Convergent Evolution of Argonaute-2 Slicer Antagonism in Two Distinct Insect RNA Viruses
Figure 6
VP1 interferes with the effector phase of the RNAi pathway.
(A) Mobility shift assays for binding of viral RNAi suppressor proteins to siRNAs. Radiolabeled siRNAs were incubated in buffer (lane 1) or with decreasing amounts of recombinant MBP-VP1ΔN284 (lanes 2–5), MBP (lanes 6–9), and MBP-NS3 (lane 10–13). Ten-fold dilutions were used, starting at 2 µM for MBP-VP1ΔN284 (lane 2) and 2.6 µM for MBP (lane 6). MBP-NS3 was tested in two-fold dilutions (highest concentration of 8 µM, lane 10). RNA mobility shifts were analyzed on an 8% native polyacrylamide gel. (B) RISC Slicer assay in Drosophila embryo lysate. Lysates were incubated with non-targeting control siRNA (Ctrl, lane 1) or with Fluc siRNA (lanes 2–4) in the absence (lane 2) or presence of recombinant MBP-VP1ΔN284 (lane 3) or MBP (lane 4). RISC cleavage products were analyzed on an 8% denaturing polyacrylamide gel. Slicer assay is representative for two independent experiments.