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Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses
RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural pro...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787807/ https://www.ncbi.nlm.nih.gov/pubmed/26304538 http://dx.doi.org/10.1093/nar/gkv838 |
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author | Lehmann, Kathleen C. Gulyaeva, Anastasia Zevenhoven-Dobbe, Jessika C. Janssen, George M. C. Ruben, Mark Overkleeft, Hermen S. van Veelen, Peter A. Samborskiy, Dmitry V. Kravchenko, Alexander A. Leontovich, Andrey M. Sidorov, Igor A. Snijder, Eric J. Posthuma, Clara C. Gorbalenya, Alexander E. |
author_facet | Lehmann, Kathleen C. Gulyaeva, Anastasia Zevenhoven-Dobbe, Jessika C. Janssen, George M. C. Ruben, Mark Overkleeft, Hermen S. van Veelen, Peter A. Samborskiy, Dmitry V. Kravchenko, Alexander A. Leontovich, Andrey M. Sidorov, Igor A. Snijder, Eric J. Posthuma, Clara C. Gorbalenya, Alexander E. |
author_sort | Lehmann, Kathleen C. |
collection | PubMed |
description | RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant non-structural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRp-associated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies. |
format | Online Article Text |
id | pubmed-4787807 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-47878072016-03-14 Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses Lehmann, Kathleen C. Gulyaeva, Anastasia Zevenhoven-Dobbe, Jessika C. Janssen, George M. C. Ruben, Mark Overkleeft, Hermen S. van Veelen, Peter A. Samborskiy, Dmitry V. Kravchenko, Alexander A. Leontovich, Andrey M. Sidorov, Igor A. Snijder, Eric J. Posthuma, Clara C. Gorbalenya, Alexander E. Nucleic Acids Res Nucleic Acid Enzymes RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant non-structural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRp-associated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies. Oxford University Press 2015-09-30 2015-08-24 /pmc/articles/PMC4787807/ /pubmed/26304538 http://dx.doi.org/10.1093/nar/gkv838 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Nucleic Acid Enzymes Lehmann, Kathleen C. Gulyaeva, Anastasia Zevenhoven-Dobbe, Jessika C. Janssen, George M. C. Ruben, Mark Overkleeft, Hermen S. van Veelen, Peter A. Samborskiy, Dmitry V. Kravchenko, Alexander A. Leontovich, Andrey M. Sidorov, Igor A. Snijder, Eric J. Posthuma, Clara C. Gorbalenya, Alexander E. Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses |
title | Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses |
title_full | Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses |
title_fullStr | Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses |
title_full_unstemmed | Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses |
title_short | Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses |
title_sort | discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the rna polymerase-containing protein of all nidoviruses |
topic | Nucleic Acid Enzymes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787807/ https://www.ncbi.nlm.nih.gov/pubmed/26304538 http://dx.doi.org/10.1093/nar/gkv838 |
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