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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The influenza virus RNA polymerase consists of a heterotrimeric complex of the PB1, PB2 and PA proteins, with the PB2 subunit responsible for recognizing 5' cap structures on the host cell RNAs used as primers for virus mRNA synthesis. To investigate further the role PB2 plays in mRNA synthesis, a set of polyclonal antisera raised against defined regions of the protein were tested for their ability to inhibit the virion transcriptase. All five sera were of sufficient titre to immunoprecipitate PB2 and four were capable of recognizing polymerase complexes containing PB1 and PA. However, only the serum raised against the carboxy terminus of PB2 (F5) substantially inhibited polymerase activity. This serum drastically reduced synthesis primed by globin mRNA, but only partially inhibited transcription primed by the dinucleotide ApG, or ApG and cap analogue. The preferential inhibition of globin-primed synthesis did not result from interference with cap recognition, as serum F5 did not reduce labelling of PB2 in a photoaffinity cap-binding assay. However, IgG and Fab fragments from F5 were found to inhibit virion endonuclease activity. This suggests that the C terminus of PB2 plays a crucial role in transcription initiation and implicates PB2 in endonuclease activity.
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PMID:Inhibition of the influenza virus RNA-dependent RNA polymerase by antisera directed against the carboxy-terminal region of the PB2 subunit. 860 68

Influenza virus-associated RNA polymerase is composed of one molecule each of three viral P proteins and carries the complete activity of capped RNA-primed vRNA-directed transcription. The RNA polymerase holoenzyme also carries capped RNA endonuclease to generate capped oligonucleotide primers for transcription and 3'-to-5' exonuclease to remove erroneously polymerized nucleotides at nascent RNA 3' termini prior to the addition of correct substrates. PB1 is the core subunit for not only RNA synthesis but also the assembly of PB2 and PA into the holoenzyme complex, while PB2 plays a key role in capped RNA cleavage. The transcriptase is converted into the RNA replicase with the full activity of replication, ie vRNA-directed cRNA synthesis and cRNA-directed vRNA synthesis, after interaction with an as yet unidentified host factor(s).
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PMID:A multi-functional enzyme with RNA polymerase and RNase activities: molecular anatomy of influenza virus RNA polymerase. 915 Aug 90

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 capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which consists of three proteins PB1, PB2 and PA. Production of primers is activated only when the 5'- and 3'-terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U-substituted RNA and UV crosslinking, we demonstrate that the 5' and 3' sequences of vRNA bind to different amino acid sequences in the same protein subunit, the PB1 protein. Mutagenesis experiments proved that these two amino acid sequences constitute the functional RNA-binding sites. The 5' sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alteration which activates two new functions of the polymerase complex. In addition to the PB2 protein subunit acquiring the ability to bind 5'-capped ends of RNAs, the PB1 protein itself acquires the ability to bind the 3' sequence of vRNA, via a ribonucleoprotein 1 (RNP1)-like motif, amino acids 249-256, which contains two phenylalanine residues required for binding. Binding to this site induces a second allosteric alteration which results in the activation of the endonuclease that produces the capped RNA primers needed for mRNA synthesis. Hence, the PB1 protein plays a central role in the catalytic activity of the viral polymerase, not only in the catalysis of RNA-chain elongation but also in the activation of the enzyme activities that produce capped RNA primers.
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PMID:RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites. 975 84

The cap-dependent endonuclease of the influenza viral RNA polymerase, which produces the capped RNA primers that initiate viral mRNA synthesis, is comprised of two active sites, one for cap binding and one for endonuclease cleavage. We identify the amino acid sequences that constitute these two active sites and demonstrate that they are located on different polymerase subunits. Binding of the 5' terminal sequence of virion RNA (vRNA) to the polymerase activates a tryptophan-rich, cap-binding sequence on the PB2 subunit. At least one of the tryptophans functions in cap binding, indicating that this active site is probably similar to that of other known cap-binding proteins. Endonuclease cleavage, which is activated by the subsequent binding of the 3' terminal sequence of vRNA, resides in a PB1 sequence that contains three essential acidic amino acids, similar to the active sites of other enzymes that cut polynucleotides to produce 3'-OH ends. These results, coupled with those of our previous study, provide a molecular map of the five known essential active sites of the influenza viral polymerase.
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PMID:The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits. 1129 40

The influenza virus polymerase complex contains two associated enzymatic activities, an endoribonuclease and a RNA-dependent RNA polymerase activity. Both activities have so far been observed only with the complete polymerase complex consisting of three subunits, PB1, PB2, and PA. This chapter describes a robust and optimized procedure for the purification of active influenza virus polymerase in complex with genomic RNA and the single-stranded RNA-binding protein nucleoprotein from influenza virus particles. It also explains the synthesis of capped RNA molecules as substrates of the influenza virus endonuclease. The enzymatic properties of influenza virus-derived endoribonuclease activity have been characterized with a model RNA substrate of 20-nucleotide length, termed G20 RNA. The rate of RNA cleavage under steady state conditions appears to be limited by product dissociation. Therefore conditions have been optimized to study the chemical step of RNA cleavage under single turnover conditions. The enzyme requires divalent metal ions for activity and can use Mn(II), Co(II), and Fe(II) efficiently at pH 7, Mg(II) with intermediate efficiency, and Ni(II) and Zn(II) with lower efficiency. The reaction progress curves show slow binding of Zn(II) and Ni(II) to the protein, suggesting a conformational change of the active site as a prerequisite for endonuclease activity in the presence of these two metal ions. Low concentrations of the detergent DOC inhibit the activity and also disrupt the trimeric polymerase complex, whereas other detergents do not have a significant effect on the activity.
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PMID:Influenza virus endoribonuclease. 1158 17

