Characterization of the catalytic center of the Ebola virus L polymerase.
Ebola virus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates. While no licensed therapeutics are available, recently there has been tremendous progress in developing antivirals. Targeting the ribonucleoprotein complex (RNP) proteins, which facilitate genome replication and t...
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ftdoajarticles:oai:doaj.org/article:64689680b86b496ba79139bb54a734c8 2023-05-15T15:15:26+02:00 Characterization of the catalytic center of the Ebola virus L polymerase. Marie Luisa Schmidt Thomas Hoenen 2017-10-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0005996 https://doaj.org/article/64689680b86b496ba79139bb54a734c8 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC5648267?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0005996 https://doaj.org/article/64689680b86b496ba79139bb54a734c8 PLoS Neglected Tropical Diseases, Vol 11, Iss 10, p e0005996 (2017) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2017 ftdoajarticles https://doi.org/10.1371/journal.pntd.0005996 2022-12-31T11:42:42Z Ebola virus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates. While no licensed therapeutics are available, recently there has been tremendous progress in developing antivirals. Targeting the ribonucleoprotein complex (RNP) proteins, which facilitate genome replication and transcription, and particularly the polymerase L, is a promising antiviral approach since these processes are essential for the virus life cycle. However, until now little is known about L in terms of its structure and function, and in particular the catalytic center of the RNA-dependent RNA polymerase (RdRp) of L, which is one of the most promising molecular targets, has never been experimentally characterized.Using multiple sequence alignments with other negative sense single-stranded RNA viruses we identified the putative catalytic center of the EBOV RdRp. An L protein with mutations in this center was then generated and characterized using various life cycle modelling systems. These systems are based on minigenomes, i.e. miniature versions of the viral genome, in which the viral genes are exchanged against a reporter gene. When such minigenomes are coexpressed with RNP proteins in mammalian cells, the RNP proteins recognize them as authentic templates for replication and transcription, resulting in reporter activity reflecting these processes. Replication-competent minigenome systems indicated that our L catalytic domain mutant was impaired in genome replication and/or transcription, and by using replication-deficient minigenome systems, as well as a novel RT-qPCR-based genome replication assay, we showed that it indeed no longer supported either of these processes. However, it still showed similar expression to wild-type L, and retained its ability to be incorporated into inclusion bodies, which are the sites of EBOV genome replication.We have experimentally defined the catalytic center of the EBOV RdRp, and thus a promising antiviral target regulating an essential aspect of the EBOV life cycle. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 11 10 e0005996 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Marie Luisa Schmidt Thomas Hoenen Characterization of the catalytic center of the Ebola virus L polymerase. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Ebola virus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates. While no licensed therapeutics are available, recently there has been tremendous progress in developing antivirals. Targeting the ribonucleoprotein complex (RNP) proteins, which facilitate genome replication and transcription, and particularly the polymerase L, is a promising antiviral approach since these processes are essential for the virus life cycle. However, until now little is known about L in terms of its structure and function, and in particular the catalytic center of the RNA-dependent RNA polymerase (RdRp) of L, which is one of the most promising molecular targets, has never been experimentally characterized.Using multiple sequence alignments with other negative sense single-stranded RNA viruses we identified the putative catalytic center of the EBOV RdRp. An L protein with mutations in this center was then generated and characterized using various life cycle modelling systems. These systems are based on minigenomes, i.e. miniature versions of the viral genome, in which the viral genes are exchanged against a reporter gene. When such minigenomes are coexpressed with RNP proteins in mammalian cells, the RNP proteins recognize them as authentic templates for replication and transcription, resulting in reporter activity reflecting these processes. Replication-competent minigenome systems indicated that our L catalytic domain mutant was impaired in genome replication and/or transcription, and by using replication-deficient minigenome systems, as well as a novel RT-qPCR-based genome replication assay, we showed that it indeed no longer supported either of these processes. However, it still showed similar expression to wild-type L, and retained its ability to be incorporated into inclusion bodies, which are the sites of EBOV genome replication.We have experimentally defined the catalytic center of the EBOV RdRp, and thus a promising antiviral target regulating an essential aspect of the EBOV life cycle. |
format |
Article in Journal/Newspaper |
author |
Marie Luisa Schmidt Thomas Hoenen |
author_facet |
Marie Luisa Schmidt Thomas Hoenen |
author_sort |
Marie Luisa Schmidt |
title |
Characterization of the catalytic center of the Ebola virus L polymerase. |
title_short |
Characterization of the catalytic center of the Ebola virus L polymerase. |
title_full |
Characterization of the catalytic center of the Ebola virus L polymerase. |
title_fullStr |
Characterization of the catalytic center of the Ebola virus L polymerase. |
title_full_unstemmed |
Characterization of the catalytic center of the Ebola virus L polymerase. |
title_sort |
characterization of the catalytic center of the ebola virus l polymerase. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2017 |
url |
https://doi.org/10.1371/journal.pntd.0005996 https://doaj.org/article/64689680b86b496ba79139bb54a734c8 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 11, Iss 10, p e0005996 (2017) |
op_relation |
http://europepmc.org/articles/PMC5648267?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0005996 https://doaj.org/article/64689680b86b496ba79139bb54a734c8 |
op_doi |
https://doi.org/10.1371/journal.pntd.0005996 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
11 |
container_issue |
10 |
container_start_page |
e0005996 |
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1766345801648308224 |