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)

BILN 2061 is a novel, specific hepatitis C virus (HCV) NS3 serine protease inhibitor discovered by Boehringer Ingelheim that has shown potent activity against HCV replicons in tissue culture and is currently under clinical investigation for the treatment of HCV infection. The poor fidelity of the HCV RNA-dependent RNA polymerase will likely lead to the development of drug-resistant viruses in treated patients. The development of resistance to BILN 2061 was studied by the in vitro passage of HCV genotype 1b replicon cells in the presence of a fixed concentration of the drug. Three weeks posttreatment, four colonies were expanded for genotypic and phenotypic characterization. The 50% inhibitory concentrations of BILN 2061 for these colonies were 72- to 1,228-fold higher than that for the wild-type replicon. Sequencing of the individual colonies identified several mutations in the NS3 serine protease gene. Molecular clones containing the single amino acid substitution A156T, R155Q, or D168V resulted in 357-fold, 24-fold, and 144-fold reductions in susceptibility to BILN 2061, respectively, compared to the level of susceptibility shown by the wild-type replicon. Modeling studies indicate that all three of these residues are located in close proximity to the inhibitor binding site. These findings, in addition to the three-dimensional structure analysis of the NS3/NS4A serine protease inhibitor complex, provide a strategic guide for the development of next-generation inhibitors of HCV NS3/NS4A serine protease.
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PMID:Mutations conferring resistance to a potent hepatitis C virus serine protease inhibitor in vitro. 1515 30

The full-length ORFs for the hepatitis C virus recombinant RF1_2k/1b (N687) and the non-recombinant 1b strain N589 were sequenced. A single recombination point was found and the sizes of the genes (C, E1, E2, p7, NS2, NS3, NS4 and NS5) were according to the parental subtypes. The PKR-eIF2alpha phosphorylation site homology domain sequence of the E2 protein was identical to those of genotype 2 strains, while the IFN-alpha-sensitivity-determining region of the NS5A protein was identical to those of interferon-resistant 1b strains. For the parental strains, two hairpin structures, HS1 and HS2, were predicted for the plus-strand up- and downstream of the crossover site, which were not present in the recombinant strain. HS2 shared similarity with the motif1 hairpin of turnip crinkle virus RNA that binds to the RNA-dependent RNA polymerase and facilitates 3'-terminal extension during recombination. This study suggests that RF1_2k/1b has emerged by homologous recombination during minus-strand synthesis via template switching because of constraints imposed by the HS1 hairpin of the 3'-parental genome.
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PMID:Full-length open reading frame of a recombinant hepatitis C virus strain from St Petersburg: proposed mechanism for its formation. 1521 69

The two sets of connected membranes induced in Kunjin virus-infected cells are characterized by the presence of NS3 helicase/protease in both, and by RNA-dependent RNA polymerase (RdRp) activity plus the associated double-stranded RNA (dsRNA) template in vesicle packets (VP), or by the absence of both the VP-specific markers in the convoluted membranes/paracrystalline arrays (CM/PC). Attempts were made to separate flavivirus-induced membranes by sedimentation or flotation analyses in density gradients of sucrose or iodixanol, respectively, after treatment of cell lysates by sonication, osmotic shock, or tryptic digestion. Only osmotic shock treatment provided suggestive evidence of separation. This was explored by flow cytometry analysis (FCA) of RdRp active membrane fractions from a sucrose gradient, using dual fluorescent labelling via antibodies to NS3 and dsRNA. FCA revealed the presence of a dual labelled membrane population indicative of VP, and in a faster sedimenting fraction a membrane population able to be labelled only in NS3, representative of CM/PC and associated (R)ER. It was postulated that osmotic shock ruptured the bounding membrane of the VP, releasing the enclosed small vesicles associated with the Kunjin virus replication complex characterized previously. Notably, the presence of the full spectrum of nonstructural proteins in some membrane fractions was not a reliable marker for RdRp activity. These experiments may provide the opportunity for isolation of relatively pure flavivirus replication complexes in their native membrane-associated state by fluorescence-activated cell sorting.
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PMID:Comparisons of physical separation methods of Kunjin virus-induced membranes. 1528 61

To determine if the cellular factors La autoantigen (La) and polypyrimidine tract-binding protein (PTB) are required for hepatitis C virus (HCV) replication, we used siRNAs to silence these factors and then monitored their effect on HCV replication using quantitative RT-PCR. In addition, we determined the influence of PTB on the activity of the 3' noncoding region (NCR) of HCV and investigated its interaction with the components of the HCV replicase complex. We found that La is essential for efficient HCV replication while PTB appears to partially repress replication. PTB does, however, block the binding of HCV RNA-dependent RNA polymerase (RdRp, NS5B) to the 3'NCR. Indirect immunofluorescence microscopy showed co-localization of cytoplasmic PTB with the HCV RdRp in hepatoma cells (Huh-7) expressing HCV proteins, while in vitro translation of viral proteins from the HCV replicon revealed the interaction of PTB isoforms with NS5B polymerase and NS3.
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PMID:Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication. 1582 7

