FIG. 1.

(A) Schematic diagram of a yeast three-hybrid system. The specific binding of MS2-RSV MΨ RNA to the protease-deleted Gag polyprotein, GagΔPR, would reconstitute the activity of the transcriptional activator and lead to the expression of a reporter gene, lacZ. DB, DB of the LexA protein; AD, AD of the Gal4 protein; UAS, binding site for the transcriptional activator upstream of the reporter gene. (B) Strategy for in vivo screening of RSV mutants with randomly mutated MΨ sequences using the yeast three-hybrid system. Pools of randomly mutagenized MΨ sequences using PCR were digested with XmaI and SphI and ligated with the XmaI-SphI fragment of the pIIIA/ms2-1 RNA hybrid expression plasmid. The ligation mixture was used to cotransform yeast cells along with the AD-GagΔPR hybrid protein expression plasmid and was plated on the plates lacking Ura and Leu to select both plasmids. The β-Gal activity of each transformant colony was qualitatively measured after colonies were transferred onto filters. Colonies with low β-Gal activities were screened to detect MΨ inserts by using PCR. The positive clones were further characterized to locate mutations in MΨ sequences.