Genetic manipulation of arterivirus alternative mRNA leader-body junction sites reveals tight regulation of structural protein expression

To express its structural proteins, the arterivirus Equine arteritis virus (EAV) produces a nested set of six subgenomic (sg) RNA species. These RNA molecules are generated by a mechanism of discontinuous transcription, during which a common leader sequence, representing the 5' end of the genomic RNA, is attached to the bodies of the sg RNAs. The connection between the leader and body parts of an mRNA is formed by a short, conserved sequence element termed the transcription-regulating sequence (TRS), which is present at the 3' end of the leader as well as upstream of each of the structural protein genes. With the exception of RNA3, only one body TRS was previously assumed to be used to join the leader and body of each EAV sg RNA. Here we show that for the synthesis of two other sg RNAs, RNA4 and RNA5, alternative leader-body junction sites that differ substantially in transcriptional activity are used. By site-directed mutagenesis of an EAV infectious cDNA clone, the alternative TRSs used to generate RNA3, -4, and -5 were inactivated, which strongly influenced the corresponding RNA levels and the production of infectious progeny virus. The relative amounts of RNA produced from alternative TRSs differed significantly and corresponded to the relative infectivities of the virus mutants. This strongly suggested that the structural proteins that are expressed from these RNAs are limiting factors during the viral life cycle and that the discontinuous step in sg RNA synthesis is crucial for the regulation of their expression. On the basis of a theoretical analysis of the predicted RNA structure of the 3' end of the EAV genome, we propose that the local secondary RNA structure of the body TRS regions is an important factor in the regulation of the discontinuous step in EAV sg mRNA synthesis.