title: An arginine-to-proline mutation in a domain with undefined functions within the helicase protein (Nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells.
abstract: Genetic manipulation of the RNA genomes by reverse genetics is a powerful tool to study the molecular biology and pathogenesis of RNA viruses. During construction of an infectious clone from a Vero cell-adapted coronavirus infectious bronchitis virus (IBV), we found that a G-C point mutation at nucleotide position 15526, causing Arg-to-Pro mutation at amino acid position 132 of the helicase protein, is lethal to the infectivity of IBV on Vero cells. When the in vitro-synthesized full-length transcripts containing this mutation were introduced into Vero cells, no infectious virus was rescued. Upon correction of the mutation, infectious virus was recovered. Further characterization of the in vitro-synthesized full-length transcripts containing the G15526C mutation demonstrated that this mutation may block the transcription of subgenomic RNAs. Substitution mutation of the Arg132 residue to a positively charged amino acid Lys affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. However, mutation of the Arg132 residue to Leu, a conserved residue in other coronaviruses at the same position, reduced the recovery rate of the in vitro-synthesized transcripts. The recovered mutant virus showed much smaller-sized plaques. On the contrary, a G-C and a G-A point mutations at nucleotide positions 4330 and 9230, respectively, causing Glu-Gln and Gly-Glu mutations in or near the catalytic centers of the papain-like (Nsp3) and 3C-like (Nsp5) proteinases, did not show detectable detrimental effect on the rescue of infectious viruses and the infectivity of the rescued viruses.
authors.affiliation: Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, 138673 Singapore.
authors.forename: Shouguo
authors.initials: S
authors.lastname: Fang
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.forename: Felicia P L
authors.initials: FP
authors.lastname: Tay
authors.forename: Beng Sern
authors.initials: BS
authors.lastname: Ng
authors.forename: Ding Xing
authors.initials: DX
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002522:Chlorocebus aethiops; D001351:Infectious bronchitis virus / Q000235:genetics* / Q000254:growth & development*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000235:genetics / Q000502:physiology; D017354:Point Mutation; D020365:RNA Helicases / Q000235:genetics* / Q000502:physiology; D012367:RNA, Viral / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014709:Vero Cells; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication / Q000235:genetics*
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins; D010447:Peptide Hydrolases; D020365:RNA Helicases