerase, providing for the selection and proliferation of the viruses with the highest fitness. We examined HCV subtype 1a whole-genome sequences to identify positions contributing to genotypic and phenotypic diversity. Phylogenetic tree reconstructions showed two distinct clades existing within the 1a subtype with each clade having a star-like tree topology and lacking definite correlation between time or place of isolation and phylogeny. Identification of significant phylogenetically informative sites at the nucleotide level revealed positions not only contributing to clade differentiation, but which are located at or proximal to codons associated with resistance to protease inhibitors (NS3 Q41) or polymerase inhibitors (NS5B S368). Synonymous/nonsynonymous substitution mutation analyses revealed that the majority of nucleotide mutations yielded synonymous amino acids, indicating the presence of purifying selection pressure across the polyprotein with pockets of positive selection also being detected. Despite evidence for divergence at several loci, certain 1a characteristics were preserved including the length of the alternative reading frame/F protein (ARF/F) gene, and a subtype 1a-specific phosphorylation site in NS5A (S349). Our analysis suggests that there may be strain-specific differences in the development of antiviral resistance to viruses infecting patients who are dependent on the genetic variation separating these two clades.
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PMID:Evidence for separation of HCV subtype 1a into two distinct clades. 2056 73

Inter-species transmission is often incriminated in the epidemiology of Pestivirus diseases. The purpose of this study was to investigate the prevalence of Pestivirus in some mountain wild ungulates and to determine their role in Pestivirus transmission, as mountain pastures are a place where cohabitations between wild and domestic ungulates are particularly high. Between 2003 and 2007, a longitudinal epidemiological study was carried out on hunted ungulates in the French Hautes-Alpes department. Pestivirus-specific antibodies against p80 protein (also named NS3) common to all Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV) were found in 45.9% (95% confidence interval [CI95%]: 40.5-51.3%) of the 343 tested chamois (Rupicapra rupicapra). In addition, mouflons (Ovis gmelinii musimon) were shown for the first time to be strongly infected (61.1%; CI95%: 38.6-83.6) by a Pestivirus. These serological ELISA results were confirmed by comparative virus neutralization tests, performed on seven Pestivirus strains by using 15 seropositive samples. The highest antibody titers were directed against 2 BDV strains (Av and 33s strains), rather than BDV-4, a strain responsible for Pyrenean-chamois epizooties. Virus neutralization tests confirm a BDV circulation in wild ungulates in the French South Alps. However, no Pestivirus RNA was detected by reverse-transcriptase polymerase chain reaction in serum and spleen samples from seronegative animals and no virus was isolated from those samples either. Efforts should be made to improve the protocol in order to be able to isolate and characterize the local strain. Finally, the oldness (age) and femaleness (gender) increase the risk of seroconversion in chamois.
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PMID:Epidemiology of Pestivirus infection in wild ungulates of the French South Alps. 2070 72

4'- Fluoro-2',3'-O-isopropylidenecytidine was synthesized via interaction of 5'-O-acetyl-4'-fluoro-2',3'-O-isopropylideneuridine with triazole and 4-chlorophenyl dichlorophosphate followed by ammonolysis. Treatment of 5'-O-acetyl-4'-fluoro-2',3'-O-isopropylidenecytidine with hydroxylamine resulted in 5'-O-acetyl-4'-fluoro-2',3'-O-isopropylidene-N(4)-hydroxycytidine. Subsequent removal of 2',3'-O-isopropylidene groups gave 5'-O-acetyl derivatives of 4'-fluorouridine, 4'-fluorocytidine and 4'-fluoro-N(4)-hydroxycytidine. 5'-O-Triphosphate of 4'-fluorouridine was obtained in three steps starting from 4'-fluoro-2',3'-O-isopropylideneuridine. The 4'-fluoro uridine 5'-O-triphospate was found to be an effective inhibitor of HCV RNA-dependent RNA polymerase, substrate for NTPase reaction, catalyzed by protein NS3 HCV (a rate of the analogue hydrolysis was similar to that of ATP) and an activator for helicase reaction (with an efficacy only three fold lower than that of ATP).
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PMID:[Synthesis and biological properties of pyrimidine 4'-fluoro nucleosides and 4'-fluoro uridine 5'-O-triphospate]. 2082 21

