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)

The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. For the molecular anatomy of this multifunctional enzyme, we have established a simultaneous expression of three P proteins in cultured insect cells using recombinant baculoviruses. For purification of P protein complexes, the PA protein was expressed as a fusion with a histidine tag added at its N terminus. By using affinity chromatography, a complex consisting of the three P proteins was isolated from nuclear extracts of virus-infected cells. The affinity-purified 3P complex showed the activities of capped RNA binding, capped RNA cleavage, viral model RNA binding, model RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. We conclude that a functional form of the viral RNA polymerase with the catalytic specificity of transcriptase is formed in recombinant baculovirus-infected insect cells. Using the viral RNA-free 3P complex, we found that the capped RNA cleavage takes place in the presence of vRNA but not of cRNA, indicating that the vRNA functions as a regulatory factor for the specificity control of viral RNA polymerase as well as a template for transcription. The structural elements of RNA directing the expression of RNA polymerase functions were analyzed using variant forms of the model RNA templates.
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PMID:Differential roles of viral RNA and cRNA in functional modulation of the influenza virus RNA polymerase. 1137 86

The isolation of infectious salmon anaemia virus (ISAV) from asymptomatic wild fish species including wild salmon, sea trout and eel established that wild fish can be a reservoir of ISAV for farmed Atlantic salmon. This report characterizes the biological properties of ISAV isolated from a disease outbreak in farmed Coho salmon in Chile and compares it with ISAV isolated from farmed Atlantic salmon in Canada and Europe. The virus that was isolated from Coho salmon tissues was initially detected with ISAV-specific RT-PCR (reverse transcription-polymerase chain reaction). The ability of the virus to grow in cell culture was poor, as cytopathology was not always conspicuous and isolation required passage in the presence of trypsin. Virus replication in cell culture was detected by RT-PCR and IFAT (indirect fluorescent antibody test), and the virus morphology was confirmed by positive staining electron microscopy. Further analysis of the Chilean virus revealed similarities to Canadian ISAV isolates in their ability to grow in the CHSE-214 cell line and in viral protein profile. Sequence analysis of genome segment 2, which encodes the viral RNA polymerase PB1, and segment 8, which encodes the nonstructural proteins NS1 and NS2, showed the Chilean virus to be very similar to Canadian strains of ISAV. This high sequence similarity of ISAV strains of geographically distinct origins illustrates the highly conserved nature of ISAV proteins PB1, NS1 and NS2 of ISAV. It is noteworthy that ISAV was associated with disease outbreaks in farmed Coho salmon in Chile without corresponding clinical disease in farmed Atlantic salmon. This outbreak, which produced high mortality in Coho salmon due to ISAV, is unique and may represent the introduction of the virus to a native wild fish population or a new strain of ISAV.
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PMID:Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in Chile. 1141 49

The first 11 nt at the 5' end of influenza virus genomic RNA were shown to be both necessary and sufficient for specific binding by the influenza virus polymerase. A novel in vitro transcription assay, in which the polymerase was bound to paramagnetic beads via a biotinylated 5'-vRNA oligonucleotide, was used to study the activities of different forms of the polymerase. Complexes composed of co-expressed PB1/PB2/PA proteins and a sub-complex composed of PB1/PA bound to the 5'-vRNA oligonucleotide, whereas PB1 expressed alone did not. The enriched 5'-vRNA/PB1/PB2/PA complex was highly active for ApG and globin mRNA primed transcription on a model 3'-vRNA template. RNA synthesis in the absence of added primers produced products with 5'-terminal tri- or diphosphate groups, indicating that genuine unprimed initiation of transcription also occurred. No transcriptase activity was detected for the PB1/PA complex. These results demonstrate a role for PA in the enhancement of 5' end binding activity of PB1, a role for PB2 in the assembly of a polymerase complex able to perform both cap-dependent and -independent synthesis and that NP is not required for the initiation of replicative transcription.
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PMID:Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase. 1178 4

