RNA signals in the 3′ terminus of the genome of Equine arteritis virus are required for viral RNA synthesis

RNA virus genomes contain cis-acting sequences and structural elements involved in virus replication. Both full-length and subgenomic negative-strand RNA synthesis are initiated at the 3' terminus of the positive-strand genomic RNA of Equine arteritis virus (EAV). To investigate the molecular mechanism of EAV RNA synthesis, the RNA secondary structure of the 3'-proximal region of the genome was analysed by chemical and enzymic probing. Based on the RNA secondary structure model derived from this analysis, several deletions were engineered in a full-length cDNA copy of the viral genome. Two RNA domains were identified that are essential for virus replication and most likely play a key role in viral RNA synthesis. The first domain, located directly upstream of the 3' untranslated region (UTR) (nt 12610-12654 of the genome), is mainly single-stranded but contains one small stem-loop structure. The second domain is located within the 3' UTR (nt 12661-12690) and folds into a prominent stem-loop structure with a large loop region. The location of this stem-loop structure near the 3' terminus of the genome suggests that it may act as a recognition signal during the initiation of minus-strand RNA synthesis.