title: Site-directed mutagenesis of the Nidovirus replicative endoribonuclease NendoU exerts pleiotropic effects on the arterivirus life cycle.
abstract: The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a Xenopus laevis endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined.
authors.affiliation: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, LUMC P4-26, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Clara C
authors.initials: CC
authors.lastname: Posthuma
authors.forename: Danny D
authors.initials: DD
authors.lastname: Nedialkova
authors.forename: Jessika C
authors.initials: JC
authors.lastname: Zevenhoven-Dobbe
authors.forename: Jeroen H
authors.initials: JH
authors.lastname: Blokhuis
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Gorbalenya
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Snijder
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D028382:Arteriviridae / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D003332:Coronaviridae / Q000235:genetics / Q000502:physiology; D006224:Cricetinae; D004722:Endoribonucleases / Q000235:genetics* / Q000502:physiology*; D007240:Equartevirus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D012367:RNA, Viral / Q000096:biosynthesis; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000502:physiology*; D010948:Viral Plaque Assay; D015678:Viral Structural Proteins / Q000032:analysis; D014779:Virus Replication / Q000235:genetics
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D015678:Viral Structural Proteins; D004722:Endoribonucleases