TTP negatively impacts EBOV minigenome activity. A, 293T cells were seeded in 12-well plates at a density of 1.5 × 105 cells per well and transfected 1 day later with EBOV minigenome plasmid 3E5E-Fluc, pCAGGS plasmids encoding EBOV NP with a full-length 3′ UTR, VP35, VP30, and L along with pCAGGS expressing T7 RNA polymerase and pMIR β-gal. pCDNA3.1-1xFlag-TTP or pCDNA3.1-1xFlag-TTP(C124R) were cotransfected in increasing quantities, as indicated. Three days posttransfection, cells were lysed and luciferase and β-gal activity was determined. Luciferase values were normalized to β-gal activity, and the normalized luciferase values for the positive control (0 = + L, no TTP plasmids) were set to one. As a negative control, the L plasmid was replaced by a plasmid encoding a fluorescent protein (− L). Data are from 3 independent experiments (each performed in triplicate). Statistical analysis was performed using GraphPad prism software, using a 1-sample t test. B, Cell lysates prepared in (A) were analyzed by western blot using antibodies against EBOV NP, FLAG, and tubulin. The shown blot is representative of 3 independent experiments (each performed in triplicate). C, Quantification of NP bands in the presence and absence of TTP or TTP(C124R) generated in (B). Protein bands were quantified using Li-COR Image Studio western blot analysis software. Shown is the NP to tubulin ratio. NP amount of the positive control (0 = + L, no TTP plasmids) was set to 1. Statistical analysis was performed using GraphPad prism software, using a 1-sample t test. * P value ≤ .05, ** P value ≤ .01, *** P value ≤ .001, **** P value ≤ .0001. Error bars depict the standard error of the mean. Abbreviations: β-gal, β-galactosidase; EBOV, Ebola virus; NP, nucleoprotein; TTP, tristetraprolin; UTR, untranslated region.
