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)

Activation of the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor (LHR) in cultured hypothalamic cells and immortalized GnRH (gonadotropin-releasing hormone) neurons (GT1-7 cells) transiently stimulates and subsequently inhibits cAMP production and pulsatile GnRH release. The marked and delayed impairment of cAMP signaling and episodic GnRH release in GT1-7 cells is prevented by pertussis toxin (PTX). This, and the LH-induced release of membrane-bound Galpha(s) and Galpha(i3) subunits, are indicative of differential G protein coupling to the LHR. Action potential (AP) firing in identified GnRH neurons also initially increased and then progressively decreased during LH treatment. The inhibitory action of LH on AP firing was also prevented by PTX. Reverse transcriptase-PCR analysis of GT1-7 neurons revealed the expression of G protein-gated inwardly rectifying potassium (GIRK) channels in these cells. The LH-induced currents were inhibited by PTX and were identified as GIRK currents. These responses indicate that agonist stimulation of endogenous LHR expressed in GnRH neurons activates GIRK channels, leading to suppression of membrane excitability and inhibition of AP firing. These findings demonstrate that regulation of GIRK channel function is a dominant factor in gonadotropin-induced abolition of pulsatile GnRH release. Furthermore, this mechanism could contribute to the suppression of pituitary function during pregnancy.
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PMID:Essential role of G protein-gated inwardly rectifying potassium channels in gonadotropin-induced regulation of GnRH neuronal firing and pulsatile neurosecretion. 1682 87

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.
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PMID:Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound. 1691 96

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.
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PMID:In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA. 1692 57

In (+) RNA coronaviruses, replication and transcription of the giant approximately 30 kb genome to produce genome- and subgenome-size RNAs of both polarities are mediated by a cognate membrane-bound enzymatic complex. Its RNA-dependent RNA polymerase (RdRp) activity appears to be supplied by non-structural protein 12 (nsp12) that includes an RdRp domain conserved in all RNA viruses. Using SARS coronavirus, we now show that coronaviruses uniquely encode a second RdRp residing in nsp8. This protein strongly prefers the internal 5'-(G/U)CC-3' trinucleotides on RNA templates to initiate the synthesis of complementary oligonucleotides of <6 residues in a reaction whose fidelity is relatively low. Distant structural homology between the C-terminal domain of nsp8 and the catalytic palm subdomain of RdRps of RNA viruses suggests a common origin of the two coronavirus RdRps, which however may have evolved different sets of catalytic residues. A parallel between the nsp8 RdRp and cellular DNA-dependent RNA primases is drawn to propose that the nsp8 RdRp produces primers utilized by the primer-dependent nsp12 RdRp.
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PMID:A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus. 1702 78

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.
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PMID:Multiple enzyme activities of flavivirus proteins. 1731 55

The organization of flaviviral replicase proteins within the membrane-bound replication complexes of West Nile (WNV), dengue (DENV) and Japanese encephalitis viruses (JEV) was probed by investigating the combined effect of detergents and trypsin on both viral replicase activity and profile of metabolically labelled viral proteins. While trypsin treatment of virus-induced membrane fractions degraded the vast majority of replicase proteins, viral RNA-dependent RNA polymerase (RdRp) activity remained completely unaffected. Solubilization of the membranes with deoxycholate (DOC) however rendered the replicase accessible to trypsin. Triton X-100 (TX100) treatment reduced RdRp activity by half in WNV but totally destroyed RdRp activity in JEV. TX100 also dissociated NS1' in addition to NS1 from NS5 and NS3 inJEV. Antibodies to NS3 coprecipitated NS1' along with NS5 only from DOC-solubilized but not from TX100-treated extracts, the former of which alone retained RdRp activity. Exogenous addition of recombinant NS1' to TX100 treated JEV-induced membranes restored the defect in the release step of RNA synthesis. Our results suggest for the first time a direct role for JEV NS1' in viral RNA synthesis in vitro.
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PMID:Organization of flaviviral replicase proteins in virus-induced membranes: a role for NS1' in Japanese encephalitis virus RNA synthesis. 1731 59

