REPLICATION AND PACKAGING OF CORONAVIRUS INFECTIOUS-BRONCHITIS VIRUS DEFECTIVE RNAS LACKING A LONG OPEN READING FRAME

The construction of a full-length clone of the avian coronavirus infec tious bronchitis virus (IBV defective RNA (D-RNA), CD-91 (9,080 nucleo tides [Z. Penzes et al., Virology 203:286-293]), downstream of the bac teriophage T7 promoter is described. Electroporation of in vitro T7-tr anscribed CD-91 RNA into IBV helper virus-infected primary chick kidne y 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 6,365 to 1,311, 1,263, o r 2,997 nucleotides in CD-44, CD-58, or CD-61, respectively. Electropo ration of in vitro T7-transcribed RNA from the three constructs into I BV helper virus-infected cells resulted in the replication and packagi ng 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 P CR 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, Ana lysis 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 replica tion. 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.