FIG 2.

SFV capsid suppresses Dicer-mediated siRNA production by sequestrating dsRNA. (A) 293T cells were cotransfected with a plasmid encoding EGFP (0.1 μg) and EGFP-specific shRNA (shEGFP) (0.3 μg), together with either empty plasmid or a plasmid encoding SFV capsid protein or NB2 (1 μg for each). At 48 hpt, the total RNAs were extracted for small Northern blotting with a DIG-labeled RNA oligonucleotide probe targeting EGFP siRNA. U6 was used as the loading control. (B) SDS-PAGE of purified recombinant SFV capsid. BSA was used as a quantity control. (C) Increasing amounts (0 to 4 μM) of MBP fusion capsid (MBP-capsid) were incubated with 0.5 μM 200-nt DIG-labeled dsRNA at 25°C for 30 min. Complexes were separated on 1.5% native-TBE agarose gel, transferred to membranes, and then incubated with anti-DIG antibody conjugated to alkaline phosphatase. MBP-FB2 and MBP were used as controls. (D) Increasing amounts (0 to 4 μM) of MBP-capsid were incubated with 1 μM 200-nt DIG-labeled dsRNA at 25°C for 30 min. The protein-dsRNA complexes were then incubated with 1 U of RNase III at 37°C for 30 min. The reaction products were subjected to 7 M urea–15% PAGE analysis.