Novel Drosophila Viruses Encode Host-Specific Suppressors of RNAi
(A) Western blot analysis of S2 cells expressing V5 epitope-tagged VP1 from D. melanogaster Nora virus (DmelNV) and D. immigrans Nora-like virus (DimmNV). S2 cells were transfected with plasmids encoding full-length VP1 (FL) and C-terminal (ΔC) or N-terminal (ΔN) deletions thereof. Expression of the VP1 constructs was analyzed by western blot using an anti-V5 (α-V5) antibody. Detection of tubulin with anti-tubulin (α-tub) antibody was used as a loading control. Molecular mass (in kDa) is indicated on the left. For DmelNV VP1ΔN284, bands of lower mobility were observed in addition to the expected 26 kDa protein, the nature of which remains unknown. Note that these additional bands are not consistently observed (Figure S2A, lane 5, and ). (B) RNAi sensor assay in S2 cells. Firefly luciferase (Fluc) and Renilla luciferase (Rluc) reporter plasmids were transfected into S2 cells, together with plasmids encoding the indicated VP1 constructs. Two days after transfection, S2 cells were soaked in either control (Ctrl) dsRNA or Fluc dsRNA, and luciferase activities were measured the next day. Fluc counts were normalized to Rluc counts, and presented as fold silencing relative to the corresponding control dsRNA treatment. (C) Hairpin-based RNAi sensor assay in S2 cells. S2 cells were transfected with plasmids coding for Fluc, Rluc, and an Rluc-hairpin RNA together with a control vector (Vector) or plasmids encoding the N-terminal deletion mutants of DmelNV VP1ΔN284 or DimmNV VP1ΔN295. Rluc counts were normalized to Fluc counts, and presented as fold silencing over non-hairpin control transfections. Bars in Panels B and C represent means and standard deviations of three independent biological replicates. One-way ANOVA followed by Dunnett's post hoc test was used to evaluate whether VP1 constructs significantly suppressed RNAi relative to the vector control (light gray bar). ** P<0.01; *** P<0.001; ns, not significant.