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

Virology. 2007 Feb 5;358(1):136-47. doi: 10.1016/j.virol.2006.08.020. Epub 2006 Sep 18.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Animals
  • Chlorocebus aethiops
  • Infectious bronchitis virus / genetics*
  • Infectious bronchitis virus / growth & development*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Hydrolases / genetics
  • Peptide Hydrolases / physiology
  • Point Mutation*
  • RNA Helicases / genetics*
  • RNA Helicases / physiology
  • RNA, Viral / metabolism
  • Sequence Homology, Amino Acid
  • Transcription, Genetic
  • Vero Cells
  • Viral Plaque Assay
  • Viral Proteins / genetics*
  • Viral Proteins / physiology
  • Virus Replication / genetics*


  • RNA, Viral
  • Viral Proteins
  • Peptide Hydrolases
  • RNA Helicases