Fig. 1.

WhNV B2 inhibits gene-specific RNAi induced by plasmid-carrying dsRNA or chemically synthesized siRNA in Pr-E cells. (A) Assay for RNA silencing of EGFP in Pr-E cells. The EGFP expression plasmid pAcEGFP was cotransfected with EGFP-specific dsRNA-expressing plasmid (anti-EGFP dsRNA) or synthesized siRNA (siRNA-EGFP) at multiple concentrations (1 to 40 nM) that were indicated above each lane. At 72 h postcotransfection, RNA was harvested from Pr-E cells to determine the reduction in EGFP transcripts and the production of siRNA by Northern blot analysis. We loaded 5 nM synthetic siRNA-EGFP as a positive control (lane 1), indicating the location of the bands corresponding to cellular siRNAs produced from EGFP-specific dsRNA. Irrelevant dsRNA-expressing plasmid (anti-Fluc dsRNA) or negative siRNA (siRNA-Fluc) was transfected as a negative control, respectively (lanes 4, 11, and 12). (B) Northern blotting shows the levels of EGFP mRNA when cotransfected with anti-EGFP dsRNA or siRNA-EGFP (40 nM) in the absence or presence of WhNV B2. Cells assayed as described for panel A were cotransfected with B2-expressing plasmid pAcWhB2-85 (lanes 5, 6, 11, and 12) or empty vector pAc (lanes 9 and 10). The mRNA levels of EGFP and the expression of B2 protein were determined by Northern blotting and Western blotting, with 18S rRNA and β-actin as loading controls, respectively.