Influenza virus RNA polymerase consists of three subunits, PB1, PB2 and PA, and catalyzes both transcription and replication of the RNA genome. PB1 is a catalytic subunit of RNA polymerization and a core of the subunit assembly. The subunit binding sites were mapped at about several hundred amino-acid size. Fine mapping of the subunit binding sites was determined. The PB1-PA binding regions were mapped within in the N-terminal 25 amino acids of PB1 and 668-692 of PA. PB1 and PB2 interacted within wider regions, 600-757 of PB1 and 51-259 of PB2. In these amino-acid spans, 206-259 of PB2 may be the most important region of PB1 binding and 718-732 of PB1 may be the most important region of PB2 binding because the binding activity was lost when the regions were lost in the subunits. The additional regions contributed to strong binding of these subunits.
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PMID:Fine mapping of the subunit binding sites of influenza virus RNA polymerase. 1200 25

The influenza A virus RNA-dependent RNA polymerase consists of three subunits-PB1, PB2, and PA. The PB1 subunit is the catalytically active polymerase, catalyzing the sequential addition of nucleotides to the growing RNA chain. The PB2 subunit is a cap-binding protein that plays a role in initiation of viral mRNA synthesis by recruiting capped RNA primers. The function of PA is unknown, but previous studies of temperature-sensitive viruses with mutations in PA have implied a role in viral RNA replication. In this report we demonstrate that the PA subunit is required not only for replication but also for transcription of viral RNA. We mutated evolutionarily conserved amino acids to alanines in the C-terminal region of the PA protein, since the C-terminal region shows the highest degree of conservation between PA proteins of influenza A, B, and C viruses. We tested the effects of these mutations on the ability of RNA polymerase to transcribe and replicate viral RNA. We also tested the compatibility of these mutations with viral viability by using reverse-genetics techniques. A mutant with a histidine-to-alanine change at position 510 (H510A) in the PA protein of influenza A/WSN/33 virus showed a differential effect on transcription and replication. This mutant was able to perform replication (vRNA-->cRNA-->vRNA), but its transcriptional activity (vRNA-->mRNA) was negligible. In vitro analyses of the H510A recombinant polymerase, by using transcription initiation, vRNA-binding, capped-RNA-binding, and endonuclease assays, suggest that the primary defect of this mutant polymerase is in its endonuclease activity.
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PMID:A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs. 1218 83

The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. The PB1 subunit plays a key role in both the assembly of three P protein subunits and the catalytic function of RNA polymerization. We have established a simultaneous expression system of three P proteins in various combinations using recombinant baculoviruses, and isolated the PA-PB1-PB2 ternary (3P) complex and two kinds of the binary (2P) complex, PA-PB1 and PB1-PB2. The affinity-purified 3P complex showed all of the catalytic properties characteristic of the transcriptase, including capped RNA-binding, capped RNA cleavage, model viral RNA binding, model viral RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. The PB1-PB2 binary complex showed essentially the same catalytic properties as does the 3P complex, whereas the PA-PB1 complex catalyzed de novo initiation of RNA synthesis in the absence of primers. Taken together we propose that the catalytic specificity of PB1 subunit is modulated to the transcriptase by binding PB2 or the replicase by interaction with PA.
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PMID:Minimum molecular architectures for transcription and replication of the influenza virus. 1227 Nov 17

The multiplication of Ulster 73 virus, an avian strain of type A influenza virus, was blocked in chick embryo fibroblast cells, CEF, by treatment with 0.5 microg/ml of chromomycin A3 whereas in LLC-MK2 cells no inhibition of replication was observed. Virus-induced polypeptide synthesis in chick embryo fibroblast cells was confined to the synthesis of PB2, PB1 and PA subunits of the RNA dependent-RNA polymerase, the nucleoprotein NP, the non-structural protein NS1, the haemagglutinin HA, the non-structural protein NS2; only the membrane M1 polypeptide synthesis was greatly inhibited. Viral unpolyadenylated cRNAs synthesis was studied at a late time of the infection, 8 hours p.i.: chromomycin A3 was able to inhibit the "novo" synthesis of complementary RNA poly(A)- and segment 7 of virion RNA. The mode of action of the drug in chick embryo fibroblast cells is discussed.
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PMID:Chromomycin A3 inhibits influenza a virus multiplication in chick embryo fibroblast cells. 1243 17

