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

Mutational analysis of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) template channel identified two residues, Trp(397) and His(428), which are required for de novo initiation but not for extension from a primer. These two residues interact with the Delta1 loop on the surface of the RdRp. A deletion within the Delta1 loop also resulted in comparable activities. The mutant proteins exhibit increased double-stranded RNA binding compared with the wild type, suggesting that the Delta1 loop serves as a flexible locking mechanism to regulate the conformations needed for de novo initiation and for elongative RNA synthesis. A similar locking motif can be found in other viral RdRps. Products associated with the open conformation of the HCV RdRp were inhibited by interaction with the retinoblastoma protein but not cyclophilin A. Different conformations of the HCV RdRp can thus affect RNA synthesis and interaction with cellular proteins.
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PMID:A locking mechanism regulates RNA synthesis and host protein interaction by the hepatitis C virus polymerase. 1844 78

Previous studies have suggested that histamine (HA) acts as an autocrine growth factor. We have explored the modulation of cell proliferation by HA using McA-RH7777 hepatoma cells. High L-histidine decarboxylase (HDC) expression and HA synthesis were found in McA-RH7777 cells. Whereas extracellular HA reached submicromolar concentrations, intracellular levels were very low, indicating that HA was secreted by the cells. McA-RH7777 cells also express H3-receptor (H3R) transcripts and proteins. Reverse transcriptase-polymerase chain reaction analysis detected only transcripts for the long isoform. Immunocytochemistry performed with a selective H3R antibody showed that most cells were immunoreactive. H3R binding sites (Bmax approximately 30 fmol/mg protein) were identified when [125I] iodoproxyfan binding was displaced by the agonist imetit. High-affinity binding also occurred at cytochrome P450 enzymes. This binding was not inhibited by HA, H3R agonists, or by a nonimidazole H3R antagonist but was displaced by imidazole H3R antagonists or by ketoconazole, a imidazole-containing cytochrome inhibitor. HA inhibited proliferation of McA-RH7777 hepatoma cells. The absence of uptake system, its much higher potency at H3Rs, and its low intracellular levels suggested that HA interacted with H3Rs rather than cytochromes. In agreement, both imidazole H3R antagonists, a nonimidazole H3R antagonist, and the HDC inhibitor alpha-monofluoromethyl histidine increased cell proliferation (up to approximately 60%), revealing a H3R-mediated inhibition by endogenous HA. Moreover, exogenous HA inhibited the increase induced by alpha-FMH or H3R antagonists with a nanomolar potency. In conclusion, our findings show that HA regulates proliferation of McA-RH7777 hepatoma cells by interacting with autoinhibitory H3Rs.
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PMID:Autoregulation of McA-RH7777 hepatoma cell proliferation by histamine H3 receptors. 1847 84

Arabidopsis thaliana acyl-CoA-binding protein 2 (ACBP2) was observed to interact with farnesylated protein 6 (AtFP6), which has a metal-binding motif (M/LXCXXC). Their interaction and expression in response to heavy metals were investigated. Yeast two-hybrid analysis and in vitro assays showed that an ACBP2 derivative lacking ankyrin repeats did not interact with AtFP6, indicating that the ankyrin repeats mediate protein-protein interaction. Autofluorescence-tagged ACBP2 and AtFP6 transiently co-expressed in tobacco (Nicotiana tabacum) were both targeted to the plasma membrane. Reverse transcriptase polymerase chain reaction and northern blot analyses revealed that AtFP6 mRNA was induced by cadmium (Cd(II)) in A. thaliana roots. Assays using metal-chelate affinity chromatography demonstrated that in vitro translated ACBP2 and AtFP6 bound lead (Pb(II)), Cd(II) and copper (Cu(II)). Consistently, assays using fluorescence analysis confirmed that (His)(6)-AtFP6 bound Pb(II), like (His)(6)-ACBP2. Arabidopsis thaliana plants overexpressing ACBP2 or AtFP6 were more tolerant to Cd(II) than wild-type plants. Plasma membrane-localized ACBP2 and AtFP6 probably mediate Pb(II), Cd(II) and Cu(II) transport in A. thaliana roots. Also, (His)(6)-ACBP2 binds [(14)C]linoleoyl-CoA and [(14)C]linolenoyl-CoA, the precursors for phospholipid repair following lipid peroxidation under heavy metal stress at the plasma membrane. ACBP2-overexpressing plants were more tolerant to hydrogen peroxide than wild-type plants, further supporting a role for ACBP2 in post-stress membrane repair.
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PMID:Arabidopsis thaliana acyl-CoA-binding protein ACBP2 interacts with heavy-metal-binding farnesylated protein AtFP6. 1882 12

During the 2007-2008 influenza season global strain surveillance for antiviral resistance revealed the sudden emergence of oseltamivir resistance in influenza A H1N1 isolates. Although oseltamivir resistance rates vary from region to region, 16% of isolates tested globally were found to be oseltamivir resistant by a histidine to tyrosine mutation of residue 275 of the neuraminidase gene of influenza A. In order to implement effective resistance testing locally a novel real-time reverse-transcriptase PCR (RT-PCR) assay was developed for the detection of the H275Y mutation. To evaluate this method, 40 oseltamivir resistant and 61 oseltamivir sensitive H1N1 influenza isolates were tested using Sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel H275Y RT-PCR assay. In comparison to Sanger sequencing, the sensitivity and specificity of the H275Y RT-PCR assay were 100% (40/40) and 100% (61/61) respectively, while the sensitivity and specificity of pyrosequencing were 100% (40/40) and 97.5% (60/61) respectively. Although all three methods were effective in detecting the H275Y mutation associated with oseltamivir resistance, the H275Y RT-PCR assay was the most rapid and could easily be incorporated into an influenza subtyping protocol.
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PMID:Development of a novel real-time reverse-transcriptase PCR method for the detection of H275Y positive influenza A H1N1 isolates. 1942 89

