A new viral vector exploiting RNA polymerase I-mediated transcription

We have developed a new viral vector system exploiting RNA-polymerase I transcription. The vector is based on the crucifer-infecting tobacco mosaic virus (crTMV) cDNA inserted into the rRNA transcriptional cassette (promoter and terminator). To visualize reproduction of the vector, the coat protein gene was replaced with the gene encoding green fluorescent protein (GFP) resulting in a PrrRNA-crTMV-GFP construct. Our results showed that agroinjection of Nicotiana benthamiana leaves with this vector results in GFP production from uncapped crTMV-GFP RNA because RNA polymerase I mediates synthesis of rRNA lacking a cap. Coexpression of the crTMV 122 kDa capping protein gene and the silencing suppressor encoded by the tomato bushy stunt virus p19 gene stimulated virus-directed GFP production more than 100-fold. We conclude that the Pol I promoter can be used to drive transcription in a transient expression system.