The NS3-NS5B interaction of classical swine fever virus (CSFV) is important for viral replication. For characterisation of the interaction between the NS3 and NS5B, a series of NS5B mutants with deletion of N-, C-terminal amino acids and quadruple alanine substitution mutations were produced. GST pull-down assays and immunoprecipitation analyses showed that NS5B and some NS5B mutants have NS3 binding activity. Further experimental data indicated that CSFV NS5B might contain two NS3 binding sites, one covering amino acids 63-99 located at the N-terminal end, another covering amino acids 611-642 at the C-terminal end. Assays for RNA-dependent RNA polymerase (RdRp) activity revealed that CSFV NS3 is able to enhance the RdRp activity of NS5B and some NS5B mutants in vitro. The enhancement might be obtained by NS3 binding to the two terminal sequences of NS5B, which could be attractive targets for drug development against CSFV.
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PMID:Characterisation of interaction between NS3 and NS5B protein of classical swine fever virus by deletion of terminal sequences of NS5B. 2123 22

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.
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PMID:Functional analysis of two cavities in flavivirus NS5 polymerase. 2134 34

HCV (Hepatitis C virus) that causes chronic liver disease. HCV NS5B RNA-dependent RNA polymerase (RbRp) and NS3 protease are able to affect virtual replication of genes. Computer-aided drug design (CADD) aims at designing new molecules with pharmacological activity. In this study, we used the Discovery Studio 2.0 program and the scoring function to estimate the Dock Score, piecewise linear potential 1 (PLP1), piecewise linear potential 2 (PLP2), and potential of mean force (PMF) score of novel compounds. In this way, novel compounds with "de novo evolution" can be found. Using the the pharmacophore features that are near the receptor pocket and the score functions to calculate scores for the ligand-receptor interaction, the new ligands were selected, developed and virtually placed in the binding site of the receptor. A new compound, EVO12, gave the best score, indicating that it may be an efficient polymerase inhibitor of HCV NS5B.
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PMID:Computational analysis of de novo evolution of hepatitis C virus NS5B polymerase inhibitors. 2147 38

Hepatitis C virus (HCV) inhibitors include direct-acting antivirals (DAAs) such as NS3 serine protease inhibitors, nucleoside and nonnucleoside polymerase inhibitors, and host-targeting antivirals (HTAs) such as cyclophilin inhibitors that have been developed in recent years. Drug-resistant HCV variants have been reported both in vitro and in the clinical setting for most classes of drugs. We report a comparative study in which the genetic barrier to drug resistance of a representative selection of these inhibitors is evaluated employing a number of resistance selection protocols. The NS3 protease inhibitors VX-950 and BILN 2061, the nucleoside polymerase inhibitor 2'-C-methylcytidine, three nonnucleoside polymerase inhibitors (thiophene carboxylic acid, benzimidazole, and benzothiadiazine), and DEB025 were included. For each drug and passage in the selection process, the phenotype and genotype of the drug-resistant replicon were determined. For a number of molecules (BILN 2061 and nonnucleoside inhibitors), drug-resistant variants were readily selected when wild-type replicon-containing cells were directly cultured in the presence of high concentrations of the inhibitor. Resistance to DEB025 could be selected only following a lengthy stepwise selection procedure. For some DAAs, the signature mutations that emerged under inhibitor pressure differed depending on the selection protocol that was employed. Replication fitness of resistant mutants revealed that the C445F mutation in the RNA-dependent RNA polymerase can restore loss of fitness caused by a number of unfit resistance mutations. These data provide important insights into the various pathways leading to drug resistance and allow a direct comparison of the genetic barriers of various HCV drugs.
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PMID:Comparative study of the genetic barriers and pathways towards resistance of selective inhibitors of hepatitis C virus replication. 2170


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