DNA-directed expression of functional flock house virus RNA1 derivatives in Saccharomyces cerevisiae, heterologous gene expression, and selective effects on subgenomic mRNA synthesis

Flock house virus (FHV), a positive-strand RNA animal virus, is the only hi gher eukaryotic virus shown to undergo complete replication in yeast, culmi nating in production of infectious virions. To facilitate studies of viral and host functions in FHV replication in Saccharomyces cerevisiae, yeast DN A plasmids were constructed to inducibly express wild-type FHV RNA1 in vivo . Subsequent translation of FHV replicase protein A initiated robust RNA1 r eplication, amplifying RNA1 to levels approaching those of rRNA, as in FHV- infected animal cells. The RNA1-derived subgenomic mRNA, RNA3, accumulated to even higher levels of > 100,000 copies per yeast cell compared to 10 cop ies or less per cell for 95% of yeast mRNAs. The time course of RNA1 replic ation and RNA3 synthesis in induced yeast paralleled that in yeast transfec ted with natural FHV virion RNA. As in animal cells, RNA1 replication and R NA3 synthesis depended on FHV RNA replicase protein A and 3 ' -terminal RNA 1 sequences but not viral protein B2. Additional plasmids were engineered t o inducibly express RNA1 derivatives with insertions of the green fluoresce nt protein (GFP) gene in subgenomic RNA3. These RNA1 derivatives were repli cated, synthesized RNA3, and expressed GFP when provided FHV polymerase in either cis or trans providing the first demonstration of reporter gene expr ession from FHV subgenomic RNA. Unexpectedly, fusing GFP to the protein A C terminus selectively inhibited production of positive- and negative-strand subgenomic RNA3 but not genomic RNA1 replication. Moreover, changing the f irst nucleotide of the subgenomic mRNA from G to T selectively inhibited pr oduction of positive-strand but not negative-strand RNA3, suggesting that s ynthesis of negative-strand subgenomic RNA3 may precede synthesis of positi ve-strand RNA3.