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)

Ribonucleoprotein complexes isolated from measles virus-infected HeLa cells contained an RNA-dependent RNA polymerase activity that catalyzed the incorporation of ribonucleotides into ribonucleic acid. The ribonucleoprotein complexes were composed of measles virus nucleoprotein, phosphoprotein, and a large protein, as well as viral RNA. The kinetics of RNA synthesis at different temperatures, time intervals, and protein, ribonucleotide, and mono- and divalent cation concentrations were analyzed. Enzyme activity was maximum at 4 h at 25 degrees C in the presence of 100 mM Na+-2.5 mM Mg2+-1 mM ribonucleotides. Actinomycin D and alpha-amanitin had no effect on the enzyme activity. Addition of cytoplasmic extracts from uninfected HeLa cells to the reaction mixture did not increase the incorporation of ribonucleotides into RNA. The in vitro synthesized RNAs were characterize by slot blot analysis and quantitated by densitometer scanning. All mRNAs coding for the structural proteins of measles virus were synthesized. Nucleoprotein RNA was the most abundant species made, followed by phosphoprotein, hemagglutinin, fusion protein, matrix protein, and large-protein RNAs. The system described here resulted in the first efficient transcription of measles virus RNA and analysis of products.
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PMID:Characterization of in vitro transcription and transcriptional products of measles virus. 366 51

An investigation was made of inhibition of transcriptase activity of influenza viruses in vitro by binding of antibody to the surface of the virion. Eight monoclonal antibodies which were directed against at least four non-overlapping antigenic regions of the haemagglutinin protein of A/Aichi/68 virus were tested for inhibitory effect. One of the antibodies directed against the B antigenic site, 22/1, inhibited transcriptase activity, while the other seven antibodies did not. Antibody from a hyperimmune rabbit serum to A/Udorn/72 (H3N2) virions inhibited the transcriptase activity of A/Udorn/72 and A/Aichi/68 (H3N2) viruses but not that of A/WSN/33 (H1N1). The antibody did not cause irreversible inactivation of the transcriptase since full activity was recovered by isolating ribonucleoprotein (RNP) cores from the inhibited virions using NP-40 treatment and subsequent centrifugation in a caesium sulphate density gradient. The antibody did not inhibit transcriptase activity of isolated RNP cores. The virion transcriptase activity was not inhibited by addition of the antiserum after the detergent treatment which is necessary for the activation of the transcriptase activity in vitro. These results suggest that the antibody blocks the activation process of the transcriptase by detergent treatment.
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PMID:Inhibition of transcriptase activity of influenza A virus in vitro by anti-haemagglutinin antibodies. 406 Aug 49

A procedure has been developed for the sequential removal and purification of the glycoprotein and membrane protein of vesicular stomatitis virus (VSV). Neither of these proteins exhibited transcriptase activity. All of the activity was recovered in the ribonucleic acid (RNA)-ribonucleoprotein complex of VSV, which also has four other minor proteins associated with it. During transcription of 41% of the RNA of a virus preparation, no dissociation of the ribonucleoprotein from the viral RNA was observed.
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PMID:Dissociation of vesicular stomatitis virus and relation of the virion proteins to the viral transcriptase. 434 41

T-particle-free stocks of temperature-sensitive mutants representing the four Glasgow complementation groups of the Indiana serotype of vesicular stomatitis virus were used to study RNA synthesis at the permissive and nonpermissive temperatures of 31 and 39 C, respectively. Mutants selected from the four Glasgow complementation groups were characterized on the basis of particle and ribonucleoprotein formation. Intracellular RNAs were further characterized by polyacrylamide gel electrophoresis. ts G22 (group II) and ts G41 (group IV), previously characterized as RNA negative at the nonpermissive temperature, synthesized low levels of RNA which could not be attributed to contaminating levels of revertants. Furthermore, the levels of synthesis could not be reduced by the addition of cycloheximide. These data suggest that ts G22 (group II) and ts G41 (group IV) contain a thermally stable, virion-encapsidated transcriptase, but fail to amplify RNA synthesis due to a thermally labile function presumably necessary for the synthesis of viral RNA. ts G31, a group III mutant, synthesized intracellular RNA at amplified levels at the nonpermissive temperature. Intracellular ribonucleoprotein complexes were isolated in copious amounts; however, no particles corresponding in size to finished virions were observed. These data suggest a thermally labile maturation factor or envelope associated structural protein to be defective in ts G31 (group III). ts G11 (group 1) showed no detectable RNA synthesis at the nonpermissive temperature. These data suggest ts G11 (group I) contains a thermally labile component involved in early transcription. This group may contain a number of mutants defective in different components of the transcription apparatus, which may not complement in vivo because of the physical improbability of subunit exchange between virion particles of the incoming inoculum.
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PMID:RNA synthesis in temperature-sensitive mutants of vesicular stomatitis virus. 435 55

The endogenous transcriptase present in purified vesicular stomatitis (VS) virions was solubilized with a Triton X-100 high-salt solution. The polymerase activity was purified on glycerol gradients and by phosphocellulose column chromatography; the viral proteins present in the active enzyme fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was demonstrated that L protein, but not NS protein, was required for in vitro RNA synthesis on the VS viral nucleocapsid template. Solubilized L protein rebinds to the ribonucleoprotein template when the transcription complex is reconstituted, and the RNA synthesized in vitro by purified L protein hybridizes to virion RNA. Cyanogen bromide peptide fingerprints indicate that the large L protein is a unique polypeptide chain. It is concluded that the L protein functions as the transcriptase, and the nucleocapsid NS protein is not essential for in vitro RNA synthesis.
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PMID:L protein requirement for in vitro RNA synthesis by vesicular stomatitis virus. 435 10

