Subgenomic mRNA transcription in tobacco necrosis virus.

Tobacco necrosis virus-D (TNV-D), a positive-strand RNA Necrovirus in the family Tombusviridae, transcribes two subgenomic (sg) mRNAs during infections. We have investigated the strategy used by TNV-D in this process and uncovered evidence that it employs a premature termination (PT) mechanism for the transcription of its sg mRNAs. Structural and mutational analysis of the TNV-D genome identified local RNA structures upstream from transcriptional initiation sites that functioned in the plus-strand as attenuation structures and mediated the production of sg mRNA-sized minus-strands. Other evidence in support of a PT mechanism included the ability to uncouple minus-strand sg RNA production from plus-strand sg mRNA synthesis and the sequence similarities observed between the sg mRNA promoter and that for the viral genome. Accordingly, our results indicate that the necrovirus TNV-D, like several other genera in the family Tombusviridae, uses a PT mechanism for transcription of its sg mRNAs.