title: Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses.
abstract: 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.
authors: Department of Medical Microbiology, Leiden University Medical Center, Leiden, 2300 RC, Leiden, The Netherlands.
authors: Kathleen C
authors: KC
authors: Lehmann
authors: Department of Medical Microbiology, Leiden University Medical Center, Leiden, 2300 RC, Leiden, The Netherlands.
authors: Anastasia
authors: A
authors: Gulyaeva
authors: Department of Medical Microbiology, Leiden University Medical Center, Leiden, 2300 RC, Leiden, The Netherlands.
authors: Jessika C
authors: JC
authors: Zevenhoven-Dobbe
authors: Department of Immunohematology and Blood transfusion, Leiden University Medical Center, Leiden, 2300 RC, Leiden, The Netherlands.
authors: George M C
authors: GM
authors: Janssen
authors: Leiden Institute of Chemistry, Leiden University, 2300 CC, Leiden, The Netherlands.
authors: Mark
authors: M
authors: Ruben
authors: Leiden Institute of Chemistry, Leiden University, 2300 CC, Leiden, The Netherlands.
authors: Hermen S
authors: HS
authors: Overkleeft
authors: Department of Immunohematology and Blood transfusion, Leiden University Medical Center, Leiden, 2300 RC, Leiden, The Netherlands.
authors: Peter A
authors: PA
authors: van Veelen
authors: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119899 Moscow, Russia.
authors: Dmitry V
authors: DV
authors: Samborskiy
authors: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119899 Moscow, Russia.
authors: Alexander A
authors: AA
authors: Kravchenko
authors: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119899 Moscow, Russia.
authors: Andrey M
authors: AM
authors: Leontovich
authors: Department of Medical Microbiology, Leiden University Medical Center, Leiden, 2300 RC, Leiden, The Netherlands.
authors: Igor A
authors: IA
authors: Sidorov
authors: Department of Medical Microbiology, Leiden University Medical Center, Leiden, 2300 RC, Leiden, The Netherlands.
authors: Eric J
authors: EJ
authors: Snijder
authors: Department of Medical Microbiology, Leiden University Medical Center, Leiden, 2300 RC, Leiden, The Netherlands.
authors: Clara C
authors: CC
authors: Posthuma
authors: Department of Medical Microbiology, Leiden University Medical Center, Leiden, 2300 RC, Leiden, The Netherlands Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119899 Moscow, Russia Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119899 Moscow, Russia a.e.gorbalenya@lumc.nl.
authors: Alexander E
authors: AE
authors: Gorbalenya
mesh_terms: D001665:Binding Sites; D017124:Conserved Sequence; D007240:Equartevirus / Q000201:enzymology / Q000502:physiology; D006151:Guanosine / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000378:metabolism; D008345:Manganese / Q000737:chemistry; D028381:Nidovirales / Q000201:enzymology* / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry; D020815:Polyproteins / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology / Q000502:physiology; D014529:Uridine / Q000737:chemistry; D014544:Uridine Triphosphate / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D009711:Nucleotides; D010710:Phosphates; D020815:Polyproteins; D014764:Viral Proteins; D006151:Guanosine; D008345:Manganese; D006160:Guanosine Triphosphate; D009713:Nucleotidyltransferases; D012324:RNA-Dependent RNA Polymerase; D014544:Uridine Triphosphate; D014529:Uridine