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)

Current therapeutic options for hepatitis C are limited, especially for genotype 1. For genotypes 2 and 3, pegylated interferon in combination with ribavirin, can lead to a sustained virological response in up to 80% of patients. Unfortunately, adverse effects of IFN and ribavirin are a major problem and the list of contraindications for HCV therapy is long, including decompensated cirrhosis of the liver and psychiatric disorders. Therefore, alternative therapeutic approaches are needed. New delivery options for IFN and ribavirin are aimed at optimising efficiency and reducing adverse effects. Recent progress in the molecular virology of HCV has identified new targets for antiviral intervention. Inhibition of HCV gene expression and replication as well as immunotherapeutic concepts aimed at enhancing the cellular immune response against HCV are being explored. Solution of the crystal structures of HCV key enzymes led to the design of specific inhibitors including compounds active against the well characterised NS3 serine protease and RNA-dependent RNA polymerase which are currently in the early phase clinical investigation. New strategies for inhibiting HCV gene expression include the use of antisense oligodeoxynucleotides and ribozymes. Immunomodulation by agents such as inosine monophosphate dehydrogenase inhibitors, thymosin-alpha 1, histamine or amantadine are being studied in combination with IFN and/or ribavirin. Immunotherapeutic vaccination with recombinant HCV E1 protein improved host immunity against HCV and thus seems to be a promising new option.
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PMID:Current therapy and new molecular approaches to antiviral treatment and prevention of hepatitis C. 1462 84

Viruses encounter the innate immune system immediately after infection of the host; specifically, soluble molecules that are both directly lethal and that initiate acquired immunity. Using the oncogenic Marek's disease alpha-herpesvirus (MDV) model, we quantified the effect of a interferon-containing supernatants (ICS), on MDV replication, gene transcription and antigen expression kinetics. We used an established cell culture system and a well-defined virulent MDV (RB-1B). RB-1B was cultured without ICS, or pretreated and then continuously treated with ICS. We compared (i) RB-1B infectivity; (ii) RB-1B growth by microscopy; (iii) numbers of cells expressing RB-1B antigens by flow cytometry; (iv) RB-1B-DNA load per cell by duplex real-time PCR, and (v) gene transcription kinetics for key MDV-life stages by duplex real-time reverse-transcriptase PCR (RT-PCR). ICS inhibited RB-1B infection, completion of productive life cycle and cell-to-cell infection. The numbers of cells expressing glycoprotein B (a kinetically late antigen) greatly decreased, but the numbers of cells expressing pp38 (a kinetically early antigen) decreased only slightly. The two greatest effects were increases in both RB-1B-DNA per infected cell and pp38 mRNA. We propose MDV has evolved to increase specific gene transcription and genome copies per cell to compensate for ICS. We speculate that the bi-directional shared pp38/origin of replication promoter, is central to this mechanism.
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PMID:Interferon-containing supernatants increase Marek's disease herpesvirus genomes and gene transcription levels, but not virion replication in vitro. 1473 37

Although randomized trials have shown enhancement of efficacy for combination therapy with interferon (IFN) alpha-2b and ribavirin compared with IFN monotherapy as first-line treatment for chronic hepatitis C, infection with genotype 1b and high viremia are still associated with significantly low response rates compared with non-1 genotypes and low viremia. We analysed amino acid sequences of the viral RNA-dependent RNA polymerase (RdRP) or nonstructural protein 5B (NS5B), responsible for ribavirin misincorporation into RNA products in patients with genotype 1b-related chronic hepatitis C and high viremia, and examined the relationship between such RdRp polymorphisms, and the initial decline in viral load induced by combination therapy with IFN-alpha and ribavirin. Substitution of glutamic acid to lysine at the 124th position (E124K) and of isoleucine to valine at the 85th position (I85V) were found to be closely associated with a potent decline of viral load and viral clearance at 8 weeks of treatment (five of five patients, coincidence rate 100%). In conclusion, our results suggest that the polymorphisms of E124K and I85V identified in NS5B protein are crucial for early viral clearance in patients with genotype 1b and high viremia by combination therapy with IFN and ribavirin, and that detection of amino acid sequence motifs might enable prediction of clinical efficacy.
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PMID:Polymorphisms of NS5B protein relates to early clearance of hepatitis C virus by interferon plus ribavirin: a pilot study. 1511 24

