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)

Eight genome RNA segments are present in both normal and von Magnustype influenza virus preparations and all species are transcribed by the virion-associated polymerase. Although the RNA polymerase activity and the amount of the three largest RNA segments are reduced in defective influenza virus preparations, these reductions do not appear to be great enough to account for the much greater loss of infectivity.
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PMID:Transcriptase activity and genome composition of defective influenza virus. 125 76

The 12 nucleotide conserved sequence at the 3' end of influenza A virion RNA is sufficient to function as a promoter in vitro. By introducing point mutations in all 12 positions of this promoter in model RNA templates and studying the efficiency of RNA synthesis in vitro, we show that only three nucleotides, residues 9, 10 and 11, are crucial for activity, although other nucleotides play a significant but less important role. Additions or deletions within the promoter are tolerated, resulting in either an increase or a decrease in promoter activity, depending on the mutation introduced; in some cases premature termination is caused. Taking these observations into account, a model for RNA polymerase binding and copying of the promoter is discussed.
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PMID:Nucleotides 9 to 11 of the influenza A virion RNA promoter are crucial for activity in vitro. 146 51

The non-structural protein NS1, encoded by genome segment 8 of influenza A virus, was expressed in Escherichia coli from cloned cDNA and purified. The NS1 protein had a specific RNA-binding activity, binding to influenza A virus minus-sense but not plus-sense RNA synthesized in vitro from cloned DNA using phage RNA polymerase. NS1 bound preferentially to the regions of RNA containing either 5'- or 3'-terminal common sequences of the genomic RNA. Binding was inhibited by virion RNA, but not by single-stranded minus-sense cDNA and oligo DNAs having the common sequences. In addition, binding was also inhibited by 28S rRNA but not 18S rRNA prepared from MDCK cells.
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PMID:Specific binding of influenza A virus NS1 protein to the virus minus-sense RNA in vitro. 153 Sep 62

Human MxA and mouse Mx1 are interferon-induced proteins capable of inhibiting the multiplication of influenza virus. MxA protein is localized in the cytoplasm, whereas Mx1 protein accumulates in the nucleus. Taking advantage of stably transfected cell lines that constitutively express either MxA or Mx1 protein, we examined the steps at which these proteins block influenza A viruses. In infected cells expressing MxA protein, all viral mRNAs synthesized as a result of primary transcription in the nucleus by the virion-associated RNA polymerase accumulated to normal levels. These primary viral transcripts were polyadenylated, were active in directing viral protein synthesis in vitro, and appeared to be efficiently transported to the cell cytoplasm. Yet viral protein synthesis and genome amplification were strongly inhibited, suggesting that MxA protein interfered with either intracytoplasmic transport of viral mRNAs, viral protein synthesis, or translocation of newly synthesized viral proteins to the cell nucleus. However, in infected cells expressing Mx1 protein, the concentrations of the longest primary transcripts encoding the three influenza virus polymerase proteins PB1, PB2, and PA were at least 50-fold reduced. Accumulation of the shorter primary transcripts encoding the other viral proteins was also inhibited but to a lesser extent. These results demonstrate that the mouse Mx1 protein interferes with primary transcription of influenza virus in the nucleus, whereas the human MxA protein inhibits a subsequent step that presumably takes place in the cytoplasm of infected cells.
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PMID:Human and mouse Mx proteins inhibit different steps of the influenza virus multiplication cycle. 154 81

A new transfection system for influenza virus was developed using the clone 76 cell line, in which the viral RNA polymerase and nucleoprotein (NP) genes can be expressed in response to dexamethasone. Ribonucleoprotein (RNP) complexes were reconstituted by expressing proteins from a chimeric NS-chloramphenicol acetyltransferase (CAT) RNA consisting of the full-length negative-strand RNA of the CAT gene positioned between the 5'- and 3'-terminal sequences of influenza virus RNA segment 8, and purifying NP from an NP gene-expressing Escherichia coli strain. When the reconstituted RNP was transfected into clone 76 cells, CAT was produced only when the synthesis of the three RNA polymerase subunits and NP was induced by treatment with dexamethasone.
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PMID:Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes. 160 55

The transcription and replication of influenza RNA can be studied in vitro by the reconstitution of functional ribonucleoprotein (RNP) complex from viral core proteins including the RNA polymerase (complex of three P protein subunits) and nucleoprotein (NP), and model templates. Here, two different core protein preparations, one based on CsCl centrifugation (CS enzyme) and the other on micrococcal nuclease treatment of viral cores (MN enzyme), were compared side-by-side. Short model RNA templates and their 3'-half molecules of both viral RNA (vRNA) and complementary RNA (cRNA) senses were reconstituted with the core protein preparations in parallel, and RNA polymerase activity was tested either in the presence or absence of ApG or globin mRNA as primers. Both enzyme preparations were active in the syntheses of short vRNA and cRNA transcripts using ApG as a primer, although the synthesis of cRNA was 2-10-fold higher (depending on the template used) than the synthesis of vRNA. The MN enzyme, however, was more active per weight of total protein than the CS enzyme, probably because of its higher content of RNA polymerase. Both enzymes failed to show primer-independent synthesis of vRNA. The differences observed in the synthesis of short transcripts using globin mRNA as a primer are discussed.
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PMID:Comparison of two reconstituted systems for in vitro transcription and replication of influenza virus. 161 40

