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)

In the presence of Mg(2+) and a specific primer, ApG or GpG, the influenza WSN virion transcriptase synthesizes large, polyadenylic acid-containing complementary RNA (cRNA) (Plotch and Krug, J. Virol., 21:24-34, 1977). After removal of its polyadenylic acid with RNase H in the presence of polydeoxythymidylic acid, the in vitro cRNA distributed into seven discrete bands during electrophoresis in acrylamide gels containing 6 M urea. The eight known segments of virion RNA (vRNA) also distributed into seven bands under these conditions as two, rather than the expected three, large-sized segments were resolved. Each of the in vitro cRNA segments migrated slightly faster than the corresponding vRNA segment. To determine whether this difference in mobility reflects a difference in size between cRNA and vRNA, the double-stranded RNA formed by annealing labeled in vitro cRNA to unlabeled vRNA was subjected to various nuclease treatments and was analyzed by gel electrophoresis. Hybrids treated with RNase T2 or a combination of RNase T2 and RNase H migrated slightly faster than those treated only with RNase H, indicating that RNase T2 removed an RNA sequence other than polyadenylic acid, most probably a short sequence of vRNA not hydrogen bonded to cRNA. These results suggest that the in vitro cRNA segments are shorter than, and thus incomplete transcripts of the corresponding vRNA segments. All eight hybrids were resolved by gel electrophoresis, indicating that all eight vRNA segments are transcribed into cRNA in vitro. We also present evidence suggesting that the ApG primer initiates in vitro transcription exactly at the 3' end of vRNA.
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PMID:Segments of influenza virus complementary RNA synthesized in vitro. 62 84

Temperature-sensitive mutants of WSN influenza virus (10, 11, 12) were tested in vitro for activity of the virion RNA-dependent RNA transcriptase at various temperatures. Temperature-sensitivity was found for virion transcriptase activity of mutants belonging to complementation/recombination group I, but not groups II, IV and V. It was not possible on the basis of the results to specify the precise biochemical lesion of mutants from group III.
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PMID:Temperature-sensitive virion transcriptase activity in mutants of WSN influenza virus. 99 15

A comparative study of the in vitro reaction kinetics of the virion RNA polymerase of influenza A strains WS and WSN was conducted to establish phenotypic differences for enzyme activity that might be exchanged as genetic markers among recombinants of these viruses. Characteristically, the RNA polymerase activity of WS virus showed an initial rate of synthesis about two- to threefold higher than that of WSN when assayed at 32 C. The two strains were also distinguishable by comparing the transcription rates of each strain at 32 and 37 C. The initial rate of WS was invariably higher at 37 than at 32 C, whereas the opposite was found with WSN. When a series of recombinants obtained from mixed infections with the WS and WSN viruses were examined for virion transcriptase activity, it was found that the two polymerase related markers behaved as properties which segregated independently of each other and of additional nonselective markers that were scored. Seven temperature-sensitive mutants of WSN virus representing distinct recombination-complementation groups were found to show a diminished transcriptase activity as compared to wild-type virus, and one of these clones (ts 24) was largely deficient for this function. None of these mutants appeared to possess a heat-liable virion polymerase.
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PMID:Virion-associated transcriptase activity of influenza recombinant and mutant strains. 115 93

Influenza WSN virus temperature-sensitive (ts) mutants were examined for defects in viral complementary RNA (cRNA) synthesis. The synthesis of viral cRNA was determined by hybridizing RNA from infected cells to radiolabeled virion RNA of known specific activity. Mutants in complementation groups I and III synthesized little, or no, cRNA at the nonpermissive temperature (39.5 C). When cells infected by these mutants were incubated for 5 h at the permissive temperature (33 C) and were then shifted to 39.5 C, net synthesis of cRNA ceased. This strongly suggests that mutants in these two complementation groups possess a ts defect in the transciptase complex. Mutants in group II and group V synthesize reduced amounts of cRNA at 39.5 C. In contrast to the group I and group III mutants, cRNA synthesis in cells infected by a group II or a group V mutant continues after a shift-up. This indicated that these mutants do not possess a ts transcriptase complex and that these mutants are most probably defective in some step in the amplification of cRNA synthesis. As will be discussed, the most likely defect in these mutants is in the synthesis of virion-type RNA. These results suggest that there are two influenza viral gene functions required for transcription and most likely two additional gene functions required for RNA replication.
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PMID:Temperature-sensitive mutants of influenza WSN virus defective in virus-specific RNA synthesis. 116 95

