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)

A novel technique is described for the production of pure, full-length influenza virus ds DNA's corresponding to each segment of the influenza virus genome, and suitable for molecular cloning and restriction endonuclease mapping. The method involves the synthesis of DNA complementary to both virion (negative strand) and messenger (positive strand) RNA, gel purification and annealing. By avoiding the use of SI nuclease, which often removes the terminal regions of DNA duplexes, the method allows transcription of the total sequence information of influenza virion and messenger RNA's into a ds DNA form.
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PMID:New procedure for the production of influenza virus-specific double-stranded DNA's. 9 12

Influenza virus polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicating that the promoter lay solely within the 15-nucleotide 3' terminus. Sequences not specific for the influenza virus termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus-sense cRNA were copied at low levels. The specificity for recognition of the virus sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap endonuclease-primed and primer-free RNA synthesis from these model templates, as well as from genomic-length RNAs. This finding indicates that the reconstituted system has catalytic properties very similar to those of native viral ribonucleoprotein complexes.
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PMID:Promoter analysis of influenza virus RNA polymerase. 258 1

Influenza virus infection has adverse effects on the metabolism of two representative RNA polymerase II transcripts in chicken embryo fibroblasts, those coding for beta-actin and for avian leukosis virus (ALV) proteins. Proviral ALV DNA was integrated into host cell DNA by prior infection with ALV. Within 1 h after influenza virus infection, the rate of transcription of beta-actin and ALV sequences decreased 40 to 60%, as determined by labeling the cells for 5 min with [3H]uridine and by in vitro, runoff assays with isolated nuclei. The transcripts that continued to be synthesized did not appear in the cytoplasm as mature mRNAs, and the kinetics of labeling of these transcripts strongly suggest that they were degraded in the nucleus. By S1 endonuclease assay, it was confirmed that nuclear ALV transcripts disappeared very early after infection, already decreasing ca. 80% by 1 h postinfection. A plausible explanation for this nuclear degradation is that the viral cap-dependent endonuclease in the nucleus cleaves the 5' ends of new polymerase II transcripts, rendering the resulting decapped RNAs susceptible to hydrolysis by cellular nucleases. In contrast to the nuclear transcripts, cytoplasmic beta-actin and ALV mRNAs, which are synthesized before infection, were more stable and did not decrease in amount until after 3 h postinfection. Similar stability of cytoplasmic host cell mRNAs was observed in infected HeLa cells, in which the levels of actin mRNA and two HeLa cell mRNAs (pHe 7 and pHe 28) remained at undiminished levels for 3 h of infection and decreased only slightly by 4.5 h postinfection. The cytoplasmic actin and pHe 7 mRNAs isolated from infected HeLa cells were shown to be translated in reticulocyte extracts in vitro, indicating that host mRNAs were not inactivated by a virus-induced modification. Despite the continued presence of high levels of functional host cell mRNAs, host cell protein synthesis was effectively shut off by about 3 h postinfection in both chicken embryo fibroblasts and HeLa cells. These results are consistent with the establishment of an influenza virus-specific translational system that selectively translates viral and not host mRNAs.
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PMID:Metabolism and expression of RNA polymerase II transcripts in influenza virus-infected cells. 609 46

RNA segment 8 of the influenza virus genome is unique in coding for two polypeptides, NS1 (Mr, approximately 25,000) and NS2 (Mr, approximately 11,000). These polypeptides are synthesized from separate mRNA species. By using cloned DNA derived from RNA segment 8 (NS DNA) the two mRNAs have been mapped on segment 8 by hybridization of mRNAs with restriction endonuclease fragments of the DNA and nuclease S1 digestion methods. These data indicate that the body of the NS1 mRNA (approximately 850 nucleotides) maps at 0.05-0.95 units of the cloned NS DNA and the body of the NS2 mRNA (approximately 340 nucleotides) maps at 0.59-0.95 unitssuggesting that the two mRNAs are 3' coterminal and share the same poly(A) addition site. These positions of the mRNAs on the viral genome segment were confirmed in hybrid-arrested translation experiments using fragments of the cloned NS DNA to inhibit the synthesis in vitro of NS1 or NS2 polypeptides. In addition, in these translation experiments the use of certain DNA fragments resulted in premature termination of the NS1 polypeptide. From these data, it could be estimated that the termination of translation of NS1 is at approximately 0.76 map unit. Thus, the coding regions of the two mRNAs overlap by approximately 144-159 nucleotides, the equivalent of approximately 48-53 amino acids. Peptide mapping experiments indicated that polypeptides NS1 and NS2 do not share methionine- or leucine-containing tryptic peptides. The results obtained indicate the translation of the NS2 mRNA occurs in a reading frame different from that used for NS1.
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PMID:Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza virus genome. 624 9

