Replication of flock house virus RNAs from primary transcripts made in cells by RNA polymerase

To develop vector systems that combine high transcription activity with biologically safe delivery vehicles, we have explored the use of RNA replication to amplify mRNAs, by using flock house virus (FHV) as a model system. The FHV RNA replicase is encoded in the larger of the two segments that comprise the viral positive-sense RNA genome. A cDNA copy of this self-replicating RNA was precisely positioned between a promoter site for cellular RNA polymerase II and a cDNA encoding a self-cleaving ribozyme from hepatitis delta virus. Transfection of this plasmid into cultured BHK cells resulted in prolonged, autonomous FHV RNA replication in the cytoplasm and substantial amplification of the RNA replicon. The replicase also amplified RNA transcribed from a second plasmid of similar design that contained a cDNA copy of the other FHV genome segment. These results constitute a significant step toward the harnessing of nodaviral RNA replica-tion as the basis of a versatile vector system. To achieve the maximum level of expression for a vectored gene, it is advantageous not only to optimize its cis-acting signals but also to use a vector that amplifies both the template and the RNA polymerase that is responsible for its transcrip-tion. However, while the ability to replicate and transcrib