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)

Noroviruses (Caliciviridae) are RNA viruses with a single-stranded, positive-oriented polyadenylated genome. To date, little is known about the replication strategy of norovirus, a so-far noncultivable virus. We have examined the initiation of replication of the norovirus genome in vitro, using the active norovirus RNA-dependent RNA polymerase (3D(pol)), homopolymeric templates, and synthetic subgenomic or antisubgenomic RNA. Initiation of RNA synthesis on homopolymeric templates as well as replication of subgenomic polyadenylated RNA was strictly primer dependent. In this context and as observed for other enteric RNA viruses, i.e., poliovirus, a protein-primed initiation of RNA synthesis after elongation of the VPg by norovirus 3D(pol) was postulated. To address this question, norovirus VPg was expressed in Escherichia coli and purified. Incubation of VPg with norovirus 3D(pol) generated VPg-poly(U), which primed the replication of subgenomic polyadenylated RNA. In contrast, replication of antisubgenomic RNA was not primer dependent, nor did it depend on a leader sequence, as evidenced by deletion analysis of the 3' termini of subgenomic and antisubgenomic RNA. On nonpolyadenylated RNA, i.e., antisubgenomic RNA, norovirus 3D(pol) initiated RNA synthesis de novo and terminated RNA synthesis by a poly(C) stretch. Interestingly, on poly(C) RNA templates, norovirus 3D(pol) initiated RNA synthesis de novo in the presence of high concentrations of GTP. We propose a novel model for initiation of replication of the norovirus genome by 3D(pol), with a VPg-protein-primed initiation of replication of polyadenylated genomic RNA and a de novo initiation of replication of antigenomic RNA.
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PMID:Protein-primed and de novo initiation of RNA synthesis by norovirus 3Dpol. 1680 11

The RNA-dependent RNA polymerase of the hepatitis C virus and the bovine viral diarrhea virus(BVDV)is able to initiate RNA synthesis denovo in the absence of a primer. Previous crystallographic data have pointed to the existence of a GTP-specific binding site (G-site) that is located in the vicinity of the active site of the BVDV enzyme. Here we have studied the functional role of the G-site and present evidence to show that specific GTP binding affects the positioning of the template during de novo initiation. Following the formation of the first phosphodiester bond, the polymerase translocates relative to the newly synthesized dinucleotide, which brings the 5'-end of the primer into the G-site, releasing the previously bound GTP. At this stage, the 3'-end of the template can remain opposite to the 5'-end of the primer or be repositioned to its original location before RNA synthesis proceeds. We show that the template can freely move between the two locations, and both complexes can isomerize to equilibrium. These data suggest that the bound GTP can stabilize the interaction between the 3'-end of the template and the priming nucleotide, preventing the template to overshoot and extend beyond the active site during de novo initiation. The hepatitis C virus enzyme utilizes a dinucleotide primer exclusively from the blunt end; the existence of a functionally equivalent G-site is therefore uncertain. For the BVDV polymerase we showed that de novo initiation is severely compromised by the T320A mutant that likely affects hydrogen bonding between the G-site and the guanine base. Dinucleotide-primed reactions are not influenced by this mutation, which supports the notion that the G-site is located in close proximity but not at the active site of the enzyme.
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PMID:Control of template positioning during de novo initiation of RNA synthesis by the bovine viral diarrhea virus NS5B polymerase. 1683 16

The RNA-dependent RNA polymerase nsP4 is an integral part of the alphavirus replication complex. To define the role of nsP4 in viral RNA replication and for a structure-function analysis, we expressed Sindbis virus nsP4 in Escherichia coli. The core catalytic domain of nsP4 (Delta97nsP4, a deletion of the N-terminal 97 amino acids), which consists of the predicted polymerase domain containing the GDD amino acid motif required for viral RNA synthesis, was stable against proteolytic degradation during expression. Therefore, the recombinant core domain and selected mutants were expressed and purified to homogeneity. We determined that Delta97nsP4 possesses terminal adenylyltransferase (TATase) activity, as it specifically catalyzed the addition of adenine to the 3' end of an acceptor RNA in the presence of divalent cations. Furthermore, Delta97nsP4 is unable to transfer other nucleotides (UTP, CTP, GTP, and dATP) to the acceptor RNA in the absence or presence of other nucleotides. Delta97nsP4 possessing a GDD-to-GAA mutation completely inactivates the enzymatic activity. However, a GDD-to-SNN mutation did not inactivate the enzyme but reduced its activity to approximately 45% of that of the wild type in the presence of Mg(2+). Investigation of the TATase of the GDD-to-SNN mutant revealed that it had TATase equivalent to that of the wild type in the presence of Mn(2+). Identification of Delta97nsP4 TATase activity suggests a novel function of the alphavirus RNA-dependent RNA polymerase in the maintenance and repair of the poly(A) tail, an element required for replication of the viral genome.
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PMID:Catalytic core of alphavirus nonstructural protein nsP4 possesses terminal adenylyltransferase activity. 1700 74

