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Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus

Here we report on the expression, purification and characterization of recombinant ebola virus RNA-dependent RNA polymerase (EBOV RdRp). Active protein complexes composed of the large L protein and viral protein VP35 were isolated from insect cells and analyzed using a short primer/template substrat...

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Autores principales: Tchesnokov, Egor P., Raeisimakiani, Parisa, Ngure, Marianne, Marchant, David, Götte, Matthias
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838098/
https://www.ncbi.nlm.nih.gov/pubmed/29507309
http://dx.doi.org/10.1038/s41598-018-22328-3
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author Tchesnokov, Egor P.
Raeisimakiani, Parisa
Ngure, Marianne
Marchant, David
Götte, Matthias
author_facet Tchesnokov, Egor P.
Raeisimakiani, Parisa
Ngure, Marianne
Marchant, David
Götte, Matthias
author_sort Tchesnokov, Egor P.
collection PubMed
description Here we report on the expression, purification and characterization of recombinant ebola virus RNA-dependent RNA polymerase (EBOV RdRp). Active protein complexes composed of the large L protein and viral protein VP35 were isolated from insect cells and analyzed using a short primer/template substrate that allowed benchmarking against related enzymes. RNA synthesis by multiprotein complexes of EBOV, influenza B, respiratory syncytial virus (RSV) and monomeric enzymes of hepatitis C and Zika (ZIKV) viruses required a 5′-phosporylated primer. The minimum length of the primer varied between two and three nucleotides in this system. The EBOV enzyme utilizes Mg(2+) as a co-factor and the D742A substitution provides an active site mutant that likely affects binding of the catalytic metal ions. Selectivity measurements with nucleotide analogues translate our assay into quantitative terms and facilitate drug discovery efforts. The related EBOV and RSV enzymes are not able to efficiently discriminate against ara-cytidine-5′-triphosphate. We demonstrate that this compound acts like a non-obligate chain-terminator.
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spelling pubmed-58380982018-03-12 Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus Tchesnokov, Egor P. Raeisimakiani, Parisa Ngure, Marianne Marchant, David Götte, Matthias Sci Rep Article Here we report on the expression, purification and characterization of recombinant ebola virus RNA-dependent RNA polymerase (EBOV RdRp). Active protein complexes composed of the large L protein and viral protein VP35 were isolated from insect cells and analyzed using a short primer/template substrate that allowed benchmarking against related enzymes. RNA synthesis by multiprotein complexes of EBOV, influenza B, respiratory syncytial virus (RSV) and monomeric enzymes of hepatitis C and Zika (ZIKV) viruses required a 5′-phosporylated primer. The minimum length of the primer varied between two and three nucleotides in this system. The EBOV enzyme utilizes Mg(2+) as a co-factor and the D742A substitution provides an active site mutant that likely affects binding of the catalytic metal ions. Selectivity measurements with nucleotide analogues translate our assay into quantitative terms and facilitate drug discovery efforts. The related EBOV and RSV enzymes are not able to efficiently discriminate against ara-cytidine-5′-triphosphate. We demonstrate that this compound acts like a non-obligate chain-terminator. Nature Publishing Group UK 2018-03-05 /pmc/articles/PMC5838098/ /pubmed/29507309 http://dx.doi.org/10.1038/s41598-018-22328-3 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Tchesnokov, Egor P.
Raeisimakiani, Parisa
Ngure, Marianne
Marchant, David
Götte, Matthias
Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus
title Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus
title_full Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus
title_fullStr Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus
title_full_unstemmed Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus
title_short Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus
title_sort recombinant rna-dependent rna polymerase complex of ebola virus
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838098/
https://www.ncbi.nlm.nih.gov/pubmed/29507309
http://dx.doi.org/10.1038/s41598-018-22328-3
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