The ribonucleoprotein-dependent RNA transcriptase in vesicular stomatitis B virions of four temperature-sensitive (ts) mutants belonging to complementation group I was analyzed in vitro at permissive (31 C) and restrictive (39 C) temperatures. The RNA-synthesizing activity of all four ts mutants was more labile at 39 C than was the transcriptive activity of wild-type (wt) virions. In order to locate the temperature-sensitive transcription defect in the mutants, wt and ts mutant virions were fractionated by Triton X-100-high salt solubilizer into a sedimentable ribonucleoprotein template and a nonsedimentable enzyme fraction, each of which alone had little or no transcriptive activity. The template- and enzyme-containing fractions of wt virions were then tested for their capacity to restore transcriptive activity at 39 C to corresponding template and enzyme preparations of ts mutant virions. Recombination of wt template and ts enzymes resulted in no significant restoration of capacity to synthesize RNA at restrictive temperature. In contrast, transcriptive function at 39 C was reconstituted by recombining the wt enzyme with the template component of ts mutants. It appears, therefore, that the enzyme, rather than the template, is the temperature-sensitive component of the transcription complex of group I vesicular stomatitis virus mutants.
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PMID:Location of the transcription defect in group I temperature-sensitive mutants of vesicular stomatitis virus. 435 28

When tested in vitro, certain temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) belonging to complementation groups I and IV appear to have defects in the virion-bound polymerase. To obtain further information concerning the nature of these defects, representative mutants were dissociated by the method of S. Emerson and R. Wagner (1972), and their supernatant (S) and pellet (P) fractions were tested for transcriptase activity when combined with the P and S fractions, respectively, of VSV-HR virions. It was found that the S fractions from group I mutants tsW4, 11, 14, 15, and 28 were defective in transcriptase activity, whereas their P fractions were as active as those of VSV-HR. On the other hand, the P fraction derived from virions of the group IV mutant tsW16B showed reduced activity at 25 C and very little activity at 38 C. These results suggest that our group I mutants, like those examined by D. Hunt and R. Wagner (1974), have a defect in the soluble transcriptase enzyme, whereas mutant tsW16B (group IV) has a defect in a sedimentable component required for transcriptase activity, possibly in the ribonucleoprotein template.
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PMID:Temperature-sensitive mutants of vesicular stomatitis virus: comparison of the in vitro RNA polymerase defects of group I and group IV mutants. 437 Sep 58

The products synthesized in vitro by an RNA-dependent RNA polymerase isolated from influenza virus-infected BHK21-F cells were analyzed by velocity sedimentation, annealing techniques, and acrylamide-agarose gel electrophoresis. Approximately 50% of the RNA synthesized in vitro remains associated with the 50 to 70S ribonucleoprotein complex containing polymerase activity; the remainder of the RNA polymerase product sediments heterogeneously with a peak at 13S. At least 90% of the in vitro product hybridizes with virion RNA. If polypeptides are labeled early in the growth cycle, both the P and NP polypeptides are detected in the ribonucleoprotein complex by acrylamide gel electrophoresis. The results suggest that the polypeptide composition and the products of the cell-associated RNA polymerase are similar to those of the RNA transcriptase associated with influenza virus particles.
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PMID:Analysis of the in vitro product of an RNA-dependent RNA polymerase isolated from influenza virus-infected cells. 485 84

The relationship between the in vitro phosphorylation of vesicular stomatitis virus (VSV) proteins and virion uncoating was examined. Activation of the VSV virion kinase with low concentrations of melittin, the active peptide component of bee venom, in the presence of gamma-[32P] ATP resulted in the phosphorylation of virion proteins. Following the in vitro phosphorylation of VSV proteins in the presence of melittin and deoxyadenosine triphosphate, the virion envelope was disrupted based on the accessibility of the internal ribonucleoprotein core (RNP) to the heavy metal stain, uranyl acetate, as determined by electron microscopic observation. The RNP structure was not observed in unphosphorylated virions treated with melittin and uranyl acetate. Phosphorylated virions treated with uranyl acetate subsequently lost the capacity for transcription whereas unphosphorylated virions treated with the stain retained transcriptase activity. These observations suggest that phosphorylation of VSV proteins may contribute to virion uncoating by disrupting the virus envelope.
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PMID:Phosphorylation of vesicular stomatitis virus proteins as a possible contributing factor in virion uncoating. 617 11

Soluble transcriptase containing the L and the NS proteins was isolated from purified vesicular stomatitis virus and its binding with the template ribonucleoprotein containing the N protein-RNA complex was studied with respect to its ability to initiate and synthesize RNA in vitro. By using u.v.-irradiated template reconstituted with soluble transcriptase, it was shown that the synthesis of leader RNA and other small initiated mRNA sequences continued while full-length mRNA synthesis decreased by 90%. In the presence of ATP and CTP, the reconstituted complex synthesized polyphosphorylated oligonucleotides which include AC, AAC and AACA which represent 5'-terminal sequences transcribed from the leader template and genes coding for mRNAs. In the presence of arabinosyl ATP, an inhibitor of RNA synthesis in vitro, the synthesis of leader RNA was found to be inhibited considerably more than other small initiated mRNA sequences. Reconstitution of RNA synthesis with soluble transcriptase and template in the presence of viral matrix (M) protein at low ionic condition resulted in virtual cessation of leader RNA synthesis, although the synthesis of small initiated N mRNA, 11 to 14 bases, continued. These results suggest that transcriptase can bind at multiple sites on the genome template and initiate RNA chains.
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PMID:Interaction of L and NS proteins of vesicular stomatitis virus with its template ribonucleoprotein during RNA synthesis in vitro. 619 59


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