The influenza A virus RNA-dependent RNA polymerase catalyzes several reactions in transcription and replication of the genome RNA. The first step in viral mRNA synthesis is the endonucleolytic cleavage of host cell mRNAs containing a cap structure to generate capped primers that are 10-14 nucleotides long which are then used to prime transcription of virus-specific mRNAs. To analyze the properties of the capped RNA-specific endonuclease associated with the influenza virus polymerase and the roles of each of the three subunits in transcription initiation, we established an in vitro assay to investigate this endonucleolytic cleavage reaction. This assay consists of an artificial RNA substrate containing a cap-0 structure at its 5' end and a partial alfalfa mosaic virus RNA 4 (AIMV RNA 4) sequence which had been shown to be cleaved by the influenza polymerase. Results showed that purified virion ribonucleoprotein complexes cleaved the RNA substrate specifically to generate a capped 14-nt RNA fragment for use as primer to initiate viral mRNA synthesis. Purified polyclonal anti-PB2 IgG inhibited the endonuclease activity, but anti-PB1 and anti-PA antibodies did not inhibit the cleavage. Partially purified trimeric polymerase expressed by recombinant baculovirus in insect cells cleaved the artificial substrate, but if one or two subunits were removed from the polymerase complex, the cleavage activity was totally lost. Our results suggest that viral PB2 protein is the endonuclease that cleaves host cell mRNA to produce the primer used to initiate transcription; however, association with the other two enzyme subunits seems to be required for this PB2 function.
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PMID:Influenza A virus RNA polymerase subunit PB2 is the endonuclease which cleaves host cell mRNA and functions only as the trimeric enzyme. 1183 24

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

Developmentally aged chicken embryo cells which hyperproduce interferon (IFN) when induced were used to quantify IFN production and its suppression by eight strains of type A influenza viruses (AIV). Over 90% of the IFN-inducing or IFN induction-suppressing activity of AIV populations resided in noninfectious particles. The IFN-inducer moiety of AIV appears to preexist in, or be generated by, virions termed IFN-inducing particles (IFP) and was detectable under conditions in which a single molecule of double-stranded RNA introduced into a cell via endocytosis induced IFN, whereas single-stranded RNA did not. Some AIV strains suppressed IFN production, an activity that resided in a noninfectious virion termed an IFN induction-suppressing particle (ISP). The ISP phenotype was dominant over the IFP phenotype. Strains of AIV varied 100-fold in their capacity to induce IFN. AIV genetically compromised in NS1 expression induced about 20 times more IFN than NS1-competent parental strains. UV irradiation further enhanced the IFN-inducing capacity of AIV up to 100-fold, converting ISP into IFP and IFP into more efficient IFP. AIV is known to prevent IFN induction and/or production by expressing NS1 from a small UV target (gene NS). Evidence is presented for an additional downregulator of IFN production, identified as a large UV target postulated to consist of AIV polymerase genes PB1 + PB2 + PA, through the ensuing action of their cap-snatching endonuclease on pre-IFN-mRNA. The products of both the small and large UV targets act in concert to regulate IFN induction and/or production. Knowledge of the IFP/ISP phenotype may be useful in the development of attenuated AIV strains that maximally induce cytokines favorable to the immune response.
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PMID:Interferon induction and/or production and its suppression by influenza A viruses. 1570 7

The RNA-dependent RNA polymerase of influenza virus is a heterotrimer formed by the PB1, PB2, and PA subunits. Although PA is known to be required for polymerase activity, its precise role is still unclear. Here, we investigated the function of the N-terminal region of PA. Protease digestion of purified recombinant influenza virus A/PR/8/34 PA initially suggested that its N-terminal region is folded into a 25-kDa domain. We then systematically introduced point mutations into evolutionarily conserved amino acids in the N-terminal region of influenza virus A/WSN/33. Most alanine-scanning mutations between residues L109 and F117 caused PA degradation, mediated by a proteasome-ubiquitin pathway, and as a consequence interfered with polymerase activity. Three further PA mutations, K102A, D108A, and K134A, were investigated in detail. Mutation K102A caused a general decrease both in transcription and replication in vivo, whereas mutations D108A and K134A selectively inhibited transcription. Both the D108A and K134A mutations completely inhibited endonuclease activity in vitro, explaining their selective defect in transcription. K102A, on the other hand, resulted in a significant decrease in both cap binding and viral RNA promoter-binding activity and consequently inhibited both transcription and replication. These results suggest that the N-terminal region of PA is involved in multiple functions of the polymerase, including protein stability, endonuclease activity, cap binding, and promoter binding.
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PMID:Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding. 1687 36


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