Key result
A 16-nucleotide cassette containing the transcription-regulating sequence was sufficient to drive subgenomic RNA synthesis in equine torovirus-infected cells.
This study provides the first formal experimental evidence that conserved motifs within the BEV intergenic sequences drive subgenomic RNA synthesis via a non-discontinuous transcription mechanism.
Establishes minimal TRS cassette for torovirus subgenomic RNA; extends models but leaves open relevance to human pathogens.
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.
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Smits et al. (2005) studied Torovirus infection. Mutational analysis of transcription-regulating sequences (TRSs) was evaluated on Synthesis of subgenomic DI RNA. A 16-nucleotide cassette containing the transcription-regulating sequence was sufficient to drive subgenomic RNA synthesis in equine torovirus-infected cells.
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