We have established a T7-based model system for hepatitis C virus (HCV) 1a strain, which involves the use of a replication-defective adenovirus that carries the gene for T7 RNA polymerase and a transcription plasmid containing full-length HCV cDNA clone. To facilitate high-level expression of HCV, sub-confluent Huh7 cells were first infected with adenovirus containing the gene for the T7 RNA polymerase and then transfected with the transcription plasmid. As a negative control, part of NS5B gene of this clone was deleted which abolishes the HCV RNA-dependent RNA polymerase and prevents replication of viral RNA. This model produces high levels of structural (core, E1, E2) and nonstructural proteins (NS5), which were detected by Western blot analysis and immunofluorescence assay. Negative-strand HCV RNA was detected only in the wild-type clone in the presence of actinomycin D, and no RNA was detected with the NS5B deleted mutant control. As a practical validation of this model, we showed that IFN alpha-2b selectively inhibits negative-strand RNA synthesis by blocking at the level of protein translation. The inhibitory effect of IFN alpha-2b is not due reduction of transcription by T7 polymerase or due to intracellular degradation of HCV RNA. This in vitro model provides an efficient and reliable means of assaying negative-strand RNA, protein processing, and testing the antiviral properties of interferon.
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PMID:Interferon alpha-2b inhibits negative-strand RNA and protein expression from full-length HCV1a infectious clone. 1512 7

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

We report that mice with a targeted null mutation in the interferon type I receptor (IFN-RI), which cannot respond to such IFNs as IFNalpha and IFNbeta, have a 30% reduction in time spent in spontaneous rapid eye movement sleep (REMS) as a consequence of a reduced number of REMS episodes. Time spent in nonrapid eye movement sleep (NREMS) was essentially unaltered in IFN-RI knockouts (KOs) compared to 129 SvEv controls. Body temperature and locomotor activity were similar in both strains of mice. Hypothalamic expression of mRNAs for molecules previously linked to sleep-wake regulation and an IFN-inducible antiviral gene, 2',5'-oligoadenylate synthetase 1a (OAS), were determined by real-time reverse-transcriptase polymerase chain reaction (RT2-PCR). The level of hypocretin A mRNA was elevated in IFN-RI KO mice compared to 129 SvEv mice, while prolactin mRNA and OAS mRNA levels were suppressed. Vasoactive intestinal peptide (VIP) and corticotropin-releasing hormone (CRH) mRNA levels were unchanged relative to controls. Serum prolactin levels were similar in both strains. Results are consistent with the hypothesis that increased hypocretin and reduced prolactin in the hypothalamus of IFN-RI KO mice are responsible for their reduced REMS. In addition, the reduced OAS expression may result in modulation of prolactin receptor signaling and thus contribute to suppression of REMS.
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PMID:Mice deficient in the interferon type I receptor have reduced REM sleep and altered hypothalamic hypocretin, prolactin and 2',5'-oligoadenylate synthetase expression. 1549 63

The sixth genomic segment of Thogoto virus (THOV) encodes two proteins, the viral matrix protein (M) and an accessory protein with an interferon (IFN)-antagonistic function named ML. M and ML are shown in this study to be structural components of the virion. Using an in vivo system based on the reconstitution of functional THOV ribonucleoprotein complexes from cloned cDNAs, it was demonstrated that M has an inhibitory effect on the viral RNA-dependent RNA polymerase (RdRP) and is essential for the formation of virus-like particles (VLPs). The functional domain responsible for the regulation of RdRP activity resides within the C-terminal half of M, while full-length M protein is required for VLP formation. The ML protein cannot complement M with respect to either RdRP downregulation or particle formation, although it is identical to M apart from a 38 aa extension at the C terminus. In contrast, ML, but not M, is able to prevent the induction of IFN-beta by double-stranded RNA. This function is contained within the C-terminal half of ML. These data suggest major structural differences between M and ML that could explain the different activities of the two proteins.
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PMID:Functional comparison of the two gene products of Thogoto virus segment 6. 1555 43