The influenza A virus promoter is recognized by the influenza A virus RNA-dependent RNA polymerase, and directs both transcription and replication of the viral RNA genome. Within the sequence of this promoter, flu strains exhibit a natural, unique variation, either a U or a C, at the fourth position from the 3' end. Promoters that contain a C residue (C4 promoter), which are invariably found in genome segments that encode the three RNA polymerase subunits (PB1, PB2 and PA), down-regulate transcription but activate genome replication. Here, we have determined the structure of the C4 promoter by NMR spectroscopy and compared it with the structure of the U4 promoter, which was determined previously. The structure of the internal loop in the C4 promoter is similar to that of the U4 promoter. However, the terminal stem of the C4 promoter is strikingly different from that of the U4 promoter. These structural data suggest that the internal loop is important for polymerase binding to the promoter, and the terminal stem is crucial for differential regulation of transcription and replication.
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PMID:A single-nucleotide natural variation (U4 to C4) in an influenza A virus promoter exhibits a large structural change: implications for differential viral RNA synthesis by RNA-dependent RNA polymerase. 1258 41

Mx proteins belong to the dynamin superfamily of high molecular weight GTPases and interfere with multiplication of a wide variety of viruses. Earlier studies show that nuclear mouse Mx1 and human MxA designed to be localized in the nucleus inhibit the transcription step of the influenza virus genome. Here we set a transient influenza virus transcription system using luciferase as a reporter gene and cells expressing the three RNA polymerase subunits, PB1, PB2 and PA, and NP. We used this reporter assay system and nuclear-localized MxA proteins to get clues for elucidating the anti-influenza virus activity of MxA. Nuclear-localized VP16-MxA and MxA-TAg NLS strongly interfered with the influenza virus transcription. Over-expression of PB2 led to a slight resumption of the transcription inhibition by nuclear MxA, whereas over-expression of PB1 and PA did not affect the MxA activity. Of interest is that the inhibitory activity of the nuclear MxA was markedly neutralized by over-expression of NP. An NP devoid of its C-terminal region, but containing the N-terminal RNA binding domain, also neutralized the VP16-MxA activity in a dose-dependent manner, whereas an NP lacking the N-terminal region did not affect the VP16-MxA activity. Further, not only VP16-MxA but also the wild-type MxA was found to interact with NP in influenza virus-infected cells. This indicates that the nuclear MxA suppresses the influenza virus transcription by interacting with not only PB2 but also NP.
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PMID:Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome. 1475 52

Influenza virus genomic RNA segments are packaged into ribonucleoprotein (RNP) structures by the PB1, PB2, and PA subunits of an RNA polymerase and a single-strand RNA-binding nucleoprotein (NP). Assembly and function of these ribonucleoproteins depend on a complex set of protein-protein and protein-RNA interactions. Here, we identify new functional domains of PB2. We show that PB2 contains two regions that bind NP and also identify a novel PB1 binding site. The regions of PB2 responsible for binding NP and PB1 show considerable overlap, and binding of NP to the PB2 fragments could be outcompeted by PB1. The binding domains of PB2 acted as trans-dominant inhibitors of viral gene expression, and consistent with the in vitro binding data, their inhibitory activity depended on the concentration of wild-type PB2, NP, and PB1. This provides evidence for functionally significant and potentially regulatory interactions between PB2 and NP.
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PMID:Functional domains of the influenza A virus PB2 protein: identification of NP- and PB1-binding sites. 1503 71


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