FIG. 5.

EBOV RNAi suppression: model for VP30. (A) The RNA binding domain of VP30 does not mediate RNAi suppression. N-terminal mutants of VP30 (VP30Δ1-40 and the R40A mutant) were tested for their ability to abrogate the reversion of GFP silencing. pGFP was transfected into HEK293 cells alone, with the nontargeting siRNA, or with siGFP; plasmids encoding the wild-type protein VP30 (pVP30-H) or the N-terminal mutants were cotransfected with pGFP and siGFP. pVR1012 empty vector was transfected into HEK293 cells as a control. Plasmids pVP30, pVP30(Δ1-40) (pΔ1-40), and pVP30(R40A) (pR40A) with a C-terminal 6×His tag were used; pCMVLacZ and siGLO were cotransfected in each transfection mixture as controls for pDNA efficiency and for siRNA delivery and stability, respectively. Total cell lysates were subjected to SDS-PAGE followed by Western blotting with antibodies to GFP, β-actin, and β-Gal. A representative experiment of three is shown. Each sample was run in duplicate. (B) Point mutations at the C terminus of VP30 do not abrogate reversion of GFP silencing. Mutations were based on the molecular docking of the VP30 C terminus and the human Dicer RNase IIIb domain. Each alanine change is represented as a vertical line. pGFP was transfected into HEK293 cells alone, with the nontargeting siRNA, or with siGFP; plasmids encoding the wild-type protein VP30 (pVP30-H) or the C-terminal mutants were cotransfected with pGFP and siGFP. pVR1012 empty vector was transfected into HEK293 cells as a control. Plasmids pVP30, pC1, pC2, and pC3 with a C-terminal 6×His tag were used; pCMVLacZ and siGLO were cotransfected in each transfection mixture as controls for pDNA efficiency and for siRNA delivery and stability, respectively. Cell extracts were subjected to SDS-PAGE followed by Western blotting with antibodies to GFP, β-actin, and β-Gal. A representative experiment of three is shown. Each sample was run in duplicate. (C) Model for EBOV VP30 interaction with the RNAi pathway. The guide strand (red) and the passenger strand (blue) are shown. Dicer (green oval) senses the duplex siRNA by length and binds to it. TRBP (yellow oval) recruits the Dicer-bound siRNA to be loaded onto the Ago2-containing complex (gray oval). The RISC unwinds duplex siRNA and cleaves the passenger strand (blue dashed line). The guide strand (red) is loaded onto the active RISC, which contains PACT (blue oval). Ago2 cleaves the targeted mRNA (Ago2-slicing activity) (scissors). (Inset) Binding to Dicer or TRBP, VP30 limits contacts between Dicer and PACT or between TRBP and PACT.