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)

An RNA-dependent RNA polymerase activity has been found associated with Uukuniemi virions. The enzyme activity is expressed only after disrupting the virions with the nonionic detergent Triton X-100 and is absolutely dependent on Mn2+, whereas Mg2+ is not required, a finding that distinguishes this polymerase from those of other enveloped minus-strand RNA viruses. Within the range pH 7.2 to 8.5 no distinct optimum was found. The optimum temperature was between 37 and 40 C. The reaction was not inhibited by actinomycin D, rifampin, or DNase, whereas RNase was completely inhibitory. The partially RNase-resistant product consisted of rather small-sized RNA, which contained sequences complementary to Uukuniemi virus RNA as shown by hybridization to the template L, M, and S RNA species of Uukuniemi virus.
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PMID:Uukuniemi virus contains an RNA polymerase. 0 May 17

In search of an anti-transcriptase, antibody was raised in rabbits to partially purified, soluble NS protein present in cytoplasmic extracts of cells infected with the Indiana serotype of vesicular stomatitis (VSInd) virus. This antiserum gave specific reactions of identity by agar immunodiffusion with both cytoplasmic and virion NS protein. NS antiserum also preferentially precipitated NS 3-H-labeled protein from infected cytoplasmic extracts, whereas anti-whole VSInd virion serum also precipitated N 3-H-labeled protein from extracts both of infected cytoplasm and virion nucleocapsids. Transcriptase activity of VSInd cytoplasmic or virion-derived nucleocapsids was effectively inhibited by ribonuclease-free immunoglubulin prepared from homologous NSInd antiserum or from anti-whole vesicular stomatitis virus serum. Transcriptase activity of heterologous New Jersey serotype (VSNJ) nucleocapsids and virions was not appreciably affected by anti-NSInd or by anti-whole VSInd virion gamma globulin. Anti-NS gamma glubulin immediately switched off RNA synthesis by actively transcribing VSInd nucleocapsids, a finding which suggests that NS antibody inhibits RNA chain elongation.
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PMID:Inhibition of viral transcriptase by immunoglobulin directed against the nucleocapsid NS protein of vesicular stomatitis virus. 4 40

Rhinovirus type 14 RNA-dependent RNA polymerase complexes were isolated from microsomal and soluble fraction of infected KB cells. Maximum activities were measured at at 6 and 7 hours post inoculation (p.i.) for microsomal and soluble polymerases, respectively. Both polymerase activities are considerably reduced by 8 to 9 hours, p.i., and interval in which the in vivo rate of synthesis of viral RNA is maximal. In vitro RNA products of RNA polymerases in both fractions consist of ribonuclease-sensitive and ribonuclease-resistant RNA of heterogeneous sizes. Detergent treatment of the microsomal RNA polymerase(s) did not affect the total amount of RNA synthesized, the proportion of ribonuclease-sensitive RNA synthesized or the size of the RNA products. The data suggest that RV14RNA polymerase complexes are intially associated with membranes but are then irreversibly released into the soluble phase of the cytoplasm; possible explanations for this phenomena are discussed.
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PMID:Rhinovirus type 14 RNA polymerase complexes. 16 77

Two newly synthesized pyrimidine derivatives were found to possess antiviral activity against Mengovirus in Fogh and Lund (FL) cells and in a cell-free system. The inhibitory effect on RNA-dependent RNA polymerase of Mengovirus-infected FL cells was assayed using 14C-UTP as precursor. Addition of 50 or 100 muM of the inhibitors in a cell-free system of crude enzyme and nucleoside triphosphate medium for 60 min incubation at 37 degrees C resulted in about 40 to 60% lower counting rates for drug-treated reaction mixtures. The analysis of the polymerase synthesis product (virus RNA extracted from the cell-free reaction mixture and deproteinized by the phenol-SDS method) was carried out by means of agarose-acrylamide gel electrophoresis. The main finding was a reduction of single-stranded Mengovirus RNA (RNase-sensitive and LiCl-precipitable). The rates of synthesis of the replicative intermediate (LiCl-precipitable) and the replicative form of RNA (LiCl-soluble) were not significantly influenced.
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PMID:Effects of pyrimidine derivatives on RNA-dependent RNA polymerase of mengovirus-infected Fogh and Lund (FL) cells. 18 80

