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)

We showed previously that cells expressing the vesicular stomatitis virus (VSV) L polymerase gene via the vaccinia-T7 RNA polymerase system accumulated 2- to 5-fold more L protein when the P protein was coexpressed (Canter et al., 1993, Virology 194, 518-529). The results presented here provide an explanation for this phenomenon. Pulse-chase analysis revealed that L was unstable with a half-life of 3 to 6 hr if expressed in the absence of P protein, but was stable for at least 16 hr when coexpressed with a 10- to 15-fold molar excess of P. The P protein, in contrast, was stable under both conditions. Stabilization correlated with formation of a P:L polymerase complex evidenced both by coimmunoprecipitation and by glycerol gradient sedimentation analyses. A mutant L protein, lacking amino acids 1638 to 1673, was not stabilized by coexpression and showed no binding to P protein. Its anomalous sedimentation, however, suggested misfolding and/or aggregation as the cause for the failure to bind P. Transcription reconstitution in vitro, using extracts from cells expressing excess of P over L protein, strongly depended on coexpression of the proteins for optimal activity. We propose that the coexpression dependence for polymerase reconstitution documented here for VSV, as well as that reported previously for the Sendai paramyxovirus, reflects the protective effect of P protein on L protein stability.
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PMID:Stabilization of vesicular stomatitis virus L polymerase protein by P protein binding: a small deletion in the C-terminal domain of L abrogates binding. 863 3

RNA polymerase of influenza virus with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the genome RNA. The RNA polymerase with transcription activity was reconstituted from three P proteins, which were separately isolated from insect cells infected with recombinant baculoviruses, each carrying cDNA for one P protein. Nuclear extracts of the insect cells infected with each of the recombinant baculoviruses or various combinations of these viruses were examined for transcription and replication activities. The nuclear extract of cells expressing all three P proteins catalyzed model template-directed RNA synthesis in the absence of primers (an indication of RNA replication), supporting the notion that the complete set of three P proteins is required for RNA replication. All the nuclear extracts containing the PB1 subunit, including the extract containing PB1 alone, were able to catalyze model template-directed dinucleotide-primed RNA synthesis (an indication of transcription). These observations not only confirm that the PB1 protein is a catalytic subunit of influenza virus RNA polymerase, but also indicate that PB1 alone is able to catalyze RNA synthesis in the absence of PB2 and PA subunits.
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PMID:Influenza virus PB1 protein is the minimal and essential subunit of RNA polymerase. 864 93

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. By transfection of various combinations of cDNA encoding wild-type and serial deletion mutants of each P protein subunit and co-immunoprecipitation with subunit-specific antibodies, the subunit-subunit contact sites on all three of the P proteins were determined. Results indicate that binary complexes are formed between PB1-PB2 and PB1-PA but not between PB2-PA. Therefore, we concluded that PB1 is the core subunit for assembly of the virus RNA polymerase. The C-terminal 158 amino acids of PB1 bound to the N-terminal 249 amino acids of PB2, while the N-terminal 140 amino acids of PB1 bound to the C-terminal two-thirds of PA. PB2-PA binding was not detected when they were expressed in the absence of the PB1 subunit.
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PMID:Molecular assembly of the influenza virus RNA polymerase: determination of the subunit-subunit contact sites. 881 Oct 14

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. Enzyme reconstitution experiments indicated that all three P proteins are required for RNA synthesis in vitro (Kobayashi, et al. Virus Res 22, 235-245, 1992). Nuclear extracts of HeLa cells infected with three kinds of the recombinant vaccinia virus, each carrying one of the three P protein cDNAs, exhibited the activity of complete replication, that is, vRNA-sense RNA-directed and cRNA-sense RNA-directed RNA synthesis in the absence of primers. The nuclear extract from cells singly infected with the virus carrying PB1 cDNA exhibited a significant level of model v-sense RNA-directed RNA synthesis activity. Thus we conclude that PB1 is the catalytic subunit of influenza virus RNA polymerase and that under certain conditions, PB1 alone is able to catalyze RNA synthesis in vitro.
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PMID:Molecular dissection of influenza virus RNA polymerase: PB1 subunit alone is able to catalyze RNA synthesis. 887 32