Influenza virus RNA polymerase catalyzes multiple step reactions in transcription and replication of the genome RNA. The core enzyme is composed of each one of the three P proteins, PB1, PB2 and PA (Honda et al. (1990) J. Biochem. 107, 624-628). For detailed analysis of the role of each P protein and of the functional domains on each P polypeptide, we expressed individual P proteins in cultured insect cells after infection with recombinant baculoviruses. PB1 and PB2 accumulated in cell nuclei whereas PA stayed in cytoplasm. Both the PB1 and PB2 proteins were purified from aggregates in the respective nuclear extract, and the PA was partially purified from the cytoplasm. RNA polymerase was reconstituted by mixing the three P proteins in a urea solution and then dialyzing against a reconstitution buffer. The reconstituted enzyme was able to transcribe model RNA templates. Minus-sense RNA was a better template than plus-sense RNA.
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PMID:Reconstitution of influenza virus RNA polymerase from three subunits expressed using recombinant baculovirus system. 162 19

In order to establish cell lines which complement the growth of temperature-sensitive (ts) mutants of influenza virus, three RNA polymerase and nucleoprotein (NP) genes each linked to the mouse mammary tumor virus LTR were cloned into the bovine papillomavirus vector DNA. After co-transfection of mouse C127 cells with these recombinant plasmids, a cell line, clone 76, in which the expression of the three polymerase and NP genes could be stimulated by dexamethasone, was established. The clone 76 cells could complement the growth of ts-mutants defective in one of the polymerase subunit genes at the nonpermissive temperature in response to dexamethasone. The results suggest that the simultaneous expression of the three polymerase genes in the same compartment of protein synthesis machinery is required for an efficient complementation of ts-mutant growth.
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PMID:Growth complementation of influenza virus temperature-sensitive mutants in mouse cells which express the RNA polymerase and nucleoprotein genes. 166 10

The influenza RNA polymerase is known to catalyse three distinct copying activities: (i) transcription of minus-sense virion RNA (vRNA) into mRNA, (ii) transcription of vRNA into full-length complementary RNA (cRNA), and (iii) transcription of cRNA to vRNA. Ever since the discovery of the conserved 13 and 12 long sequences at each end of all the influenza RNA segments, these have been good candidates for promoters of transcription. By devising a new, simple method for preparing influenza polymerase complex capable of transcribing in vitro added short model RNA templates without interference from endogenous viral RNA, we have now tested the promoter hypothesis. We conclude that the 13 long and the 12 long 3' conserved sequences of cRNA and vRNA of influenza A virus are by themselves sufficient to promote vRNA and cRNA synthesis in vitro. Using our new method, we also show that chloramphenicol acetyl transferase (CAT) activity can be detected in MDBK (bovine kidney) cells, after transfection of influenza polymerase assembled with a negatively stranded CAT RNA, even in the absence of helper virus. As in a previously described method (Luytjes et al., 1989), CAT activity is amplified by helper virus and can be rescued in infectious recombinant virus.
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PMID:A new method for reconstituting influenza polymerase and RNA in vitro: a study of the promoter elements for cRNA and vRNA synthesis in vitro and viral rescue in vivo. 172

Influenza viruses were disrupted layer by layer with the nonionic detergent NP-40 at fixed pH. Treatment of the virions with NP-40 at neutral or mildly alkaline pH (6.8-8.0) yielded viral core structures containing M1 protein. The matrix M1 protein was selectively extracted from cores at acidic pH 3.0-4.5 with citrate, acetate, and phosphate buffers or with morpholinoethanesulfonic acid. The resulting M1 protein sedimented in a glycerol gradient with a coefficient of 2.8 S and most likely existed as a monomeric form of the 27,000-Da polypeptide. An antigenic map of the monomeric protein M1 tested with a panel of monoclonal anti-M1 antibodies was found to be similar to those of the assembled M1 protein in whole virions. The isolated M1 protein retained biological properties and inhibited the RNA polymerase activity of viral RNP. This transcription-inhibition function of M1 monomers was specifically restricted by one of the monoclonal antibodies studied.
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PMID:Isolation of matrix protein M1 from influenza viruses by acid-dependent extraction with nonionic detergent. 172 9


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