Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses

Nucleic Acids Res. 2015 Sep 30;43(17):8416-34. doi: 10.1093/nar/gkv838. Epub 2015 Aug 24.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Conserved Sequence
  • Equartevirus / enzymology
  • Equartevirus / physiology
  • Guanosine / chemistry
  • Guanosine Triphosphate / metabolism
  • Manganese / chemistry
  • Nidovirales / enzymology*
  • Nidovirales / genetics
  • Nucleotides / metabolism
  • Nucleotidyltransferases / chemistry*
  • Nucleotidyltransferases / metabolism
  • Phosphates / chemistry
  • Polyproteins / chemistry
  • Polyproteins / metabolism
  • Protein Structure, Tertiary
  • RNA-Dependent RNA Polymerase / chemistry*
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism
  • SARS Virus / enzymology
  • SARS Virus / physiology
  • Uridine / chemistry
  • Uridine Triphosphate / metabolism
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism
  • Virus Replication

Substances

  • Nucleotides
  • Phosphates
  • Polyproteins
  • Viral Proteins
  • Guanosine
  • Manganese
  • Guanosine Triphosphate
  • Nucleotidyltransferases
  • RNA-Dependent RNA Polymerase
  • Uridine Triphosphate
  • Uridine