Journal
NUCLEIC ACIDS RESEARCH
Volume 43, Issue 17, Pages 8416-8434Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkv838
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Funding
- European Union Seventh Framework program through the EUVIRNA project (European Training Network on (+) RNA virus replication and Antiviral Drug Development [264286]
- SILVER project [260644]
- Netherlands Organization for Scientific Research (NWO) through TOP-GO [700.10.352]
- Leiden University Fund
- Leiden University Medical Center
- Moscow State University (MoBiLe)
- Netherlands Organisation for Scientific Research
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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.
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