A soluble RNA-dependent RNA polymerase was isolated from poliovirus-infected HeLa cells and was shown to copy poliovirus RNA in vitro. The enzyme was purified from a 200,000-X-g supernatant of a cytoplasmic extract of infected cells. The activity of the enzyme was measured throughout the purification by using a polyadenylic acid template and oligouridylic acid primer. The enzyme was partially purified by ammonium sulfate precipitation, glycerol gradient centrifugation, and phosphocellulose chromatography. The polymerase precipitated in a 35% saturated solution of ammonium sulfate, sedimented at about 7S on a glycerol gradient, and eluted from phosphocellulose with 0.15 M KC1. The polymerase was purified about 40-fold and was shown to be totally dependent on exogenous RNA for activity and relatively free of contaminating nuclease. The partially purified polymerase was able to use purified polio virion RNA as well as a template. Under the reaction conditions used, the polymerase required an oligouridylic acid primer and all four ribonucleside triphosphates for activity. The optimum ratio of oligouridylic acid molecules to poliovirus RNA molecules for priming activity was about 16:1. A nearest-neighbor analysis of the in vitro RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA rendered it resistant to RNase digestion, thus suggesting that the product RNA was complementary to the virion RNA template.
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PMID:Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro. 23 68

Reovirus mRNA's containing a 5'-terminal methylated cap structure (m(7)GpppG(m)) were shown to be effective primers for influenza viral RNA transcription in vitro catalyzed by the influenza virion transcriptase. Priming activity required the presence of methyl groups in the cap since reovirus mRNA's with 5'-terminal GpppG were inactive as primers. Both the cap and internal nucleotides were physically transferred from radiolabeled reovirus mRNA to influenza viral complementary RNA (cRNA) during transcription in vitro. By using reovirus mRNA's with methyl-(3)H-labeled caps as primers, we showed that the influenza viral cRNA synthesized in the presence of unlabeled nucleoside triphosphates contained [methyl-(3)H]m(7)GpppG(m), identical to that found in the reovirus mRNA primer. To demonstrate transfer of internal residues, reovirus mRNA's synthesized in the presence of all four alpha-(32)P-labeled ribonucleoside triphosphates were used as primers. The resulting influenza viral cRNA was (32)P-labeled. Diethyl-aminoethyl-Sephadex chromatography of the RNase T2 digest of this cRNA demonstrated (32)P radiolabel in both internal residues (charge -2) and the cap (charge -4.6). Approximately 25 internal nucleotides along with the cap of reovirus mRNA were transferred to each chain of influenza viral cRNA. Gel electrophoretic analysis indicated that the segments of influenza viral cRNA primed by reovirus mRNA were approximately the same size as those primed by a different mRNA, globin mRNA, strongly suggesting that the influenza virion transcriptase complex transfers approximately the same number of nucleotides plus the cap from different mRNA primers to the 5' end of influenza viral RNA transcripts.
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PMID:Cap and internal nucleotides of reovirus mRNA primers are incorporated into influenza viral complementary RNA during transcription in vitro. 51 5

In the presence of Mg(2+) and a specific primer, ApG or GpG, the influenza WSN virion transcriptase synthesizes large, polyadenylic acid-containing complementary RNA (cRNA) (Plotch and Krug, J. Virol., 21:24-34, 1977). After removal of its polyadenylic acid with RNase H in the presence of polydeoxythymidylic acid, the in vitro cRNA distributed into seven discrete bands during electrophoresis in acrylamide gels containing 6 M urea. The eight known segments of virion RNA (vRNA) also distributed into seven bands under these conditions as two, rather than the expected three, large-sized segments were resolved. Each of the in vitro cRNA segments migrated slightly faster than the corresponding vRNA segment. To determine whether this difference in mobility reflects a difference in size between cRNA and vRNA, the double-stranded RNA formed by annealing labeled in vitro cRNA to unlabeled vRNA was subjected to various nuclease treatments and was analyzed by gel electrophoresis. Hybrids treated with RNase T2 or a combination of RNase T2 and RNase H migrated slightly faster than those treated only with RNase H, indicating that RNase T2 removed an RNA sequence other than polyadenylic acid, most probably a short sequence of vRNA not hydrogen bonded to cRNA. These results suggest that the in vitro cRNA segments are shorter than, and thus incomplete transcripts of the corresponding vRNA segments. All eight hybrids were resolved by gel electrophoresis, indicating that all eight vRNA segments are transcribed into cRNA in vitro. We also present evidence suggesting that the ApG primer initiates in vitro transcription exactly at the 3' end of vRNA.
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PMID:Segments of influenza virus complementary RNA synthesized in vitro. 62 84

