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)

Measles virus (MV) expresses at least 3 proteins from the phosphoprotein (P) cistron. Alternative translation initiation directs synthesis of the C protein from the +1 reading frame, while so-called RNA editing generates a second population of mRNAs which express the V protein from the -1 reading frame which lies within and overlaps the larger P reading frame. While the P protein has been demonstrated to be an essential cofactor for the L protein in the formation of active transcriptase complexes, the functions of the V and C proteins remain unknown. In order to investigate these functions, we have expressed the MV P, V and C proteins as GST fusions in E. coli for affinity purification and use in an in vitro binding assay with other viral and cellular proteins. The P protein was found to interact with L, NP, and with itself. These interactions were mapped to the carboxy-terminal half of the protein which is absent in the V protein. In contrast, both the V and C proteins failed to interact with any other viral proteins, but were each found to interact specifically with one or more cellular proteins. Appropriate aspects of these results were confirmed in vivo using the yeast two-hybrid system. These observations suggest that the V and C proteins may be involved in modulation of the host cellular environment within MV-infected cells. Such activity would be distinct from their previously proposed role in the possible down-regulation of virus-specific RNA transcription and replication.
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PMID:Protein interactions entered into by the measles virus P, V, and C proteins. 857 62

The phosphoprotein (P) of vesicular stomatitis virus was previously shown to assemble into a homomultimer upon phosphorylation by casein kinase II. It thus acquired transcriptional activity, including the ability to bind to the other two transcriptional components, the polymerase L and the N-RNA template. This multimer has now been found to be a trimer using a His-tag dilution method. Trimer stability was assessed using a variation of this method, by measuring the rate of exchange of monomers between preformed tagged and untagged trimers at different values of pH and ionic strength. Exchange rates increased with increasing ionic strength and were similar at pH 6, 8, and 10, but the trimer was completely dissociated at pH 4. This suggests that the trimer is stabilized by electrostatic interactions, probably involving carboxylate and guanidino groups. Addition of viral L protein stabilized the P trimers, completely preventing subunit exchange under transcription conditions. The association constants (Kass) for trimerization of partially active D and A substitution mutants were also determined by His-tag dilution and found to correlate well with transcriptional activity, further confirming that the active species is the trimer. Circular dichroism spectra were identical for phosphorylated and unphosphorylated wild-type P protein and for D and A mutants known to be predominantly trimeric and monomeric, respectively.
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PMID:The transcriptional form of the phosphoprotein of vesicular stomatitis virus is a trimer: structure and stability. 893 54

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

Transcription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent RNA polymerase and transcriptional cofactor P. The P protein is activated by phosphorylation, an event initiated by cellular kinases. The kinase used differs among this group of RNA viruses; vesicular stomatitis virus and respiratory syncytial virus utilize casein kinase II (CKII), whereas human parainfluenza virus type 3 utilizes PKC isoform zeta (PKC-zeta) for activation of its P protein. To identify the cellular kinase(s) involved in the phosphorylation of the canine distemper virus (CDV) P protein, we used recombinant CDV P in phosphorylation assays with native kinase activities present in CV1 cell extracts or purified CKII and PKC isoforms. Here, we demonstrate that the CDV P protein is phosphorylated by two cellular kinases, where PKC-zeta has the major and CKII the minor activities. In contrast, the P protein of another member of the morbillivirus genus, measles virus, is phosphorylated predominantly by CKII, whereas PKC-zeta has only minor activity. Selective inhibition of PKC-zeta activity within CV1 cells eliminated permissiveness to CDV replication, indicating an in vivo role for PKC-zeta in the virus replication cycle. The broad tissue expression of PKC-zeta parallels the pantropic nature of CDV infections, suggesting that PKC-zeta activity is a determinant of cellular permissiveness to CDV replication.
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PMID:Phosphorylation of canine distemper virus P protein by protein kinase C-zeta and casein kinase II. 918 3

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

The phosphoprotein (P) of vesicular stomatitis virus (VSV) serotypes New Jersey [P(NJ)] and Indiana [P(I)] contains a highly conserved carboxy-terminal domain which is required for binding to the cognate N-RNA template as well as to form a soluble complex with the nucleocapsid protein N in vivo. We have shown that the deletion of 11 amino acids from the C terminal end of the P(I) protein abolishes both the template binding and the complex forming activity with the N protein. Within this region, there are conserved basic amino acid residues (R260 and K262) that are potential candidates for such interactions. We have generated mutant P proteins by substitution of these basic amino acid residues with alanine and studied their role in both transcription and replication. We have found that the R260A mutant failed to bind to the N-RNA template, whereas the K262A mutant bound efficiently as the wild-type protein. The R260A mutant, as expected, was unable to support mRNA synthesis in vitro in a transcription reconstitution reaction as well as transcription in vivo of a minigenome using a reverse genetic approach. However, the K262A mutant supported low level of transcription (12%) both in vitro and in vivo, suggesting that direct template binding of P protein through the C-terminal domain is necessary but not sufficient for optimal transcription. Using a two-hybrid system we have also shown that both R260A and K262A mutants interact inefficiently with the L protein, suggesting further that the two point mutants display differential phenotype with respect to binding to the template. In addition, both R260A and K262A mutants were shown to interact efficiently with the N protein in vivo, indicating that these mutants form N-P complexes which are presumably required for replication. This contention is further supported by the demonstration that these mutants support efficient replication of a DI RNA in vivo. Since the transcription defective P mutants can support efficient replication, we propose that the transcriptase and the replicase are composed of two distinct complexes containing (L-P2-3) and L-(N-P), respectively.
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PMID:Basic amino acid residues at the carboxy-terminal eleven amino acid region of the phosphoprotein (P) are required for transcription but not for replication of vesicular stomatitis virus genome RNA. 937 14

The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites (Ser-226, Ser-227, and Ser-233) were altered to alanines either individually or in various combinations. Analyses of the mutant proteins for their ability to support replication of a VSV minigenomic RNA suggest that phosphorylation of either Ser-226 or Ser-227 is necessary for optimal replication activity of the protein. The mutant protein (P226/227) in which both of these residues were altered to alanines was only about 8% active in replication compared to the wild-type (wt) protein. Substitution of alanine for Ser-233 did not have any adverse effect on replication activity of the protein. In contrast, all the mutant proteins showed activities similar to that of the wt protein in transcription. These results indicate that phosphorylation of the carboxy-terminal domain II residues of P protein are required for optimal replication activity but not for transcription activity. Furthermore, substitution of glutamic acid residues for Ser-226 and Ser-227 resulted in a protein that was only 14% active in replication but almost fully active in transcription. Taken together, these results, along with our earlier studies, suggest that phosphorylation of residues at two different domains in the P protein regulates its activity in transcription and replication of the VSV genome.
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PMID:Optimal replication activity of vesicular stomatitis virus RNA polymerase requires phosphorylation of a residue(s) at carboxy-terminal domain II of its accessory subunit, phosphoprotein P. 1036 10


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