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)

We studied the antiviral activity and the mechanism of action of a new antiviral agent and kanamycin derivative, 1-N-eicosanoyl-3"-N-trifluoroacetyl kanamycin A (ETKA), against influenza A virus. From yield reduction assays with VERO cells, ETKA showed a significant antiviral activity with negligible cytotoxic effect. In the presence of 20 micrograms/ml of ETKA at which VERO cell growth was not inhibited, virus titer was suppressed to 11.2% of control, and at 100 micrograms/ml virus production was suppressed to more than 99%. ETKA markedly inhibited viral protein synthesis when cells were pretreated with the drug before infection, but there was no inhibition when the drug was added 15 min post-infection. ETKA did not inhibit virus adsorption and penetration. Nor did it affect the activity of viral RNA polymerase in vitro. We found that the drug had a direct inactivating effect on influenza A virus under acidic conditions. These results suggest that ETKA exerts its antiviral action mainly in the early stage, prior to uncoating by direct inactivation of the virus due to the acidic environment of the endocytic vesicle. Aerosol treatment with the drug protected mice against a lethal influenza A virus infection.
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PMID:Inhibition of influenza A virus replication by a kanamycin derivative. 188 75

DNA-directed RNA polymerase is responsible for gene expression. Despite its importance, many details of its function and higher-order structure still remain unknown. We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St. The most remarkable similarity is that the catalytic sites of the RNases are shared with the eukaryotic RNA polymerase II subunits of Drosophila melanogaster and Saccharomyces cerevisiae. Several amino acids conserved among the RNases and the RNase-like domains of the RNA polymerase subunits are located in the neighborhood of the catalytic sites of barnase, whose three-dimensional structure has been resolved. This observation suggests the functional importance of the RNase-like domain of the RNA polymerase subunits and indicates that the RNase-like domain may have RNase activity. The location of the RNase-like domain relative to the region necessary for RNA polymerization is similar to the relative proximity of 5'----3' or 3'----5' exonuclease and the region of polymerase activity of DNA polymerase I. The RNase-like domain might work in proofreading, as in RNA-directed RNA polymerase of influenza virus, or it may contribute to RNA binding through an unknown function.
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PMID:RNase-like domain in DNA-directed RNA polymerase II. 192 68

To determine the function(s) of the PB2 protein of influenza A virus, six temperature-sensitive (ts) mutants of A/Udorn/72 (H3N2) virus, each carrying a ts mutation in the PB2 gene, were analysed for virus RNA and protein synthesis. One of the mutants, ICRC27, exhibited unique phenotypes and was characterized in detail. At the non-permissive temperature, 40 degrees C, the accumulation of mRNA for each genome segment was reduced severely, leading to delayed and reduced synthesis of viral proteins, complementary and viral RNAs (cRNAs and vRNAs). At the permissive temperature, 34 degrees C, the mutant virus produced several-fold greater concentrations of both mRNAs and cRNAs of PB2, PB1 and PA segments than wild-type virus. The synthesis of the three polymerase proteins and the induction of RNA polymerase activity were also greatly increased. By contrast, the expression of the haemagglutinin (HA) gene was severely suppressed. The over-production of the polymerase mRNAs was not observed during primary transcription, i.e. in the presence of cycloheximide. The ts+ revertants of ICRC27 did not exhibit the ts defects and also lost most of the non-ts phenotypes at 34 degrees C. These observations indicate that the PB2 protein participates not only in the synthesis of viral RNAs, but also in the regulation of viral gene expression, i.e. in the down-regulation of the three polymerase genes and the up-regulation of the HA gene during secondary transcription.
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PMID:Involvement of the influenza A virus PB2 protein in the regulation of viral gene expression. 194 Aug 63