We propose a mechanism for the priming of influenza viral RNA transcription by capped RNAs in which specific 5'-terminal fragments are cleaved from the capped RNAs by a virion-associated endonuclease. These fragments would serve as the actual primers for the initiation of transcription by the initial incorporation by the initial incorporation of a G residue at their 3' end. We show that virions and purified viral cores contain a unique endonuclease that cleaves RNAs containing a 5' methylated cap structure (m7GpppXm) preferentially at purine residues 10 to 14 nucleotides from the cap, generating fragments with 3'-terminal hydroxyl groups. RNAs containing the 5'-terminal structure GpppG could not be cleaved to produce these specific fragments. Consistent with our proposed mechanism, those capped fragments that function as primers could be linked to a G residue in transcriptase reactions containing alpha-32P-GTP as the only ribonucleoside triphosphate. The pattern of G and C incorporation onto these primer fragments suggests that this incorporation is directed by the second and third bases at the 3' end of the virion RNA template, which has the sequence 3' UCG. Primer fragments with a 3'-terminal A residue were used more efficiently than those with a 3'-terminal G residue, indicating a preference for generating an AGC sequence in the viral mRNA complementary to the 3' end of the virion RNA. Cleavage of the RNA primer and initiation of transcription are not necessarily coupled, because a 5' fragment isolated from one reaction could be used as a primer when added to a second reaction. Uncapped ribopolymer inhibitors of viral RNA transcription inhibited the cleavage of capped RNAs.
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PMID:A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. 626 60

We have cloned and expressed the hemagglutinin (HA) gene of a human influenza virus (A/WSN/33) in monkey kidney cells by linking it to deleted simian virus 40 (SV40) genomes that contain the entire early gene region, the origin of replication, and late leader sequences. The HA gene (1775 base pairs long) was originally inserted by the dG . dC tailing technique into the multicopy plasmid of Escherichia coli, pBR322, using cDNA made from viral RNA. The cloned gene was further modified by treatment with nuclease Bal 31 to remove the dG . dC tails and some of the untranslated sequences and recloned in E. coli after addition of BamHI restriction endonuclease linkers. A number of SV40 and HA recombinants (SV--HA) were constructed by inserting recloned HA DNA into the late gene region of SV40. The SV--HA recombinants, when complemented in a lytic infection of monkey cells by the helper function of SV40 early deletion mutants expressed influenza HA as detected by immunofluorescence and immunoprecipitation of in vivo-labeled proteins using either heterogeneous anti-influenza rabbit antibodies or monoclonal antibodies against HA. Furthermore, the WSN HA expressed by the SV--HA recombinants was also glycosylated and possessed the same molecular weight (approximately 70,000) as the uncleaved HA of WSN virus in monkey cells.
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PMID:Human influenza virus hemagglutinin is expressed in monkey cells using simian virus 40 vectors. 628 58

Analogues of the mRNA 5'-terminal methyl cap structure were found to stimulate the influenza virion RNA-dependent RNA polymerase. The single nucleotide analogue m7GMP was incorporated into RNA during transcription in vitro, and the stimulatory effect was not additive with the primer ApG, suggesting that m7GMP stimulates the virion polymerase by priming virus-specific mRNA synthesis, as has been shown for ApG. By contrast, stimulation by m7G(5')ppp(5')m6AM2-O was additive with that by ApG, and we could not demonstrate incorporation of the similar analogue m7G(5')ppp(5')Am2-O into RNA during transcription. We propose that these dinucleotide cap analogues stimulate the virion polymerase by allosteric modulation, independent of priming. This stimulation can be abolished by mutation, without loss of other activities associated with the cap-dependent endonuclease.
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PMID:Capped mRNAs may stimulate the influenza virion polymerase by allosteric modulation. 653 99

