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

Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands

Correspondence
Eric J. Snijder
e.j.snijder{at}lumc.nl

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.

The predicted RNA secondary structures of the deletion mutants produced in this study are available as supplementary material in JGV Online.

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