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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Disodium phosphonoacetate (PAA) was found to inhibit the replication of African swine fever virus (ASFV). The action of this compound has been compared with the inhibitory capacity of iododeoxyuridine (IDU) upon ASFV growing in Vero cells. The study was done by the immunofluorescence technique in order to detect formations of cytoplasmic virus antigens and inclusion bodies; both were found to be inhibited by IDU and PAA. At 100 microgram/ml, IDU blocked completely the multiplication of ASFV and with PAA, a few scattered cells showed positive fluorescence. The infectivity of the virus was reduced 1--5 log depending upon drug concentrations and time of exposure to the drugs. Inhibition of ASFV replication by PAA suggests that this virus, like other herpesviruses, involves a virus-specific DNA polymerase in its replication mechanism.
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PMID:Effect of disodium phosphonoacetate and iododeoxyuridine on the multiplication of African swine fever virus in vitro. 37 73

Phosphonoacetic acid (PAA) inhibits the multiplication of African swine fever (ASF) virus in VERO cells. The observed inhibition of the in vivo DNA synthesis could be related to the in vitro inhibition of a virus-induced DNA polymerase activity present in cytoplasmic extracts from infected VERO cells.
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PMID:Inhibition of African swine fever (ASF virus replication by phosphonoacetic acid. 65 Jan 76

African swine fever virus (ASFV) induces the synthesis of a virus-specific DNA polymerase, which is inhibited by phosphonoacetic acid and cytosine arabinoside. In contrast to all other alpha-like DNA polymerases of DNA viruses, ASFV-specific DNA polymerase is resistant to aphidicolin. Concentrations of the drug as high as 160 microM had no effect on virus production or plaquing efficiency. The resistance of ASFV DNA polymerase to aphidicolin was confirmed by analyzing the effect of the drug on viral DNA synthesis. A moderate inhibition of viral DNA synthesis was observed when aphidicolin was added immediately after virus adsorption but normal synthesis occurred, with a peak at 10 hr p.i., when the drug was added at 2 or 4 hr p.i. This suggests that a very early phase of ASFV DNA replication is sensitive to aphidicolin and is probably catalyzed by a different enzyme. An in vitro assay of DNA polymerase activity was used to assay the sensitivity of the virus-specific DNA polymerase to inhibitors. In correspondence to the results observed in vivo, phosphonoacetic acid strongly inhibited the enzyme activity, whereas aphidicolin had no effect. Resistance to aphidicolin was independent of the concentration of dCTP used in the assay. Three independent ASFV mutants resistant to phosphonoacetic acid showed the same resistance to aphidicolin as wild type virus.
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PMID:African swine fever virus-induced DNA polymerase is resistant to aphidicolin. 141 23

We describe a new method for obtaining DNA fragments starting at a desired point where there is no recognition sequence for any known restriction endonuclease. A single-stranded DNA containing the fragment of interest is annealed to a synthetic oligonucleotide hybridizing at the 5' end of the required fragment. Then, a partially double-stranded DNA is synthesized using the Klenow fragment of DNA polymerase I in the presence of the four deoxynucleoside triphosphates. The remaining single-stranded regions are removed by digestion with a single-strand nuclease, and the resulting 5' blunt-ended fragment is finally released by digestion with a restriction endonuclease at any site downstream its 3' end. The usefulness of the method was exemplified here by insertion of an epidermal growth factor-like African swine fever virus gene immediately downstream of the ribosome binding site of an expression vector.
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PMID:A general method to cleave a known DNA sequence at any site. 166 37

A cell-free system that catalyzes DNA replication was prepared from cytoplasmic extracts of Vero cells infected with African swine fever virus (ASFV). The cells were permeabilized with lysolecithin and disrupted by mild mechanical action and the nuclei were removed by low-speed centrifugation. Extracts prepared from infected cells at the time of maximal DNA replication incorporated [alpha-32P]dTTP into acid-insoluble material that was sensitive to DNase and resistant to RNase. The reaction was inhibited by phosphonoacetic acid, an inhibitor of ASFV-specific DNA polymerase. Extracts from mock-infected cells had a negligible activity. Micrococcal nuclease-treated extracts were able to replicate added virion DNA or viral replicative DNA. An increase in the mass of DNA detected by ethidium bromide staining and by dot blot hybridization with ASFV DNA showed that the incorporation was due to true replication. Plasmid DNA was also replicated, which indicates that ASFV-specific DNA polymerase does not require a virus-specific origin of replication. The pattern of fragments generated by EcoRI digestion of the in vitro product was characteristic of viral replicative DNA. Hybridization with a recombinant plasmid containing a terminal fragment of ASFV DNA confirmed the presence of dimer terminal ASFV fragments presumably generated from concatemeric replicative intermediates.
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PMID:In vitro DNA replication by cytoplasmic extracts from cells infected with African swine fever virus. 221 42

Bovine interferon-alpha I1 (IFN-alpha I1) and porcine interferon-gamma (IFN-gamma) inhibited African swine fever virus replication in both porcine monocytes and alveolar macrophages. The most potent antiviral activity was observed with IFN-gamma-treated alveolar macrophages. The production of both a virulent (CC83) and a non-virulent (BA71) isolate of the virus was inhibited. Bovine tumour necrosis factor alpha did not show antiviral activity in either monocytes or alveolar macrophages. Rather, an increase of African swine fever virus production in tumour necrosis factor alpha-treated monocytes was found. An analysis of viral protein synthesis in IFN-alpha I1- and IFN-gamma-treated alveolar macrophages showed an inhibition of synthesis of some viral proteins. The inhibition of late proteins was very pronounced in IFN-gamma-treated cells, and it was probably a consequence of the inhibition of African swine fever virus DNA polymerase activity.
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PMID:Effect of interferon-alpha, interferon-gamma and tumour necrosis factor on African swine fever virus replication in porcine monocytes and macrophages. 314 9