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

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

The effect on retroviruses of two transition metal complexes of known antiviral activity, 4-methyl-2-amino-pyridine-palladium-chloride (MAP) and cis-dichloro-diammine-platinum(II) (cis-DDP) has been investigated. The experiments included the evaluation of the action of compounds on virus particle-associated reverse transcriptase in exogenous assays, on virus propagation in persistently infected cell cultures and on virus infectivity in mice. In disrupted viruses and in the absence of excess protein, the reverse transcriptase was inhibited by MAP but not by cis-DDP. The same results were obtained when examining the activity of the virus-associated RNA polymerase of influenza virus A/WSN. Both compounds did not inhibit the replication of retroviruses in cell cultures, except at high dose levels which exerted toxic action on both cells and virus formation. The leukemogenicity of Rauscher murine leukemia virus was strongly inhibited when the virus had been incubated with MAP before inoculation. A similar treatment with cis-DDP did not influence viral leukemogenicity. Despite somewhat different results with both compounds tested, we conclude from the present results that the above mentioned compounds cannot be considered as antiretroviral drugs.
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PMID:[The biological effects of coordination compounds of transitional metals. 6. Effect of 4-methyl-2-aminopyridine-palladium chloride and cis-dichlorodiammine-platinum(II) on retroviruses and the virus-associated RNA polymerase of the influenza virus]. 243 60

A cDNA encoding the entire amino acid sequence of the matrix (M1) protein of influenza A/WSN/33 virus was cloned, sequenced, and expressed in a vaccinia virus system consisting of the T7 bacteriophage RNA polymerase and a plasmid carrying the M1 gene flanked by T7 polymerase promoter and terminator sequences. The transiently expressed M1 gene product comigrated on SDS-polyacrylamide gels with the endogenous WSN virus M1 protein and was recognized in Western blot analysis by three epitope-specific monoclonal antibodies directed to the M1 protein. The nucleotide sequence and the predicted amino acid sequence of the cloned WSN virus M1 coding region was found to be more than 97% homologous to that of the M1 gene of influenza virus A/PR/8/34 reported by G. Winter and S. Fields (Nucleic Acids Res. 8, 1965-1974, 1980).
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PMID:Transient expression and sequence of the matrix (M1) gene of WSN influenza A virus in a vaccinia vector. 335 9

An investigation was made of inhibition of transcriptase activity of influenza viruses in vitro by binding of antibody to the surface of the virion. Eight monoclonal antibodies which were directed against at least four non-overlapping antigenic regions of the haemagglutinin protein of A/Aichi/68 virus were tested for inhibitory effect. One of the antibodies directed against the B antigenic site, 22/1, inhibited transcriptase activity, while the other seven antibodies did not. Antibody from a hyperimmune rabbit serum to A/Udorn/72 (H3N2) virions inhibited the transcriptase activity of A/Udorn/72 and A/Aichi/68 (H3N2) viruses but not that of A/WSN/33 (H1N1). The antibody did not cause irreversible inactivation of the transcriptase since full activity was recovered by isolating ribonucleoprotein (RNP) cores from the inhibited virions using NP-40 treatment and subsequent centrifugation in a caesium sulphate density gradient. The antibody did not inhibit transcriptase activity of isolated RNP cores. The virion transcriptase activity was not inhibited by addition of the antiserum after the detergent treatment which is necessary for the activation of the transcriptase activity in vitro. These results suggest that the antibody blocks the activation process of the transcriptase by detergent treatment.
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PMID:Inhibition of transcriptase activity of influenza A virus in vitro by anti-haemagglutinin antibodies. 406 Aug 49

In vitro reaction conditions have been determined for the maximal synthesis of product ribonucleic acid by the influenza (WSN) virion ribonucleic acid polymerase. The reaction requires the presence of all four triphosphates, Mg(2+) and Mn(2+) ions, monovalent cations, nonionic detergent, and ribonucleoside triphosphates at concentrations above certain threshold values. The optimum pH for the reaction is around 8.0 to 8.2 and the kinetics of product synthesis are linear through at least 6 hr when incubated at 31 to 33 C.
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PMID:Transcription of the influenza ribonucleic acid genome by a virion polymerase. I. Optimal conditions for in vitro activity of the ribonucleic acid-dependent ribonucleic acid polymerase. 432 17


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