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Journal of Virology, July 2005, p. 8275-8281, Vol. 79, No. 13
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.13.8275-8281.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Torovirus Non-Discontinuous Transcription: Mutational Analysis of a Subgenomic mRNA Promoter

Saskia L. Smits, Arno L. W. van Vliet, Katja Segeren, Hamid el Azzouzi, Maarten van Essen, and Raoul J. de Groot*

Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands

Received 20 December 2004/ Accepted 8 March 2005

Toroviruses (order Nidovirales) are enveloped positive-strand RNA viruses of mammals. The prototype torovirus, equine torovirus strain Berne (Berne virus [BEV]), uses two different transcription strategies to produce a 3'-coterminal nested set of subgenomic (sg) mRNAs. Its mRNA 2 carries a leader sequence derived from the 5' end of the genome and is produced via discontinuous transcription. The remaining three sg mRNAs, 3 to 5, are colinear with the 3' end of the genome and are made via non-discontinuous RNA synthesis. Their synthesis is supposedly regulated by short conserved sequence motifs, 5'-ACN3-4CUUUAGA-3', within the noncoding intergenic regions that precede the M, HE, and N genes (A. L. van Vliet, S. L. Smits, P. J. Rottier, and R. J. de Groot, EMBO J. 21:6571-6580, 2002). We have now studied the—for nidoviruses unusual—non-discontinuous transcription mechanism in further detail by probing the role of the postulated transcription-regulating sequences (TRSs). To this end, we constructed a synthetic defective interfering (DI) RNA, carrying a 24-nucleotide segment of the intergenic region between the HE and N genes. We demonstrate that this DI RNA, when introduced into BEV-infected cells, directs the synthesis of a sg DI RNA species; in fact, a 16-nucleotide cassette containing the TRS already proved sufficient. Synthesis of this sg DI RNA, like that of mRNAs 3 to 5 of the standard virus, initiated at the 5'-most adenylate of the TRS. An extensive mutational analysis of the TRS is presented. Our results provide first and formal experimental evidence that the conserved motifs within the BEV intergenic sequences indeed drive sg RNA synthesis.

* Corresponding author. Mailing address: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands. Phone: 31-30-2531463/2485. Fax: 31-30-2536723. E-mail: R.Groot{at}vet.uu.nl.

Journal of Virology, July 2005, p. 8275-8281, Vol. 79, No. 13
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.13.8275-8281.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Pasternak, A. O., Spaan, W. J. M., Snijder, E. J. (2006). Nidovirus transcription: how to make sense...?. J. Gen. Virol. 87: 1403-1421 [Abstract] [Full Text]  


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