An RNA-dependent RNA polymerase activity has been found copurifying with measles virus infectivity and complement-fixing antigen in three Vero cell-grown variants of measles virus: the attenuated Edmonston B strain, the natural non-attenuated Edmonston strain, and a subacute sclerosing panencephalitis isolate, IP-3. Incubation of purified measles virions with immunoglobulin G derived from sera of monkeys hyperimmunized against measles specifically removes activity sedimenting in the density region of measles virions. The requirements of the reaction, which is RNase sensitive, are similar to those reported for other paramyxovirus-associated activities, including detergent, divalent cation, ribonucleoside triphosphates, and a reducing agent. The size classes of RNA synthesized correspond to those found in measles-infected cells, including 50, 35, and 16 to 20S. The product RNA of the Edmonston B virus-stimulated reaction was rendered RNase resistant by annealing with RNA extracted from purified Edmonston B virions. RNA from uninfected Vero cells was ineffective in the annealing reaction.
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PMID:Characterization of an RNA-dependent RNA polymerase activity associated with measles virus. 64 73

In the presence of Mg(2+) and a specific dinucleotide primer (ApG or GpG), the influenza virion transcriptase synthesizes the eight discrete segments of complementary RNA (cRNA) containing polyadenylic acid (Plotch and Krug, J. Virol. 21:24-34, 1977). Virions were examined for their ability to cap and methylate cRNA containing di- or triphosphorylated 5' termini. By using the primers ppApG, pppApG, or ppGpG, viral cRNA was synthesized in vitro with [alpha-(32)P]-GTP and S-[methyl-(3)H]adenosylmethionine as labeled precursors. DEAE-Sephadex chromatography of the RNase T2 digest of the cRNA product demonstrated no (3)H incorporation at all and the absence of a (32)P-labeled cap structure. The 5' terminus of ppApG-primed cRNA could be capped and methylated by enzymes from vaccinia virus, indicating that the two 5'-terminal phosphates derived from the primer were preserved in the product cRNA. The cap structure formed by the vaccinia enzymes and released by RNase T2 digestion as m(7)GpppA(m)pGp was radioactively labeled at its 3'-terminal phosphate only when [alpha-(32)P]CTP was used as the labeled precursor during transcription. This indicates that the 5'-terminal sequence of the cRNA is ppApGpC and that, therefore, ppApG most probably initiates transcription exactly at the 3' GpCpU(OH) terminus of the virion RNA templates. Virions were also tested for their ability to cap and methylate ppApG in the absence of transcription. No such activities were detected, whereas under the same conditions the vaccinia virus enzymes successfully capped and methylated this compound. Consequently, these experiments, together with those reported earlier, have not detected in influenza virions any capping and methylating enzymes active on the 5'-initiated termini of viral cRNA chains synthesized in vitro, whether these termini possess one, two, or three phosphates. Some mechanism for capping and methylation of viral cRNA must, however, exist, because the viral mRNA (cRNA) synthesized in the infected cell contains 5'-terminal methylated cap structures (Krug et al., J. Virol. 20:45-53, 1976). Possible mechanisms are discussed.
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PMID:Absence of detectable capping and methylating enzymes in influenza virions. 70 57

A fraction containing membrane-bound tobacco mosaic virus RNA replicase was isolated form tobacco mosaic virus-infected tobacco callus cultures. The replicase activity reached a maximum 60 h after inoculation and then declined. The enzyme activity was insensitive to actinomycin D and DNase. The corresponding fraction from healthy callus contained essentially no activity. The viral RNA synthesis in vitro proceeded linearly for 30 min and required the four nucleotide triphosphates and Mg2+ ions. Mn2+ was a poor substitute for Mg2+. During RNA synthesis the product was at least 70% resistant to RNase in 2X SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digested by RNase in 0.1X SSC. Analysis of the product by polns) that appeared to be replicative form and a partially RNase-resistant structure similar to replicative intermediate form. Washing the membrane-bound replicase with Mg2+-deficient buffer solubilized enzyme. The solubulized enzyme was further purified by DEAE-Sephadex column chromatography. The DEAE-purified enzyme was nearly completely dependent upon tobacco mosaic virus RNA for activity. Analysis of the product on a sucrose gradient revealed a double-stranded RNA with sedimentation of 16S and smaller heterogeneous RNase-sensitive products.
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PMID:In vitro replication of tobacco mosaic virus RNA in tobacco callus cultures: solubilization of membrane-bound replicase and partial purification. 83 35


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