We previously found that hepatitis C virus (HCV) core protein (Core) activated the interferon (IFN)-inducible 40/46 kDa 2'-5'-oligoadenylate synthetase (2'-5'-OAS) gene through an IFN-stimulated response element (ISRE) in non-neoplastic human hepatocyte PH5CH8 cells. Here, we found that Core and NS5B synergistically enhanced the 2'-5'-OAS gene promoter activity through ISRE. Further analysis revealed that amino acid positions 12 and/or 13 of Core and RNA-dependent RNA polymerase activity of NS5B were essential for the activation of the 2'-5'-OAS gene promoter. Interestingly, we observed that the activation by Core or NS5B was still partially enhanced by even the NS5B or Core mutant lacking the activating ability, respectively, suggesting an indirect interaction between Core and NS5B. Furthermore, we showed that the activation by NS5B could be explained by NS5B's induction of IFN-beta, however, IFN-beta was not induced by Core. Moreover, we showed that the synergistic effect of Core and NS5B was not invalidated by NS3-4A, although NS3-4A significantly inhibited the activation by combination of Core and NS5B. Taken together, our findings reveal that NS5B/Core and NS3-4A exhibit conflicting effects (activation and inhibition) on the IFN system in PH5CH8 cells, and suggest that such effects may promote the distraction of the host defense system to lead to persistent infection.
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PMID:Hepatitis C virus proteins exhibit conflicting effects on the interferon system in human hepatocyte cells. 1613 43

Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific anti-hepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.
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PMID:Mutations conferring resistance to a hepatitis C virus (HCV) RNA-dependent RNA polymerase inhibitor alone or in combination with an HCV serine protease inhibitor in vitro. 1618 12

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.
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PMID:Nuclear localization of flavivirus RNA synthesis in infected cells. 1669 25

The global prevalence of hepatitis C virus (HCV) infection and serious health consequences associated with chronic state of the disease have become a significant health problem worldwide. Currently, there is no vaccine to prevent the disease and no specific antiviral drug directed against HCV infection. The current standard of care, interferon-based therapies, both alone or in combination with ribavirin, has demonstrated limited success and is associated with undesirable side effects. Thus, the treatment of the chronic HCV infection represents an unmet medical need. With advances in the understanding of HCV replication and the crystal structures of the virally encoded enzymes, the HCV NS3/4A serine protease and the NS5B RNA-dependent RNA polymerase have emerged as ideal targets toward the control of the disease and the development of new anti-HCV agents. In this review, we will summarize the current treatment options, and outline the approaches toward discovery of small molecule antivirals against the virally encoded enzymes. The current clinical studies of promising lead compounds are also reviewed.
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PMID:Recent development of therapeutics for chronic HCV infection. 1682 88

Recently, nitric oxide (NO) has been shown to suppress dengue virus (DENV) RNA and protein accumulation in infected cells. In this report, the potential target of the inhibitory effect of NO was studied at the molecular level. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), showed an inhibitory effect on RNA accumulation at around 8-14 h post-infection, which corresponded to the step of viral RNA synthesis in the DENV life cycle. The activity of the viral replicase isolated from SNAP-treated DENV-2-infected cells was suppressed significantly compared with that of the negative-control N-acetyl-DL-penicillamine (NAP)-treated cells. Further investigations on the molecular target of NO action showed that the activity of recombinant DENV-2 NS5 in negative-strand RNA synthesis was affected in the presence of 5 mM SNAP in in vitro RNA-dependent RNA polymerase (RdRp) assays, whereas the RNA helicase activity of DENV-2 NS3 was not inhibited up to a concentration of 15 mM SNAP. These results suggest that the inhibitory effect of NO on DENV infection is partly via inhibition of the RdRp activity, which then downregulates viral RNA synthesis.
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PMID:Antiviral action of nitric oxide on dengue virus type 2 replication. 1696 59

Flaviviruses are enveloped viruses with a single-stranded, 10.7kb positive-sense RNA genome. The genomic RNA, which has a 5' cap but no poly(A) tail, is translated as a single polyprotein that is then cleaved into three structural proteins and seven non-structural (NS) proteins by both viral and host proteases. The NS proteins include an RNA-dependent RNA polymerase (NS5), a helicase/protease (NS3), and other proteins that form part of the viral replication complex. Sequences and structures in the 5' and 3' untranslated regions (UTR) and capsid gene, including the cyclization sequences, the upstream AUG region, and the terminal 3' stem-loop, regulate translation, RNA synthesis and viral replication. We have also found that an RNA hairpin structure in the capsid coding region (cHP) influences start codon selection and viral replication of the flavivirus dengue virus (DENV). Peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs) were used to further dissect the role of conserved regions of the 5' and 3' UTRs; several P-PMOs were shown to specifically inhibit DENV translation and/or RNA synthesis and, hence, are potentially useful as antiviral agents. Regarding the mechanism of DENV translation, we have shown that DENV undergoes canonical cap-dependent translation initiation as well as a non-canonical mechanism when cap-dependent translation is suppressed. Although much remains to be elucidated about the molecular biology of flavivirus infection, progress is being made towards defining the cis and trans factors that regulate flavivirus translation and replication.
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PMID:Molecular biology of flaviviruses. 1731 52


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