We investigated the modulating actions of the nonselective K(+) channel blocker 4-aminopyridine (4-AP) on amyloid beta (Abeta(1-42))-induced human microglial signaling pathways and functional processes. Whole-cell patch-clamp studies showed acute application of Abeta(1-42) (5 mum) to human microglia led to rapid expression of a 4-AP-sensitive, non-inactivating outwardly rectifying K(+) current (I(K)). Intracellular application of the nonhydrolyzable analog of GTP, GTPgammaS, induced an outward K(+) current with similar properties to the Abeta(1-42)-induced I(K) including sensitivity to 4-AP (IC(50) = 5 mm). Reverse transcriptase-PCR showed a rapid expression of a delayed rectifier Kv3.1 channel in Abeta(1-42)-treated microglia. Abeta(1-42) peptide also caused a slow, progressive increase in levels of [Ca(2+)](i) (intracellular calcium) that was partially blocked by 4-AP. Chronic exposure of human microglia to Abeta(1-42) led to enhanced p38 mitogen-activated protein kinase and nuclear factor kappaB expression with factors inhibited by 4-AP. Abeta(1-42) also induced the expression and production of the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha, the chemokine IL-8, and the enzyme cyclooxygenase-2; 4-AP was effective in reducing all of these pro-inflammatory mediators. Additionally, toxicity of supernatant from Abeta(1-42)-treated microglia on cultured rat hippocampal neurons was reduced if 4-AP was included with peptide. In vivo, injection of Abeta(1-42) into rat hippocampus induced neuronal damage and increased microglial activation. Daily administration of 1 mg/kg 4-AP was found to suppress microglial activation and exhibited neuroprotection. The overall results suggest that 4-AP modulation of an Abeta(1-42)-induced I(K) (candidate channel Kv3.1) and intracellular signaling pathways in human microglia could serve as a therapeutic strategy for neuroprotection in Alzheimer's disease pathology.
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PMID:Broad-spectrum effects of 4-aminopyridine to modulate amyloid beta1-42-induced cell signaling and functional responses in human microglia. 1709 87

The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Early results suggested that the RNA polymerase "switched" from a transcriptase to a replicase during the viral life cycle in response to the expression of viral proteins. However, recently an alternative model suggesting that replication of influenza virus is regulated by stabilization of the replicative intermediates was proposed. According to this model, the virion-associated polymerase is capable of synthesizing both mRNA and cRNA. We now demonstrate that virion-derived viral ribonucleoproteins (vvRNPs) synthesize both mRNA and cRNA in vitro in the absence of non-virion-associated RNA polymerase or nucleoproteins. The authenticity of the in vitro-transcribed mRNA and cRNA was confirmed by terminal sequence analysis. The addition of non-virion-associated polymerase or NP had no effect on vvRNP activity. De novo synthesis of cRNA was found to be more sensitive than the capped primer-dependent synthesis of mRNA to the concentration of ATP, CTP, and GTP. We conclude that vvRNPs intrinsically possess both transcriptase and replicase activities and that there is no switch in the synthesis of mRNA to cRNA during the influenza virus life cycle.
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PMID:Influenza virion-derived viral ribonucleoproteins synthesize both mRNA and cRNA in vitro. 1716 11

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.
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PMID:Unconventional mechanism of mRNA capping by the RNA-dependent RNA polymerase of vesicular stomatitis virus. 1721 73