Influenza virus, a negative strand RNA virus, cannibalizes host cell, capped RNA polymerase II transcripts in the nucleus via a process termed "cap-snatching". The viral transcriptase enzyme; which is composed of a complex of the three viral polymerase (P) proteins, contains a cap-dependent endonuclease that cleaves capped cellular RNAs in the nucleus 10-13 nucleotides from their 5' ends. The resulting capped RNA fragments are required as primers for the initiation of viral mRNA synthesis. In the 18 year since the discovery of "cap-snatching" it has not been determined how the viral transcriptase exhibits selectivity and "snatches" caps from cellular, but not viral, mRNAs. Here we elucidate the surprising mechanism of this selectivity: the complex of the same three viral P proteins that catalyzes "cap-snatching" is also responsible for selectivity protecting the 5' ends of viral, but not cellular, mRNAs from "cap-snatching". The viral P protein complex is able to acquire these two very different functions because this complex lacks any detectable activity unless it binds to one or more specific RNA sequences. Here we demonstrate that the viral P protein complex binds to the common sequence in all the viral mRNAs that is immediately 3' to the 5' sequence that is "snatched" from host cell RNAs. This binding activates the cap-binding activity of the P protein complex, thereby enhancing its binding to the capped viral mRNA. We show that these P protein complexes protect the 5' ends of viral mRNAs from endonucleolytic cleavage by the viral transcriptase, whereas the 5' ends of nonviral mRNAs are not protected.
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PMID:Surprising function of the three influenza viral polymerase proteins: selective protection of viral mRNAs against the cap-snatching reaction catalyzed by the same polymerase proteins. 895 65

The phosphoproteins (P) of nonsegmented negative strand RNA viruses are viral RNA polymerase subunits involved in both transcription and replication during the virus life cycle. Phosphorylation of P proteins in several negative strand RNA viruses by specific cellular kinases was found to be required for P protein function. In the present study, using bacterially expressed unphosphorylated P protein of Sendai virus, a mouse parainfluenza virus, we have shown that the major cellular kinase that phosphorylates P protein in vitro is biochemically and immunologically indistinguishable from protein kinase C (PKC) zeta isoform. PKC zeta was packaged into the Sendai virion and remained associated with purified viral ribonucleoprotein, where it phosphorylated both the P and the nucleocapsid protein in vitro. When PKC zeta-specific inhibitory pseudosubstrate peptide was introduced into LLC-MK2 cells prior to Sendai virus infection, production of progeny virus was dramatically attenuated, and kinetic analysis revealed that primary transcription was repressed. These data indicate that phosphorylation of the Sendai virus P protein by PKC zeta plays a critical role in the virus life cycle.
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PMID:Phosphorylation of Sendai virus phosphoprotein by cellular protein kinase C zeta. 919 69

The genome of influenza virus is composed of eight RNA segments of negative polarity. The RNA-dependent RNA polymerase is associated with each viral RNA (vRNA) segment and in virus-infected cells, involved in both transcription, i.e. vRNA-directed synthesis of viral mRNA, and two step reactions of vRNA replication, i.e. vRNA-dependent synthesis of complementary RNA (cRNA) and cRNA-dependent synthesis of vRNA. The RNA polymerase is composed of three viral proteins, PB1, PB2 and PA. PB1 is the core subunit for not only the RNA synthesis but also the assembly of PB2 and PA into this multifunctional enzyme complex. PB1 alone is able to catalyze vRNA-dependent RNA synthesis, but PB2 is required for capped RNA-dependent transcription, both together forming the transcriptase. The third P protein, PA, and an as yet unidentified host factor(s) are involved in the conversion of RNA polymerase from transcriptase to replicase. The functional map is being made for both PB1 and PB2 proteins.
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PMID:The molecular anatomy of influenza virus RNA polymerase. 922 27

