title: Replication and packaging of coronavirus infectious bronchitis virus defective RNAs lacking a long open reading frame.
abstract: The construction of a full-length clone of the avian coronavirus infectious bronchitis virus (IBV) defective RNA (D-RNA), CD-91 (9,080 nucleotides [Z. Penzes et al., Virology 203:286-293]), downstream of the bacteriophage T7 promoter is described. Electroporation of in vitro T7-transcribed CD-91 RNA into IBV helper virus-infected primary chick kidney cells resulted in the production of CD-91 RNA as a replicating D-RNA in subsequent passages. Three CD-91 deletion mutants were constructed--CD-44, CD-58, and CD-61--in which 4,639, 3,236, and 2,953 nucleotides, respectively, were removed from CD-91, resulting in the truncation of the CD-91 long open reading frame (ORF) from 6,465 to 1,311, 1,263, or 2,997 nucleotides in CD-44, CD-58, or CD-61, respectively. Electroporation of in vitro T7-transcribed RNA from the three constructs into IBV helper virus-infected cells resulted in the replication and packaging of CD-58 and CD-61 but not CD-44 RNA. The ORF of CD-61 was further truncated by the insertion of stop codons into the CD-61 sequence by PCR mutagenesis, resulting in constructs CD-61T11 (ORF: nucleotides 996 to 1,058, encoding 20 amino acids), CD-61T22 (ORF: nucleotides 996 to 2,294, encoding 432 amino acids), and CD-61T24 (ORF: nucleotides 996 to 2,450, encoding 484 amino acids), all of which were replicated and packaged to the same levels as observed for either CD-61 or CD-91. Analysis of the D-RNAs showed that the CD-91- or CD-61-specific long ORFs had not been restored. Our data indicate that IBV D-RNAs based on the natural D-RNA, CD-91, do not require a long ORF for efficient replication. In addition, a 1.4-kb sequence, corresponding to IBV sequence at the 5' end of the 1b gene, may be involved in the packaging of IBV D-RNAs or form part of a cis-acting replication element.
authors.affiliation: Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, United Kingdom.
authors.forename: Z
authors.initials: Z
authors.lastname: Pénzes
authors.forename: C
authors.initials: C
authors.lastname: Wroe
authors.forename: T D
authors.initials: TD
authors.lastname: Brown
authors.forename: P
authors.initials: P
authors.lastname: Britton
authors.forename: D
authors.initials: D
authors.lastname: Cavanagh
mesh_terms: D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D003673:Defective Viruses / Q000235:genetics*; D017353:Gene Deletion; D001351:Infectious bronchitis virus / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D012367:RNA, Viral; D017384:Sequence Deletion; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral