FIG. 3.

Northern blot hybridization analysis of plasmid-initiated NoV RNA2 replication in yeast cells. Panel A is a schematic of a DNA plasmid pN2 for RNA polII-mediated transcription of NoV RNA2 and its expected DNA-dependent and RNA-dependent transcription products; symbols for sequence elements are as described for Fig. 2A. The open box in RNA2 represents the capsid ORF. The relative positions of several RNA2 derivatives are indicated: a mutation of the translational start codon (AUG−), a derivative in which nt 53 to 476 were deleted (N2Δ), an N2Δ deletion derivative encoding the yeast HIS3 ORF (N2H), and an N2Δ deletion derivative encoding mammalian codon-optimized GFP (N2G). Panel B shows a Northern blot of positive-sense RNAs. Total RNA was extracted from induced yeast containing a plasmid encoding WT NoV RNA1 and the indicated RNA2 plasmid at 0 and 1 dpi and analyzed by Northern blot hybridization as described for Fig. 1, except that only riboprobes specific for positive-sense RNA2 were used. For each construct, the position of the full-length RNA is indicated with a solid arrowhead at the right, while the open arrowhead indicates the primary transcript before cleavage by the HDV ribozyme (see Materials and Methods).