FIG. 2.

Accumulation of plus- and minus-strand genomic RNAs by RUB SP. The synthesis of replicon genome in the absence or presence of RUB SP was investigated by use of an RNase protection assay (A) and Northern blot analysis (B). (A) Intracellular RNA from transfected Vero cells with replicons with or without SP RNA cotransfection was harvested 18 h (for detecting minus-strand RNA; upper panel) or 72 h (for detecting plus-strand RNA; lower panel) after transfection and subjected to an RNase protection assay using ³⁵S-labeled RNA probes. The RNase-protected fragments corresponding to the rG, rSG, vG, and vSG RNAs are indicated. (B) Intracellular RNA prepared from infected (RUB inf) or uninfected (MI) cells transfected with specific replicons with or without RUB SP gene was prepared at 18 h after transfection. RNA was denatured by dimethyl sulfoxide and glyoxal and electrophoresed on an 0.85% agarose gel followed by hybridization using a DIG-labeled pGEM-GFP₄₀₀ probe of positive polarity. The RNA species corresponding to rG and vG RNA are indicated at the right.