Novel Drosophila Viruses Encode Host-Specific Suppressors of RNAi
(A) RNAi reporter assay based on hairpin-induced silencing of an Rluc reporter. The experiment was performed as described in the legend to Figure 2C, except that a non-specific control dsRNA (Ctrl) or dsRNA targeting the coding sequence or the 3′UTR of Dmel AGO2 (AGO2 CDS and AGO2 3′UTR, respectively) was co-transfected along with the reporter plasmids. Bars represent means and standard deviations of three biological replicates. One-way ANOVA followed by Dunnett's post hoc test was used to evaluate loss of silencing by AGO2 dsRNA compared to control dsRNA treated samples (light gray bars). (B) Rescue of endogenous AGO2 knockdown by D. immigrans AGO2 and suppression thereof by DimmNV VP1. Endogenous AGO2 expression was reduced by dsRNA targeting the AGO2 3′UTR, which was transfected along with luciferase reporter plasmids, Rluc hairpin plasmid, and control plasmid (Vector) or expression plasmids encoding D. melanogaster AGO1 (Dmel AGO1), AGO2 (Dmel AGO2), or D. immigrans AGO2 (Dimm AGO2). Control vector (Ctrl, white bars) or a plasmid encoding D. immigrans Nora virus VP1 (DimmNV VP1ΔN295, black bars) was co-transfected to analyze the ability of DimmNV VP1 to suppress Dimm and Dmel AGO2-mediated silencing. Data are presented as fold silencing relative to the corresponding vector control transfection. Bars represent means and standard deviations of three biological replicates. One-way ANOVA followed by Dunnett's post hoc test was used to evaluate whether AGO expression rescued silencing relative to the vector control in the absence of VP1 (light gray bar). A Student's T-test was used to analyze whether loss of silencing by expression of DimmNV VP1 was significant. * P<0.05; *** P<0.001.