Fig 3.

Effect of a Pkc1 inhibitor (cercosporamide) on viral RNA accumulation in yeast. (A) (Top) Northern blot analysis was used to detect DI-72 (+)repRNA accumulation in a yeast strain treated with cercosporamide (0.5 μg/ml) to inhibit Pkc1p function. rec, recombinant RNA; deg, partially degraded. (Bottom) Ethidium-bromide stained gel of total RNA extracts of the samples used for Northern blotting above. (B to D) Northern blot analysis of TBSV repRNA replication in yeast expressing the wt p33 or the nonphosphorylatable p33-A₂₀₅A₂₁₀A₂₁₁ (B) or partially phosphorylatable p33-A₂₁₀A₂₁₁ (C) and in p33-D₂₀₅ mutants from the ADH1 promoter (D), while wt p92 and DI-72 repRNA were expressed from the CUP1 promoter and the GAL1 promoter, respectively. Yeast was cultured for 36 h at 23°C in the presence of cercosporamide (1.0 μg/ml), 2% galactose, and 50 μM CuSO₄.