Appropriate RNAs are transcribed and amplified and proteins are expressed after transfection into cells of in vitro-reconstituted RNA-protein complexes and infection with influenza virus as the helper. This system permits us to study the signals involved in transcription of influenza virus RNAs. For the analysis we used a plasmid-derived RNA containing the reporter gene for chloramphenicol acetyltransferase (CAT) flanked by the noncoding sequences of the NS RNA segment of influenza A/WSN/33 virus. Mutations were then introduced into both the 5' and 3' ends, and the resulting RNAs were studied to determine their transcription in vitro and their CAT expression activity in the RNA-protein transfection system. The results reveal that a stretch of uninterrupted uridines at the 5' end of the negative-strand RNA is essential for mRNA synthesis. Also, a double-stranded RNA "panhandle" structure generated by the 5'- and 3'-terminal nucleotides appears to be required for polyadenylation, since opening up of these base pairs diminished mRNA synthesis and eliminated expression of CAT activity by the mutant RNAs. Finally, it was shown that this double-stranded RNA structural requirement is not sequence specific, since a synthetic GC clamp can replace the virus-coded RNA duplex. The data suggest that the viral RNA polymerase adds poly(A) by a slippage (stuttering) mechanism which occurs when it hits the double-stranded RNA barrier next to the stretch of uridines.
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PMID:The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure. 203 59

A system for the expression of a foreign gene derived from negative polarity RNA was developed using influenza virus, a negative-stranded RNA virus. From cDNA for the influenza virus RNA genome segment 8, the region coding for the nonstructural protein was deleted and replaced by the chloramphenicol acetyltransferase (CAT) gene. The resulting DNA sequence was placed under the control of the promoter of T7 RNA polymerase such that the antisense RNA to CAT mRNA was produced when transcribed by T7 RNA polymerase. Transfection of HeLa cells with this antisense CAT RNA in the presence of the helper ribonucleoprotein cores led to no significant production of the CAT. In contrast, when the RNA was covered with purified nucleoprotein prior to transfection, the CAT gene was efficiently expressed. This indicated that the viral RNA polymerase transcribed the RNA transfected as the RNA-nucleoprotein complexes. In addition, this system was used for analysis of the cis-acting region in transcription and the promoter structure of the viral RNA genome.
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PMID:In vivo analysis of the promoter structure of the influenza virus RNA genome using a transfection system with an engineered RNA. 205 14

The temperature-sensitive mutant ts-1 of influenza virus A/WSN/33 carries mutations in the gene encoding RNA polymerase PB2 subunit. Effect of temperature on various steps of viral RNA synthesis was examined using disrupted virions of ts-1 mutant. The initiation of RNA synthesis with dinucleotide ApG primer was not affected by elevated temperature, whereas that with primer RNA containing 5'-terminal cap-1 structure was temperature-sensitive. The result supports the previous notion deduced from the UV-crosslinking experiments, that PB2 is involved in the cap-1 dependent initiation of RNA synthesis. In addition, the ts-1 mutant showed a defect in RNA chain elongation. Nucleotide sequence analysis of RNA segment 1 of ts-1 mutant revealed that the amino acid number 417 is essential for the recognition of cap-1 structures and/or the interaction with catalytic unit of the RNA polymerase.
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PMID:Characterization of a temperature-sensitive mutant in the RNA polymerase PB2 subunit gene of influenza A/WSN/33 virus. 222 91

Series of (1,4)-Naphthoquinono(3,2-c)-1H-pyrazoles and 2-substituted amino-1,4-naphthoquinones have been synthesised and studied for their possible anticancer activity (animal tumours, Walker 256 carcinosarcoma), Influenza RNA transcriptase activity, antibiotic activity (C. neoformans, T. mentagraphytes, M. canis, A. niger, and C. albicans).
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PMID:Synthesis and pharmacological studies of some (1,4)-naphthoquinono[3,2-c]-1H-pyrazoles, 2-substituted amino-1,4-naphthoquinones, and related compounds. 224 32

