title: Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites.
abstract: Proteolytic processing of the polyprotein encoded by mRNA 1 is an essential step in coronavirus RNA replication and gene expression. We have previously reported that an open reading frame (ORF) 1a-specific proteinase of the picornavirus 3C proteinase group is involved in processing of the coronavirus infectious bronchitis virus (IBV) 1a/1b polyprotein, leading to the formation of a mature viral protein of 100 kDa. We report here the identification of a novel 10-kDa polypeptide and the involvement of the 3C-like proteinase in processing of the ORF 1a polyprotein to produce the 10-kDa protein species. By using a region-specific antiserum, V47, raised against a bacterial-viral fusion protein containing IBV sequence encoded between nucleotides 11488 and 12600, the 10-kDa polypeptide was detected in lysates from both IBV-infected and plasmid DNA-transfected Vero cells. Coexpression, deletion, and mutagenesis studies showed that this novel polypeptide was encoded by ORF 1a from nucleotide 11545 to 11878 and was cleaved from the 1a polyprotein by the 3C-like proteinase domain. Evidence presented suggested that a previously predicted Q-S (Q3783 S3784) dipeptide bond encoded by ORF 1a between nucleotides 11875 and 11880 was responsible for the release of the C terminus of the 10-kDa polypeptide and that a novel Q-N (Q3672 N3673) dipeptide bond encoded between nucleotides 11542 and 11547 was responsible for the release of the N terminus of the 10-kDa polypeptide.
authors.affiliation: Institute of Molecular Agrobiology, National University of Singapore. imaliudx@leonis.nus.sg
authors.forename: D X
authors.initials: DX
authors.lastname: Liu
authors.forename: H Y
authors.initials: HY
authors.lastname: Xu
authors.forename: T D
authors.initials: TD
authors.lastname: Brown
mesh_terms: D000818:Animals; D001665:Binding Sites; D002522:Chlorocebus aethiops; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D001351:Infectious bronchitis virus / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C087637:gene 1 protein, Coronavirus; D003546:Cysteine Endopeptidases; C000706208:3C-like proteinase, Avian infectious bronchitis virus; D000086782:Coronavirus 3C Proteases