[35S]Methionine-labeled proteins from total or cytoplasmic extracts of Vero cells infected with African swine fever virus were chromatographed on native and denatured DNA-cellulose and DNA-binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), by DNA binding to Western blots, or by two-dimensional electrophoresis. Thirteen virus-specific DNA-binding proteins were detected by one-dimensional analysis. Major species have molecular mass 44,000 (44K), 38K, 20K, 18K, 14K, 13K, and 12K. The remaining DNA-binding proteins are proteins with molecular mass 130K, 110K, 35K, 33K, 17K, and 14.5K. When viral DNA used in the binding assay the results were very similar but the 13K protein did not bind viral DNA. Seven other minor virus-specific DNA-binding proteins could be detected by two-dimensional analysis. This technique also enabled the assignment of virus-specific proteins. Seven of the virus-specific DNA-binding proteins are structural proteins. Twelve are late proteins, the remaining being early proteins synthesized before viral DNA replication. Most of the virus-specific DNA-binding proteins bind both to double-stranded and to single-stranded DNA. The 110K, 29K, and 18K DNA-binding proteins bind only to single-stranded DNA. Two virus-specific enzymatic activities, DNA polymerase and RNA polymerase, were present in the fractions separated by DNA-cellulose chromatography. The virus-specific single-stranded DNA nuclease did not bind to DNA.
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PMID:DNA-binding proteins specified by African swine fever virus. 368 26

The DNA polymerase gene of African swine fever virus (ASFV) was mapped by marker rescue experiments using a phosphonoacetic acid-resistant mutant and hybridization with an oligonucleotide probe designed from the most conserved motif of family B DNA polymerases. Viral DNA fragments mapping in this region were cloned and sequenced. An open reading frame coding for a 1244 amino acid long peptide with a molecular mass of 142.5 kDa was determined from the sequence. A unique feature of ASFV DNA polymerase is the presence of 13 tandem repeats of the sequence Ala-Gly-Asp-Pro near the carboxyl end of the molecule. Comparison with 30 sequences of alpha-like DNA polymerases of cellular and viral origin showed that ASFV DNA polymerase has all the conserved motifs of family B DNA polymerases. A 3.9 kb transcript was detected by Northern hybridization and the transcription initiation and termination sites were mapped by S1 analysis and primer extension. Late transcription was initiated at a site different from the early transcription initiation site. A 145 kDa protein, consistent with the size of the gene, was identified by an in situ enzyme assay after gel electrophoresis of infected cell extracts.
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PMID:Genetic identification and nucleotide sequence of the DNA polymerase gene of African swine fever virus. 812 6

African swine fever virus (ASFV) growth and plaque formation were inhibited by phosphonoacetic acid (PAA) concentrations of 200 micrograms/ml or more. One spontaneous mutant and two mutants isolated from mutagenized virus were resistant to PAA inhibition and showed practically normal viral DNA synthesis in the presence of PAA. DNA polymerase activity present in the cytoplasmic fraction from cells infected with the mutants required 10-fold higher concentrations of PAA for inhibition compared to equivalent inhibition of the wild-type enzyme. Like wild-type virus, the PAA-resistant mutants were resistant to inhibition by aphidicolin. Marker rescue analysis with mutant DNA fragments covering different regions of the ASFV DNA polymerase gene mapped the mutations within a fragment which was cloned and sequenced. A single nucleotide and amino acid change was assigned to each mutant. Two of the PAA-resistant mutations lie within the highly conserved region II common to alpha-like DNA polymerases, which has been implicated in pyrophosphate binding and probably also in dNTP binding. The other mutation was localized to within a region of moderate homology among viral DNA polymerases close to one of the motifs allegedly considered as constituting the 3'-5' exonuclease active site.
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PMID:Functional and molecular characterization of African swine fever virus mutants resistant to phosphonoacetic acid. 852 40

In order to examine genetic relatedness among viruses that infect microalgae, DNA polymerase gene (DNA pol) fragments were amplified and sequenced from 13 virus clones that infect three genera of distantly related microalgae (Chlorella strains NC64A and Pbi, Micromonas pusilla and Chrysochromulina spp.). Phylogenetic trees based on DNA pol sequences and hybridization of total genomic DNA showed similar branching patterns. Genetic relatedness calculated from the hybridization and sequence data showed good concordance (r=0.90), indicating that DNA pol sequences can be used to determine genetic relatedness and infer phylogenetic relationships among these viruses. The phylogenetic tree inferred from the deduced amino acid sequences of DNA pol from 24 dsDNA viruses, including phycodnaviruses, herpesviruses, poxviruses, baculoviruses, and African swine fever virus corresponded well with groupings based on the International Committee on Taxonomy of Viruses. Microalgal viruses are more closely related to each other than to the other dsDNA viruses and form a distinct phyletic group, suggesting that they share a common ancestor and belong to the Phycodnaviridae. Moreover, the Phycodnaviridae are more closely related to the Herpesviridae than to other virus families for which DNA pol sequences are available.
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PMID:Evolutionary relationships among large double-stranded DNA viruses that infect microalgae and other organisms as inferred from DNA polymerase genes. 862 26


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