Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses
| Autor: | Dmitry V. Samborskiy, Eric J. Snijder, Mark Ruben, Peter A. van Veelen, George M.C. Janssen, Anastasia A. Gulyaeva, Igor A. Sidorov, Alexander E. Gorbalenya, Hermen S. Overkleeft, Kathleen C. Lehmann, Andrey M. Leontovich, Alexander A. Kravchenko, Jessika C. Zevenhoven-Dobbe, Clara C. Posthuma |
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| Rok vydání: | 2015 |
| Předmět: |
viruses
Protein domain RNA-dependent RNA polymerase Uridine Triphosphate Nidovirales Biology Virus Replication medicine.disease_cause Phosphates Viral Proteins 03 medical and health sciences chemistry.chemical_compound Equartevirus RNA polymerase Genetics medicine Binding site Uridine Conserved Sequence Polyproteins 030304 developmental biology Coronavirus Manganese 0303 health sciences Messenger RNA Binding Sites Guanosine Nucleic Acid Enzymes Nucleotides 030302 biochemistry & molecular biology RNA RNA-Dependent RNA Polymerase Nucleotidyltransferases Molecular biology Protein Structure Tertiary Severe acute respiratory syndrome-related coronavirus chemistry Biochemistry Nucleic acid Guanosine Triphosphate |
| Zdroj: | Nucleic Acids Research Nucleic Acids Research, 43(17), 8416-8434 |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkv838 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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