The interferon-induced murine Mx1 protein, which is localized in the nucleus, most likely specifically blocks influenza virus replication by inhibiting nuclear viral mRNA synthesis, including the mRNA synthesis catalyzed by inoculum (parental) virion nucleocapsids (R. M. Krug, M. Shaw, B. Broni, G. Shapiro, and O. Haller, J. Virol. 56:201-206, 1985). We tested two possible mechanisms for this inhibition. First, we determined whether the transport of parental nucleocapsids into the nucleus was inhibited in murine cells expressing the nuclear Mx1 protein. To detect the Mx1 protein, we prepared rabbit antibodies against the Mx1 protein with a CheY-Mx fusion protein expressed in bacteria. The fate of parental nucleocapsids was monitored by immunofluorescence with an appropriate dilution of monoclonal antibody to the nucleocapsid protein. The protein synthesis inhibitor anisomycin was added to the cells 30 min prior to infection, so that the only nucleocapsids protein molecules in the cells were those associated with nucleocapsids of the parental virus. These nucleocapsids were efficiently transported into the nuclei of murine cells expressing the Mx1 protein, indicating that this protein most likely acts after the parental nucleocapsids enter the nucleus. The second possibility was that the murine Mx1 protein might act in the nucleus to inhibit viral mRNA synthesis indirectly via new cap-binding activities that sequestered cellular capped RNAs away from the viral RNA transcriptase. We show that the same array of nuclear cap-binding proteins was present in Mx-positive and Mx-negative cells treated with interferon. Interestingly, a large amount of a 43-kDa cap-binding activity appeared after interferon treatment of both Mx-positive and Mx-negative cells. Hence, the appearance of new cap-binding activities was unlikely to account for the Mx-specific inhibition of viral mRNA synthesis. These results are most consistent with the possibility that the Mx1 protein acts directly to inhibit the viral transcriptase in the nucleus.
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PMID:Parental influenza virion nucleocapsids are efficiently transported into the nuclei of murine cells expressing the nuclear interferon-induced Mx protein. 224 97

A cDNA clone of the influenza virus NS (non-structural protein) gene in a vector carrying a bacteriophage T7 RNA polymerase promoter was manipulated so as to reiterate the initiation site to give two in-frame AUG codons 57 nucleotide residues apart. Each initiation site was in either a preferred context (...AUAAUGG...) or a less favourable context (...UUUAUGG...) and the four possible permutations were constructed. When capped mRNA transcripts of these clones were translated in the rabbit reticulocyte lysate system, products from initiation at both AUG codons were observed. At low RNA concentrations the frequency of initiation at the 5'-proximal AUG codon rather than the second was higher when the first AUG codon was in the preferred context, in qualitative agreement with the scanning ribosome model. However, a completely unexpected finding was that the ratio of initiation at the first AUG codon to initiation at the second decreased with increasing mRNA concentration, irrespective of the particular context involved. Several lines of evidence indicated that the increased frequency of initiation at the second AUG codon was not due solely to the lower density of ribosome loading per mRNA at high RNA concentrations, and may therefore be the result of high RNA concentrations out-titring the capacity of endogenous reticulocyte factors responsible for preferential initiation at the 5'-proximal AUG codon. The effect of supplementing the system with purified initiation factors was examined. Only eIF-2 was capable of decreasing the frequency of initiation at the second AUG codon and promoting use of the first AUG at high mRNA concentrations; eIF-3, 4A, 4B, 4C + 4D, 4F and 5 were inactive.
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PMID:Selection of the 5'-proximal translation initiation site is influenced by mRNA and eIF-2 concentrations. 229 14

The anti-influenza virus activity of polysaccharides and other high molecular weight fractions from pine cone extract (PCE) of Pinus parviflora Sieb. et Zucc. was investigated. None of the fractions affected the growth of MDCK cells. The acidic PCE substances markedly suppressed the growth of the influenza virus in MDCK cells. Significant inhibition of both the viral protein synthesis in infected cells and virion-associated RNA-dependent RNA polymerase activity was observed with these acidic fractions. Although amantadine inhibited virus plaque formation as effectively as PCE fractions, it was less effective in inhibiting the RNA polymerase activity. These results suggest that PCE, which has been shown to contain antitumor substance(s), also contains anti-influenza virus substance(s).
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PMID:Inhibition of influenza virus infection by pine cone antitumor substances. 233 67


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