Nonsegmented negative strand RNA viruses package an RNA-dependent RNA polymerase composed of two subunits, a large protein L and a phosphoprotein P, for transcription and replication of their genome RNAs. The RNA polymerase activity resides within the L protein, while the P protein acts as a transcription factor or transactivator of the polymerase. Since P protein is heavily phosphorylated and phosphorylation is known to regulate function of many viral as well as cellular proteins, the role of phosphorylation of P protein in the gene expression of this group of RNA viruses has recently been investigated. Through expression in bacteria the P protein was produced in large quantity in the nonphosphorylated form and involvement of cellular kinase(s) in its phosphorylation was studied. Casein kinase II and/or protein kinase C have been shown to play a critical role in the activation of P protein in transcription. These findings have opened up a new avenue for studying an important regulatory step in virus gene expression that may lead to the development of an effective antiviral agent.
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PMID:Role of cellular kinases in the gene expression of nonsegmented negative strand RNA viruses. 922 28

Phosphorylation by casein kinase II at three specific residues (S-60, T-62, and S-64) within the acidic domain I of the P protein of Indiana serotype vesicular stomatitis virus has been shown to be critical for in vitro transcription activity of the viral RNA polymerase (P-L) complex. To examine the role of phosphorylation of P protein in transcription as well as replication in vivo, we used a panel of mutant P proteins in which the phosphate acceptor sites in domain I were substituted with alanines or other amino acids. Analyses of the alanine-substituted mutant P proteins for the ability to support defective interfering RNA replication in vivo suggest that phosphorylation of these residues does not play a significant role in the replicative function of the P protein since these mutant P proteins supported replication at levels > or = 70% of the wild-type P-protein level. However, the transcription function of most of the mutant proteins in vivo was severely impaired (2 to 10% of the wild-type P-protein level). The level of transcription supported by the mutant P protein (P(60/62/64)) in which all phosphate acceptor sites have been mutated to alanines was at best 2 to 3% of that of the wild-type P protein. Increasing the amount of P(60/62/64) expression in transfected cells did not rescue significant levels of transcription. Substitution with other amino acids at these sites had various effects on replication and transcription. While substitution with threonine residues (P(TTT)) had no apparent effect on transcription (113% of the wild-type level) or replication (81% of the wild-type level), substitution with phenylalanine (P(FFF)) rendered the protein much less active in transcription (< 5%). Substitution with arginine residues led to significantly reduced activity in replication (6%), whereas glutamic acid substituted P protein (P(EEE)) supported replication (42%) and transcription (86%) well. In addition, the mutant P proteins that were defective in replication (P(RRR)) or transcription (P(60/62/64)) did not behave as transdominant repressors of replication or transcription when coexpressed with wild-type P protein. From these results, we conclude that phosphorylation of domain I residues plays a major role in in vivo transcription activity of the P protein, whereas in vivo replicative function of the protein does not require phosphorylation. These findings support the contention that different phosphorylated states of the P protein regulate the transcriptase and replicase functions of the polymerase protein, L.
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PMID:Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription but not for replication. 934 67

The genome of influenza virus is composed of eight RNA segments of negative polarity. The RNA-dependent RNA polymerase is associated with each viral RNA (vRNA) segment and after infection, involved in both transcription (vRNA-directed synthesis of viral mRNA) and vRNA replication (vRNA-dependent synthesis of complementary RNA(cRNA) and cRNA-dependent synthesis of vRNA). The RNA polymerase is composed of three viral proteins, PB1, PB2 and PA. PB1 is the core subunit for not only the RNA synthesis but also the assembly of PB2 and PA into this multifunctional enzyme complex. PB1 alone is able to catalyze vRNA-dependent RNA synthesis, but PB2 is required for capped RNA-dependent transcription, both together forming the transcriptase. The third P protein, PA, and an as yet unidentified host factor(s) are involved for the conversion of RNA polymerase from transcriptase to replicase. The functional map including both subunit-subunit contact sites and catalytic sites for capped RNA endonuclease and RNA polymerization is being made for both PB1 and PB2 proteins.
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PMID:[Transcription and replication of influenza virus genome]. 936 Mar 71


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