Isolation and characterization of an arterivirus defective interfering RNAgenome

Equine arteritis virus (EAV), the type member of the family Arteriviridae, is a single-stranded RNA virus with a positive-stranded genome of approxima tely 13 kb. EAV uses a discontinuous transcription mechanism to produce a n ested set of six subgenomic mRNAs from which its structural genes are expre ssed. We have generated the first documented arterivirus defective interfer ing (DI) RNAs by serial undiluted passaging of a wild-type EAV stock in BHK -21 cells. A cDNA copy of the smallest DI RNA (5.6 kb) was cloned. Upon tra nsfection into EAV-infected BHK-21 cells, transcripts derived from this clo ne (pEDI) were replicated and packaged. Sequencing of pEDI revealed that th e DI RNA was composed of three segments of the EAV genome (nucleotides 1 to 1057, 1388 to 1684, and 8530 to 12704) which were fused in frame with resp ect to the replicase reading frame. Remarkably, this DI RNA has retained al l of the sequences encoding the structural proteins By insertion of the chl oramphenicol acetyltransferase reporter gene in the DI RNA genome, we were able to delimitate the sequences required for replication/DI-based transcri ption and packaging of EBV DI RNAs and to reduce the maximal size of a repl ication-competent EAV DI RNA to approximately 3 kb.