Multiple widely spaced elements determine the efficiency with which a distal cistron is expressed from the polycistronic pregenomic RNA of figwort mosaic caulimovirus

The polycistronic expression mechanism of the plant pararetrovirus figwort mosaic caulimovirus (FMV) depends upon cis-acting elements present in its pregenomic RNA and a trans-acting protein (P6) which is expressed from a monocistronic subgenomic RNA. Using transient expression of FMV-derived polycistronic reporter constructs in Nicotiana edwardsonii cell suspension protoplasts, we further analyzed the cis-acting elements involved in polycistronic expression. A cis-acting element located within the first 74 nucleotides of the 7,954-nucleotide pregenomic RNA appears to be essential for P6 to transactivate expression of an internal cistron. Expression of this internal cistron, in the presence of P6, is greatly enhanced by the combined presence of two cis-acting elements located at the 3' end of the polycistronic RNA. Surprisingly, deletion of the most upstream of these two 3' cis-acting elements exposed a negative-acting element located internally on the polycistronic RNA, at the 3' end of open reading frame I. The action of both this negative-acting internal element and the positive-acting 3' elements is more pronounced when the large 5' untranslated leader region is present. This indicates that the 5' untranslated leader region is central to regulation of the FMV gene expression mechanism. Although a limited set of elements suffices to direct polycistronic expression in this eukaryotic system, a complex interplay between elements is involved in the spatial regulation of the genes present on the pregenomic RNA of FMV.