Fig. 6.

In vitro RNA-binding properties of the WhNV B2 protein and its mutants. (A) Gel shift assay for WT and mutated B2 proteins. In vitro-transcribed EGFP-derived 500-bp dsRNA (top lanes) and synthesized siRNA-EGFP (bottom lanes) at a concentration of 0.1 μM were incubated with 30 μM (100 μM for siRNA) MBP-B2, MBP, or its mutants as indicated above each lane. After 30 min of incubation at 25°C, products were separated on 1.2% TBE-agarose gels, and the RNA was visualized by staining with ethidium bromide. (B) RNase III cleavage protection assay for the B2 WT and its mutants. After incubation with 30 μM MBP-B2, MBP, or its mutants as indicated above, 0.1 μM 500-bp dsRNA was incubated with 1 U of RNase III at 37°C for 30 min. The synthesized siRNA-EGFP was loaded as a positive control (lane 1), indicating the location of the bands corresponding to the RNase III digestion products. (C) RNase III cleavage protection assay for the B2 protein at multiple concentrations. The experimental conditions used were identical to those indicated for panel B, except that the concentration of MBP-B2 was increased from 0 μM to 30 μM, as indicated above each lane, which was positively correlated with the increasing ratios of protected dsRNA to RNase III-processed siRNA.