FIG. 2.

Three EBOV products suppress siRNA-mediated RNA silencing. (A) Mammalian RNAi-based GFP reporter assay to screen EBOV proteins working as SRSs. pGFP was transfected into HEK293 cells alone, with the nontargeting siRNA, or with siGFP; plasmids encoding EBOV proteins were cotransfected with pGFP and siGFP. pVR1012 empty vector was transfected into HEK293 cells as a control. Plasmids pVP30, pVP35, and pVP40 with a C-terminal FLAG epitope are indicated by -F. 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, NP, FLAG, β-actin, and β-Gal. A representative experiment of six is shown. Each sample was run in triplicate. (B) GFP expression quantified by densitometric analysis (ImageJ program) and normalized to β-Gal expression. Results represent the means ± SD from six independent experiments. Samples were analyzed with multiple-comparison repeated-measures one-way ANOVA with Student-Newman-Keuls t test (GraphPad Prism) to compute P values. Transfected β-Gal was used to normalize GFP expression; β-actin was used as a loading control. (C) siRNA treatment inhibits GFP expression without inducing a type I IFN (IFN-α/β) response. HEK293 cells were transfected with pGFP in the absence of siRNA, with nontargeting siRNA, with siGFP alone, or together with plasmids encoding viral proteins. Levels of IFN-α/β produced from HEK293 cells were measured by ELISA. Poly(I·C) and poly(dA-dT) were transfected with Lipofectamine into HEK293 cells and used as positive controls. One representative measurement of three is shown. (D) Reversion of GFP silencing is dose dependent. HEK293 cells were transiently transfected with pGFP alone or along with the nontargeting siRNA, with the siGFP, or with increasing amounts of each plasmid encoding the viral proteins (gray triangle). Total cell lysates were subjected to SDS-PAGE followed by Western blotting. EBOV VP30, VP35, and VP40 expression was detected with the anti-FLAG antibody, and NP expression was detected with a monoclonal antibody against NP. (E) EBOV RNAi suppressors block viral siRNA-mediated silencing. The plasmid pGP was transfected into HEK293 cells alone, with the nontargeting siRNA, with siGP4 targeting the GP mRNA alone, or with pVP30, pVP35, pVP40, or pNP. pVR1012 empty vector was transfected as a control. GP expression was measured by Western blotting using a GP antibody and normalized to β-Gal expression. β-Actin was used as a loading control. (F) EBOV proteins coexpressed with GFP in the absence of siRNA do not enhance reporter gene expression. pGFP and pLac, a second plasmid carrying a reporter gene used as a transfection control, were cotransfected into HEK293 cells alone or together with plasmids encoding EBOV proteins. pVR1012 empty vector was transfected into HEK293 cells as a control. Total cell lysates were subjected to SDS-PAGE followed by Western blotting with antibodies to GFP, β-actin, and β-Gal. GFP and β-Gal expression was normalized to endogenous β-actin expression (loading control). One experiment of two is shown (sample in triplicate).