Kathleen C. Lehmann 1 , Anastasia Gulyaeva 1 , Jessika C. Zevenhoven-Dobbe 1 , George M. C. Janssen 2 , Mark Ruben 3 , Hermen S. Overkleeft 3 , Peter A. van Veelen 2 , Dmitry V. Samborskiy 4 , Alexander A. Kravchenko 4 , ‡ , Andrey M. Leontovich 4 , Igor A. Sidorov 1 , Eric J. Snijder 1 , Clara C. Posthuma 1 , Alexander E. Gorbalenya 1 , 4 , 5 , *
24 August 2015
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.