FIG. 3.

Effect of CP on sgRNA transcription-mediated inhibition of RNA3 replication. (A) Schematic diagram of expression cassettesfor RNA3 derivatives and CP mRNA used in this experiment and not introduced in Fig. 2. A solid box used for the CP ORF indicates a translationally active wt CP ORF; a dashed box represents a translationally inactive CP ORF due to a frameshift mutation. CP-trans mRNA has the BMV CP ORF and yeast 5′- and 3′-UTRs and is expressed under control of the GAL1 promoter and ADH1 polyadenylation sequences. (B) Northern blot and Western blot analyses of BMV RNA3-specific products and CP, respectively. Yeast expressing BMV 1a, 2a, and RNA3 derivatives plus empty plasmid or CP-trans plasmid were grown in galactose medium, collected, and divided for RNA (as for Fig. 2) or protein analysis. CP expression was analyzed by Western blotting using total cell lysate and polyclonal anti-CP serum. Ethidium bromide-stained rRNA is shown below the Northern blots as a loading control. (C) Effect of CP expression on the relative accumulation of positive- and negative-strand RNA3 derivatives. The histogram compares accumulation of the positive strand (black bars) and negative strand (gray bars) for the RNA3 derivatives shown in panel B.