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

An in vitro system was applied to analyze the replication of a satellite RNA of Bamboo mosaic virus (BaMV), designated satBaMV RNA, using solubilized membrane-bound RNA-dependent RNA polymerase (RdRp) complexes isolated from BaMV-infected Nicotiana benthamiana. After removal of endogenous templates, the RdRp complexes of BaMV catalyzed RNA synthesis upon the addition of the full-length positive (+)- or negative (-)-strand satBaMV RNA transcripts used as templates. Both (+)- and (-)-satBaMV RNA products were detected when only the (+)-satBaMV RNA was used as a template in the in vitro RdRp assays, which further demonstrated the capability of the RdRp preparation to complete the replication cycles of satBaMV RNAs. In addition, use of 5' rapid amplification of cDNA ends and DNA sequencing showed that the BaMV RdRp preparation could specifically recognize the promoter sequences in the (-)-satBaMV RNA for accurate initiation of (+)-satBaMV RNA synthesis. The results suggested that the same enzyme complexes could be used for the replication of both BaMV genomic and satBaMV RNAs. The soluble and template-dependent RdRp could be further used in mechanistic studies, such as those analyzing the cis-elements and candidate host factors required for satBaMV RNA replication in vitro.
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PMID:In vitro replication of Bamboo mosaic virus satellite RNA. 1853 84

RNA-dependent RNA polymerase activity and protein synthesis in brome mosaic virus (BMV)-infected and uninfected protoplasts were investigated during the course of viral replication. In protoplast homogenates, the membrane-bound RNA polymerase activity resistant to actinomycin D was enhanced by BMV infection up to 30-fold that found in mock-inoculated protoplasts. The activity was first detected 7 to 8 hr postinfection. It reached a maximum at around 30 hr postinfection, then decreased gradually while virus and the viral antigen in the protoplasts, as measured by infectivity and fluorescent antibody staining, respectively, continued to increase. The bound enzyme activity was not enhanced by inoculation with BMV RNA 3 and/or RNA 4. Four proteins with molecular weights of 120, 110, 36, and 19.5 x 103 were observed in BMV-infected protoplasts from three plant species (a systemic host, a local lesion host, and a nonhost of BMV).
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PMID:RNA polymerase activity and protein synthesis in brome mosaic virus-infected protoplasts. 1863 24

A membrane-bound RNA-dependent RNA polymerase from Cowpea mosaic virus (CPMV)-infected cowpea leaves (Vigna unguiculata) has been purified 15,000-fold by DEAE-Sepharose CL-6B chromatography, affinity chromatography on poly(U)-Sepharose 4B, and glycerol gradient centrifugation. Particularly, poly(U)-Sepharose 4B chromatography was a very efficient purification step and, in addition, achieved the separation of a host-encoded terminal uridylyl transferase activity from the RNA polymerase activity. On glycerol gradient centrifugation, the polymerase activity sedimented as a homogeneous peak with a rate corresponding to a molecular weight of 120,000. Analysis of the protein composition of the gradient fractions revealed that only one polypeptide with a molecular weight of 130,000 cosedimented with the polymerase activity, suggesting a monomeric enzyme. The most purified enzyme preparations from CPMV infected leaves did not contain polypeptides encoded by RNA from CPMV B-component which presumably carries functions essential for CPMV replication. Using the same purification procedure, an RNA-dependent RNA polymerase has also been purified from mock-inoculated leaves, which appeared to be identical to the RNA polymerase from infected leaves. This host enzyme was strongly stimulated in cowpea leaves infected with CPMV. The role, if any, of the RNA-dependent RNA polymerase from cowpea leaves in CPMV-RNA replication is discussed in view of the recent evidence for virus encoded functions involved in CPMV multiplication.
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PMID:Purification of a host-encoded RNA-dependent RNA polymerase from cowpea mosaic virus-infected cowpea leaves. 1863 12


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