The replication of the foot-and-mouth disease virus (FMDV) genome is critically dependent upon the activity of a virally encoded RNA-dependent RNA polymerase (RdRp). In this study, four mutant RdRps of FMDV were isolated from viral quasi-species treated with ribavirin, of which two were single mutants (L123F and T381A) and two were double mutants (T291I/T381I and L123F/F244L). The mutant proteins were expressed in Escherichia coli and purified by His-bind resin chromatography. In combination with real-time RT-PCR, an in vitro RNA replication system that uses genome RNA/VPg as template-primers was used to determine polymerase activity. Mutant L123F exhibited a 0.6-fold decrease (p < 0.001) in polymerase activity relative to wild-type RdRp, whereas the activity of L123F/F244L and T381A was undetectable. Surprisingly, the activity of T291I/T381I yielded a 0.7-fold increase (p < 0.001) as compared to wild-type. In order to study the structure-function relationship of RdRp, all structures of the RdRp-RNA template-primer complex were obtained through homology modeling and molecular docking. The VPg1 orientation in the RdRp-VPg1 complexes was determined and analyzed with mathematical methods. Our results reveal that the orientation of VPg after binding to the polymerase determines the FMDV RdRp catalytic activity, which provides a basis for the rational design of novel antiviral agents.
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PMID:Structure-function analysis of mutant RNA-dependent RNA polymerase complexes with VPg. 1991 26

This investigation examines the role of the SaeR/S 2-component system in USA300, a prominent circulating clone of community-associated methicillin-resistant Staphylococcus aureus. Using a saeR/S isogenic deletion mutant of USA300 (USA300DeltasaeR/S) in murine models of sepsis and soft-tissue infection revealed that this sensory system is critical to pathogenesis of USA300 during both superficial and invasive infection. Oligonucleotide microarray and real-time reverse-transcriptase polymerase chain reaction identified numerous extracellular virulence genes that are down-regulated in USA300DeltasaeR/S. Unexpectedly, an up-regulation of mecA and mecR1 corresponded to increased methicillin resistance in USA300DeltasaeR/S. 5'-RACE analysis defined transcript start sites for sbi, efb, mecA, lukS-PV, hlb, SAUSA300_1975, and hla, to underscore a conserved consensus sequence within promoter regions of genes under strong SaeR/S transcriptional regulation. Electrophoretic mobility shift assay experiments illustrated direct binding of SaeR(His) to promoter regions containing the conserved consensus sequence. Collectively, the findings of this investigation demonstrate that SaeR/S directly interacts with virulence gene promoters to significantly influence USA300 pathogenesis.
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PMID:SaeR binds a consensus sequence within virulence gene promoters to advance USA300 pathogenesis. 2000 58

Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae. The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5' overhangs showing 5'-to-3' polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg(2+) binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5' overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.
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PMID:NTPase and 5' to 3' RNA duplex-unwinding activities of the hepatitis E virus helicase domain. 2007 63

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5'-phosphorylated viral mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional mRNA capping reaction catalyzed by the RNA:GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to GDP to produce the capped RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the RNA transfer reaction. To locate the active site of the PRNTase domain in the L protein, the covalent RNA attachment site was mapped. We found that the 5'-monophosphate end of the RNA is linked to the histidine residue at position 1,227 (H1227) of the L protein through a phosphoamide bond. Interestingly, H1227 is part of the histidine-arginine (HR) motif, which is conserved within the L proteins of the NNS RNA viruses including rabies, measles, Ebola, and Borna disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing antiviral agents.
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PMID:Histidine-mediated RNA transfer to GDP for unique mRNA capping by vesicular stomatitis virus RNA polymerase. 2016 4

All RNA viruses encode an RNA-dependent RNA polymerase (RdRP) that is required for replication of the viral genome. Nuclear inclusion b (NIb) gene codes for the RdRp in Potyviridae viruses. In this study, expression, solubilization and purification of NIb protein of Cardamom mosaic virus (CdMV) is reported. The objective of the present study was to express and purify the NIb protein of CdMV on a large scale for structural characterization, as the structure of the RdRp from a plant virus is yet to be determined. However, the expression of NIb protein with hexa-histidine tag in Escherichia coli led to insoluble aggregates. Out of all the approaches [making truncated versions to reduce the size of protein; replacing an amino acid residue likely to be involved in hydrophobic intermolecular interactions with a hydrophilic one; expressing the protein along with chaperones; expression in Origami cells for proper disulphide bond formation, in E. coli as a fusion with maltose-binding protein (MBP) and in Nicotiana tabacum] to obtain the RdRp in a soluble form, only expression in E. coli as a fusion with MBP and its expression in N. tabacum were successful. The NIb expressed in plant or as a fusion with MBP in E. coli can be scaled up for further work.
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PMID:Optimized expression, solubilization and purification of nuclear inclusion protein b of cardamom mosaic virus. 2108 22

In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. Reduced antigenicity of the latter suggested that C-terminal truncation affects 8A3B.6 epitope construction probably via inhibition of VP7 trimer structure formation.
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PMID:Expression and characterization of bluetongue virus serotype 21 VP7 antigen: C-terminal truncated protein has significantly reduced antigenicity. 2118 84


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