Biochemical characterization of arterivirus nonstructural protein 11 reveals the nidovirus-wide conservation of a replicative endoribonuclease

J Virol. 2009 Jun;83(11):5671-82. doi: 10.1128/JVI.00261-09. Epub 2009 Mar 18.

Abstract

Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biocatalysis
  • Conserved Sequence
  • Endoribonucleases / genetics
  • Endoribonucleases / isolation & purification
  • Endoribonucleases / metabolism*
  • Enzyme Activation / drug effects
  • Genome, Viral / genetics
  • Manganese / pharmacology
  • Molecular Sequence Data
  • Mutation / genetics
  • Nidovirales / chemistry
  • Nidovirales / genetics
  • Nidovirales / metabolism*
  • RNA, Viral / biosynthesis*
  • RNA, Viral / genetics*
  • Sequence Alignment
  • Substrate Specificity
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*

Substances

  • RNA, Viral
  • Viral Nonstructural Proteins
  • Manganese
  • Endoribonucleases