title: Dependence of coronavirus RNA replication on an NH2-terminal partial nonstructural protein 1 in cis.
abstract: Genomes of positive (+)-strand RNA viruses use cis-acting signals to direct both translation and replication. Here we examine two 5'-proximal cis-replication signals of different character in a defective interfering (DI) RNA of the bovine coronavirus (BCoV) that map within a 322-nucleotide (nt) sequence (136 nt from the genomic 5' untranslated region and 186 nt from the nonstructural protein 1 [nsp1]-coding region) not found in the otherwise-identical nonreplicating subgenomic mRNA7 (sgmRNA7). The natural DI RNA is structurally a fusion of the two ends of the BCoV genome that results in a single open reading frame between a partial nsp1-coding region and the entire N gene. (i) In the first examination, mutation analyses of a recently discovered long-range RNA-RNA base-paired structure between the 5' untranslated region and the partial nsp1-coding region showed that it, possibly in concert with adjacent stem-loops, is a cis-acting replication signal in the (+) strand. We postulate that the higher-order structure promotes (+)-strand synthesis. (ii) In the second examination, analyses of multiple frame shifts, truncations, and point mutations within the partial nsp1-coding region showed that synthesis of a PEFP core amino acid sequence within a group A lineage betacoronavirus-conserved NH2-proximal WAPEFPWM domain is required in cis for DI RNA replication. We postulate that the nascent protein, as part of an RNA-associated translating complex, acts to direct the DI RNA to a critical site, enabling RNA replication. We suggest that these results have implications for viral genome replication and explain, in part, why coronavirus sgmRNAs fail to replicate.
  
  
  OBJECTIVE
  cis-Acting RNA and protein structures that regulate (+)-strand RNA virus genome synthesis are potential sites for blocking virus replication. Here we describe two: a previously suspected 5'-proximal long-range higher-order RNA structure and a novel nascent NH2-terminal protein component of nsp1 that are common among betacoronaviruses of group A lineage.
authors.affiliation: Department of Biomedical and Diagnostic Sciences, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee, USA.
authors.forename: Yu-Pin
authors.initials: YP
authors.lastname: Su
authors.affiliation: Department of Biomedical and Diagnostic Sciences, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee, USA.
authors.forename: Yi-Hsin
authors.initials: YH
authors.lastname: Fan
authors.affiliation: Department of Biomedical and Diagnostic Sciences, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee, USA dbrian@utk.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Brian
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence / Q000235:genetics; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D045744:Cell Line, Tumor; D018352:Coronavirus Infections / Q000235:genetics / Q000821:virology; D017938:Coronavirus, Bovine / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins