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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purified preparations of molluscum contagiosum virus contain a DNA-dependent RNA polymerase (EC 2.7.7.6) with similar but not identical properties to those of the enzyme found in vaccinia virions. The ultraviolet inactivation kinetics of the RNA polymerase from both viruses were similar, displaying fast and slow components. Ultraviolet irradiation destroyed the interfering capacities of molluscum and inactivated vaccinia virions, and the interferon-inducing capacity of molluscum virus slowly and with first-order kinetics. Inactivation studies of the interferon-inducing capacity of vaccinia virus were complicated by cytotoxic effects. Electron microscopical studies showed all stages of virus growth in vaccinia-infected mouse embryo cells; molluscum virus appeared to be degraded in lysosome-like bodies. In preliminary studies, marked changes in cytoplasmic RNA synthesis and in patterns of polypeptide synthesis were found in vaccinia-infected but not in molluscum-infected mouse embryo cells.
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PMID:Molluscum contagiosum -- a defective poxvirus? 1 Dec 75

An abortive infection of a rabbit cornea cell line (RC-60) by vesicular stomatitis virus (VSV), yielding less than 1 PFU/cell, was converted to a productive infection, yielding 1,900 PFU/cell, when cells were superinfected with vaccinia. Studies on the synthesis of VSV-directed RNA in RC-60 cells suggest that the abortive infection by VSV alone may be due in part to (i) a limited production of 40S virion RNA and (ii) a markedly reduced activity of virion-bound transcriptase activity in RC-60 cells compared to the activity in mouse L cells, a permissive host for VSV. No recognizable VSV structures, except a small amount of viral core structures, were produced by the abortive infection. In contrast, double infection of RC-60 cells with VSV and vaccinia in the presence of hydroxyurea resulted in the production of infective B particles of VSV. Although the function supplied by vaccinia responsible for the productive replication of VSV in double infected RC-60 cells has not been identified, metabolic inhibitor studies indicate that continuous vaccinia-dependent RNA synthesis is required for maximal production of infective VSV. The possibility is considered that vaccinia may supply a product or function required for VSV replication which is ordinarily supplied by the host but which is lacking in RC-60 cells.
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PMID:Abortive infection of a rabbit cornea cell line by vesicular stomatitis virus: conversion to productive infection by superinfection with vaccinia virus. 16 5

The participation of host RNA polymerase II in the vaccinia life cycle was examined by comparing efficiency of multiplication after treating the Ama+ sensitive and Ama 102 drug resistant lines with alpha-amanitin. In the latter, resistance is due to a mutation in RNA polymerase II. The toxin profoundly reduces synthesis of virus-specified polypeptides and morphopoeisis in Ama+ but not in Ama 102 rat myoblasts without appreciably altering vaccinia DNA replication in either cell type. This implicates RNA polymerase II in the expression of late virus functions. Circumstantial evidence from a model system indicates that gamma irradiation of the host prior to infection might disrupt transcription into functional mRNA from the nucleus. Irradiation does not, however, alter the capability of the host to support vaccinia multiplication fully. Therefore, ongoing host nuclear transcription may not be required by this virus. The above results are consistent with the ability of cytoplasts to produce small quantities of mature progeny. Our studies lead us to hypothesize that RNA polymerase II or a subunit of the host enzyme may participate directly in late transcription of the vaccinia genome.
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PMID:Biogenesis of poxviruses: role for the DNA-dependent RNA polymerase II of the host during expression of late functions. 22 96

