Insertion of a new transcriptional unit into the genome of mouse hepatitisvirus

The subgenomic mRNAs of the coronavirus mouse hepatitis virus (MHV) are com posed of a leader sequence, identical to the 5' 70 nucleotides of the genom e, joined at distant downstream sites to a stretch of sequence that is iden tical to the 3' end of the genome. The points of fusion occur at intergenic sequences (IGSs), loci on the genome that contain a tract of sequence homo logous to the 3' end of the leader RNA. We have constructed a mutant of MHV -A59 containing an extra IGS inserted into the genome immediately downstrea m of the 3'-most gene, that encoding the nucleocapsid (N) protein. We show that in cells infected with the mutant, there is synthesis of an additional leader-containing subgenomic RNA of the predicted size. Our study demonstr ates that (i) an IGS can be a sufficient cis-acting element to dictate MHV transcription, (ii) the relative efficiency of an IGS must be influenced by factors other than the nucleotides immediately adjacent to the 5'AAUCUA AA C3' core consensus sequence or its position relative to the 3' end of the g enome, (iii) a downstream IGS can exert a polar attenuating effect on upstr eam IGSs, and (iv) unknown factors prevent the insertion of large exogenous elements between the N gene and the 3' untranslated region of MHV. These r esults confirm and extend conclusions previously derived from the analysis of defective interfering RNAs.