We have previously shown that synthesis of poliovirus protein 3CD in uninfected HeLa cell extracts induces an increased association with membranes of the cellular Arf GTPases, which are key players in cellular membrane traffic. Arfs cycle between an inactive, cytoplasmic, GDP-bound form and an active, membrane-associated, GTP-bound form. 3CD promotes binding of Arf to membranes by initiating recruitment to membranes of guanine nucleotide exchange factors (GEFs), BIG1 and BIG2. GEFs activate Arf by replacing GDP with GTP. In poliovirus-infected cells, there is a dramatic redistribution of cellular Arf pools that coincides with the reorganization of membranes used to form viral RNA replication complexes. Here we demonstrate that Arf translocation in vitro can be induced by purified recombinant 3CD protein; thus, concurrent translation of viral RNA is not required. Coexpression of 3C and 3D proteins was not sufficient to target Arf to membranes. 3CD expressed in HeLa cells was retained after treatment of the cells with digitonin, indicating that it may interact with a membrane-bound host factor. A F441S mutant of 3CD was shown previously to have lost Arf translocation activity and was also defective in attracting the corresponding GEFs to membranes. A series of other mutations were introduced at 3CD residue F441. Mutations that retained Arf translocation activity of 3CD also supported efficient growth of virus, regardless of their effects on 3D polymerase elongation activity. Those that abrogated Arf activation by 3CD generated quasi-infectious RNAs that produced some plaques from which revertants that always restored the Arf activation property of 3CD were rescued.
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PMID:Activation of cellular Arf GTPases by poliovirus protein 3CD correlates with virus replication. 1756 96

Nucleoside 5'-triphosphates (NTPs) play key roles in biology and medicine. However, these compounds are notoriously difficult to synthesize. We describe a one-pot method to prepare NTPs from nucleoside 5'-H-phosphonate monoesters via pyridinium phosphoramidates, and we used this approach to synthesize ATP, UTP, GTP, CTP, ribavirin-TP, and 6-methylpurine ribonucleoside-TP (6MePTP). Poliovirus RNA-dependent RNA polymerase efficiently employed 6MePTP as a substrate, suggesting that the cognate nucleoside, a poorly understood antiviral agent, may damage viral RNA.
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PMID:One-pot synthesis of nucleoside 5'-triphosphates from nucleoside 5'-H-phosphonates. 1839 12

The NS5B protein of classical swine fever virus (CSFV) is an important enzyme bearing a unique RNA-dependent RNA polymerase (RdRp) activity. The RdRp plays a crucial role in the viral replication cycle and in forming a replicase complex. However, the initiating synthesis mechanism of the CSFV RNA polymerase is unclearly described at present. Our aim is to reveal the RdRp-GTP docking sites and the effective modules of GTP initially bound to the polymerase in starting initiation of replication according to a well predicted CSFV RdRp model. Based on some known crystal structures of RNA polymerase, computational methods were used to establish the model of a CSFV RdRp. An analogous mechanism of CSFV RNA polymerase in de novo initiation was subsequently represented through docking a GTP into the structure model. The unique GTP binding pocket of the polymerase was pointed out: five residues E227, S408, R427, K435, and R439 involved in steady hydrogen bonds and two residues C407 and L232 involved in hydrophobic contact with the GTP. From a genetic evolutionary point of view, three residues C407, S408 and R427 have been suggested to be of particular importance by analysis of residue conservation. It is suggested that these crucial residues should have very significant function in the de novo initiation of the rigorous CSFV polymerase model, which can lead us to design experiments for studying the mechanism of viral replication and develop valid anti-viral drugs.
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PMID:The GTP binding sites interacted with RNA-dependent RNA polymerase of classical swine fever virus in de novo initiation. 1843 Sep 87

The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Previous studies in our laboratory have shown that virion-derived viral ribonucleoprotein complexes synthesize both mRNA and cRNA in vitro and early in the infection cycle in vivo. Our continued studies showed that de novo synthesis of cRNA in vitro is more sensitive to the concentrations of ATP, CTP, and GTP than capped-primer-dependent synthesis of mRNA. Using rescued recombinant influenza A/WSN/33 viruses, we now demonstrate that the 3'-terminal sequence of the vRNA promoter dictates the requirement for a high nucleoside triphosphate (NTP) concentration during de novo-initiated replication to cRNA, whereas this is not the case for the extension of capped primers during transcription to mRNA. In contrast to some other viral polymerases, for which only the initiating NTP is required at high concentrations, influenza virus polymerase requires high concentrations of the first three NTPs. In addition, we show that base pair mutations in the vRNA promoter can lead to nontemplated dead-end mutations during replication to cRNA in vivo. Based on our observations, we propose a new model for the de novo initiation of influenza virus replication.
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PMID:Role of initiating nucleoside triphosphate concentrations in the regulation of influenza virus replication and transcription. 1846 55


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