The host-cell derived RNA primer sequences at the 5' termini of mRNAs of influenza A and B viruses, obtained from sequences of 29 cDNA clones, have been compared. This has been done for clones of five different genome segments from four strains of influenza A and B virus. The results indicate that host RNA primers containing a 3'-terminal Py-G-C-A sequence before the presumed endonuclease cleavage site are preferred for use as primers in influenza virus mRNA synthesis. Primer-extension analyses of the 5'-terminal heterogeneous sequences of in vivo synthesized mRNAs confirm the preference for G-C-A-terminated primer fragments, with some differences noted between the transcripts of types A and B influenza virus genome segments.
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PMID:A specific sub-set of host-cell mRNAs prime influenza virus mRNA synthesis. 653 8

The first step in influenza viral mRNA synthesis is the endonucleolytic cleavage of heterologous RNAs containing cap 1 (m(7)GpppNm) structures to generate capped primers that are 10 to 13 nucleotides long, which are then elongated to form the viral mRNA chains. We examined the temperature sensitivity of these steps in vitro by using two WSN virus temperature-sensitive mutants, ts1 and ts6, which have a defect in the genome RNA segment coding for the viral PB2 protein. For these experiments, it was necessary to employ purified viral cores rather than detergent-treated virions to catalyze transcription, as preparations of detergent-treated virions contain destabilizing or inhibitory activities which render even the transcription catalyzed by wild-type virus temperature sensitive. Using purified wild-type viral cores, we found that the rates of endonucleolytic cleavage of capped primers and of overall transcription were similar at 39.5 and 33 degrees C, the in vivo nonpermissive and permissive temperatures, respectively. In contrast, the activities of the cap-dependent endonucleases of ts1 and ts6 viral cores at 39.5 degrees C were only about 15% of those at 33 degrees C. The steps in transcription after endonucleolytic cleavage of the capped RNA primer were largely, if not totally, temperature insensitive, indicating that the mutations in the PB2 protein found in ts1 and ts6 virions affect only the endonuclease step. The temperature-sensitive defect is most likely in the recognition of the 5'-terminal cap 1 structure that occurs as a required first step in the endonuclease reaction: the cap-dependent binding of a specific capped primer fragment to ts1 viral cores was temperature sensitive under conditions in which binding to wild-type viral cores was not affected by increasing the temperature from 33 to 39.5 degrees C. Thus, our results establish that the viral PB2 protein functions in cap recognition during the endonuclease reaction.
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PMID:Influenza virus temperature-sensitive cap (m7GpppNm)-dependent endonuclease. 682 15

Catalytic properties of the capped RNA-specific endonuclease associated with the influenza virus RNA polymerase were analyzed with use of synthetic hetero- and homopolymers containing 32P-labeled CAP structures at their 5' termini. The endonuclease displays its intrinsic activity provided that substrate RNA contains both the CAP-1 structure (m7GpppGm) and either A or U residues at 9 to 11 nucleotides distant from the CAP structure. Independent recognition of multiple RNA signals by the endonuclease was further supported by the findings that dinucleotide ApG, free CAP structures and RNA without the CAP structure inhibited the endonuclease activity to different extents. In the presence of four species of ribonucleoside 5'-triphosphates, the endonucleolytically cleaved fragments with the CAP-1 structure were incorporated into polynucleotides, supporting the concept that they are used as the primers for the transcription. The initial nucleotide linked to the primers was a G residue, the nucleotide complementary to the second base of the 3' termini of the vRNA segments.
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PMID:RNA polymerase of influenza virus. IV. Catalytic properties of the capped RNA endonuclease associated with the RNA polymerase. 685 61


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