Expression of reporter genes from the defective RNA CD-61 of the coronavirus infectious bronchitis virus.

Stirrups, K; Shaw, K; Evans, S; Dalton, K; Casais, R; Cavanagh, D; Britton, P

The defective RNA (D-RNA) CD-61, derived from the Beaudette strain of the avian coronavirus infectious bronchitis virus (IBV), was used as an RNA vector for the expression of two reporter genes, luciferase and chloramphenicol acetyltransferase (CAT). D-RNAs expressing the CAT gene were demonstrated to be capable of producing CAT protein in a helper-dependent expression system to about 1.6 microgram per 10(6) cells. The reporter genes were expressed from two different sites within the CD-61 sequence and expression was not affected by interruption of the CD-61-specific ORF. Expression of the reporter genes was under the control of a transcription-associated sequence (TAS) derived from the Beaudette gene 5, normally used for the transcription of IBV subgenomic mRNA 5. The Beaudette gene 5 TAS is composed of two tandem repeats of the IBV canonical consensus sequence involved in the acquisition of a leader sequence during the discontinuous transcription of IBV subgenomic mRNAs. It is demonstrated that only one canonical sequence is required for expression of mRNA 5 or for the expression of an mRNA from a D-RNA and that either sequence can function as an acceptor site for acquisition of the leader sequence.