We have recently demonstrated that globin mRNAs are effective primers for influenza viral RNA transcription in vitro catalyzed by the virion transcriptase [Bouloy, M., Plotch, S. J. & Krug, R. M. (1978) Proc. Natl. Acad. Sci. USA 75, 4886-4890]. Here, we present direct evidence that the 5'-terminal methylated cap of the globin mRNAs is transferred to viral complementary RNA (cRNA) during transcription. Chemical (beta-elimination) or enzymatic removal of the cap of globin mRNAs eliminated essentially all their priming activity. Much of this activity could be restored by recapping the beta-eliminated globin mRNAs with the vaccinia virus guanylyl and methyl transferases. Globin mRNAs containing (32)P label only in the cap (m(7)G(32)pppm(6)A(m)-) were prepared by recapping beta-eliminated globin mRNAs with the vaccinia virus enzymes, [alpha-(32)P]GTP, and unlabeled S-adenosylmethionine. By using this labeled globin mRNA as primer and unlabeled nucleoside triphosphates as precursors, the viral cRNA segments that were synthesized were shown to contain a (32)P-labeled 5'-terminal cap structure. Gel electrophoretic analysis indicated that the globin mRNA-primed cRNA segments were 10-15 nucleotides longer at their 5' end than ApG-primed cRNA segments, which initiate exactly at the 3' end of the virion RNA templates. This suggests that, in addition to the cap, about 10-15 other nucleotides are also transferred from the globin mRNA to viral cRNA. A mechanism for the priming of influenza viral cRNA synthesis by globin mRNA is proposed.
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PMID:Transfer of 5'-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro. 28 3

Adenosine (beta,gamma-imido)triphosphate (AMP-PNP) and guanosine (beta,gamma-imido)triphosphate (GMP-PNP) are analogs of ATP and GTP with non-hydrolyzable gamma-phosphates. Although both AMP-PNP and GMP-PNP were used in place of ATP and GTP by Escherichia coli RNA polymerase to transcribe vaccinia virus DNA, only GMP-PNP was used by the transcriptase present within vaccinia virus cores. AMP-PNP specifically prevented initiation of transcription, since RNA initiated in the presence of ATP, GTP, and CTP was subsequently elongated by incubating the washed cores in the presence of AMP-PNP, GTP, CTP, and UTP. The RNA formed in this manner, however, was (i) several times longer than normal transcripts, indicating a defect in chain termination and/or cleavage of nascent RNA, (ii) was not polyadenylylated (although free polyadenylic acid formed), and (iii) was not extruded from the virus cores. Nearest neighbor analysis demonstrated that AMP-PNP was incorporated adjacent to all four nucleotides, and hybridization to restriction endonuclease fragments of vaccinia virus DNA indicated that the high-molecular-weight RNA was transcribed from representative fractions of the entire genome. The possibility of a block in processing rather than or in addition to a block in chain termination was suggested by the cleavage of the high-molecular-weight RNA within the core after replacement of AMP-PNP with ATP. Cleavage of purified high-molecular-weight RNA by a soluble endoribonuclease extracted from vaccinia virus cores, however, was not dependent upon ATP, nor was it inhibited by AMP-PNP. The latter results suggest that AMP-PNP blocks a step preceding cleavage.
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PMID:Multiple roles for ATP in the synthesis and processing of mRNA by vaccinia virus: specific inhibitory effects of adenosine (beta,gamma-imido) triphosphate. 69 Nov 15

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

Although the bulk of RNA synthesized in vitro by vaccinia virus is 8 to 12 S, a small amount of high molecular weight RNA can be detected. This RNA is virion-associated and is not extruded from the virus as high molecular weight RNA. It is sensitive to pancreatic RNase digestion in high salt, has a density in neutral CS2SO4 of 1.68 g ml-1 and remains large after digestion with DNase or denaturation in dimethyl sulfoxide. In the presence of high concentrations of virus in the in vitro RNA polymerase reaction, pulse-labeling experiments indicate an RNA sedimenting heterogeneously between 20 and 30 S. Pulse-chase experiments indicate that a fraction of this high molecular weight RNA can be chased into RNA sedimenting at 8 to 12 S. Cleavage into smaller fragments is not dependent on continued RNA synthesis but does require ribonucleoside triphosphates. In the presence of ethidium bromide, the RNA is not cleaved.
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PMID:In vitro synthesis of a high molecular weight virion-associated RNA by vaccinia. 83 1

In vaccinia virus infected cells the appearance of a late enzyme RNA polymerase was prevented by MPB, an inhibitor of nucleolar RNA synthesis, although inductions of the early enzymes thymidine kinase and DNA polymerase were not affected. It is inferred the nucleoli may be involved in the replication of vaccinia virus.
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PMID:Failure of poxvirus replication in the presence of an inhibitor of nucleolar RNA synthesis. 85 97

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.
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PMID:Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells. 130 73

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.
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PMID:Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli. 130 92


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