A novel nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), [(1R)-5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyano[3,4-b]indol-1-yl] acetic acid (HCV-371), was discovered through high-throughput screening followed by chemical optimization. HCV-371 displayed broad inhibitory activities against the NS5B RdRp enzyme, with 50% inhibitory concentrations ranging from 0.3 to 1.8 microM for 90% of the isolates derived from HCV genotypes 1a, 1b, and 3a. HCV-371 showed no inhibitory activity against a panel of human polymerases, including mitochondrial DNA polymerase gamma, and other unrelated viral polymerases, demonstrating its specificity for the HCV polymerase. A single administration of HCV-371 to cells containing the HCV subgenomic replicon for 3 days resulted in a dose-dependent reduction of the steady-state levels of viral RNA and protein. Multiple treatments with HCV-371 for 16 days led to a >3-log10 reduction in the HCV RNA level. In comparison, multiple treatments with a similar inhibitory dose of alpha interferon resulted in a 2-log10 reduction of the viral RNA level. In addition, treatment of cells with a combination of HCV-371 and pegylated alpha interferon resulted in an additive antiviral activity. Within the effective antiviral concentrations of HCV-371, there was no effect on cell viability and metabolism. The intracellular antiviral specificity of HCV-371 was demonstrated by its lack of activity in cells infected with several DNA or RNA viruses. Fluorescence binding studies show that HCV-371 binds the NS5B with an apparent dissociation constant of 150 nM, leading to high selectivity and lack of cytotoxicity in the antiviral assays.
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PMID:Novel nonnucleoside inhibitor of hepatitis C virus RNA-dependent RNA polymerase. 1556 61

It is unclear whether the current antiviral treatment for chronic hepatitis C virus (HCV) infection results in complete elimination of the virus, or whether small quantities of virus persist. Our study group comprised 17 patients with chronic HCV who had sustained virological response (SVR) after interferon/ribavirin treatment. Serum and peripheral blood mononudear cells were collected 2 to 3 times at 3- to 6-month intervals starting 40 to 109 months (mean, 64.2 +/- 18.5 months) after the end of therapy. In addition, lymphocyte and macrophage cultures were established at each point. In 11 patients, frozen liver tissue samples were available from follow-up biopsies performed 41 to 98 months (mean, 63.6 +/- 16.7 months) after therapy. Presence of HCV RNA was determined by sensitive reverse-transcriptase polymerase chain reaction, and concentration of positive and negative strands was determined by a novel quantitative real-time reverse transcriptase polymerase chain reaction. Only 2 of 17 patients remained consistently HCV RNA negative in all analyzed compartments. HCV RNA was detected in macrophages from 11 patients (65%) and in lymphocytes from 7 patients (41%). Viral sequences were also detected in 3 of 11 livers and in sera from 4 patients. Viral replicative forms were found in lymphocytes from 2 and in macrophages from 4 patients. In conclusion, our results suggest that in patients with SVR after therapy, small quantities of HCV RNA may persist in liver or macrophages and lymphocytes for up to 9 years. This continuous viral presence could result in persistence of humoral and cellular immunity for many years after therapy and could present a potential risk for infection reactivation.
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PMID:Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C. 1594 Jun 51

The nonstructural 5B (NS5B) protein of hepatitis C virus possesses RNA-dependent RNA polymerase activity and plays an essential role in viral replication. The mutations in NS5B were determined and the correlation with viral load and response to interferon (IFN) were assessed. The entire NS5B region in 33 patients and its thumb domain in 62 patients was sequenced. The number of amino acid substitutions in the NS5B protein, that in thumb domain and the substitution at aa 389 was correlated with viral load and the response to IFN. Multivariate analysis selected only mutation in IFN sensitivity determining region (ISDR) as a factor associated with the viral load and response to IFN. The number of substitutions in the thumb domain and the substitution at aa 389 correlated with the number of substitutions in the ISDR. These results suggest that mutations in NS5B, especially in the thumb domain and at aa 389, have an important effect on viral load and the response to IFN, although they were dependent on mutations in ISDR. Further studies on the relationship between NS5B and NS5A (ISDR) are necessary to elucidate the mechanism of the correlation with viral load and the response to IFN.
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PMID:Mutations in the nonstructural region 5B of hepatitis C virus genotype 1b: their relation to viral load, response to interferon, and the nonstructural region 5A. 1571 88


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