Fig 3. TRIM25 antiviral effect against EBOV trVLPs is independent of RIG-I and downstream pro-inflammatory signal transduction.
(A, B, H) U87-MG- and/or HEK293T-based CRISPR cells lines were treated with increasing amounts of IFN-I, and lysed 24 hours later for protein analysis. Protein levels of HSP90 (A, B and H), RIG-I (A), FL-MAVS/mini-MAVS (B) and TBK1 (H) were determined by western blot on LacZ CRISPR control cells and corresponding CRISPR knock-out (KO) cells lines. (C, I) HEK293T-based CRISPR cell lines depicted in the figures were transfected with plasmids expressing EBOV RNP proteins and TIM1 together with either GFP (grey bars) or TRIM25 (blue bars), and later infected with a fixed amount of EBOV trVLPs. 24 hours post-infection cells were lysed and trVLP reporter activities measured. Protein levels of HSP90 and TRIM25 were determined by western blot at time of infection. (D) EBOV trVLP reporter activities on U87-MG LacZ CRISPR (grey) and U87-MG RIG-I CRISPR KO (blue) target cells (p1), transfected with TIM1 and EBOV RNP proteins, and pre-treated overnight with increasing amounts of IFN-I prior to infection. Luciferase activities measured 24 hours post-infection. (E) EBOV trVLP reporter activities on U87-MG LacZ CRISPR (grey), U87-MG FL-MAVS KO (blue) and FL-MAVS/miniMAVS CRISPR DKO (red) target cells (p1), transfected with TIM1 and EBOV RNP proteins, and pre-treated overnight with increasing amounts of IFN-I prior to infection. Luciferase activities measured 24 hours post-infection. (F) Protein levels of HSP90 and MAVS determined by western blot lysates from HEK293T LacZ control cells, and HEK293T-MAVS DKO cell lines engineered to stably express CRISPR-resistant variants of both MAVS isoforms (MAVSCR), miniMAVS (M1ACR) or FL-MAVS (M142ACR). (G) HEK293T LacZ CRISPR and engineered HEK293T-MAVS DKO cell lines from (F) were co-transfected with plasmids expressing EBOV RNP components and TIM1 together with either GFP (grey bars) or TRIM25 (blue bars), and later infected with EBOV trVLPs. Reporter activities were measured 24 hours later. (J) U87-MG LacZ CRISPR and U87-MG TBK1 CRISPR KO cells were transfected with RNP proteins and TIM1, followed by a IFN-I pre-treatment prior to infection with a fixed amount of EBOV trVLPs. EBOV trVLP reporter activities in p1 were measured 24 hours after infection. (K) Fold activation of a firefly luciferase NF-kB reporter in the depicted HEK293T-based CRISPR cells lines transiently transfected with TRIM25 compared to control GFP vector. Cells were harvested 48 hours post-transfection and FLuc reporter values normalised to control Renilla luciferase activity in the same lysates. All the represented EBOV trVLP Renilla reporter activities are normalized to control Firefly luciferase values obtained in the same lysates. *p > 0.05, **p > 0.01 and ***p > 0.001 as determined by two-tailed paired t-test. All error bars represent ± SEM of at least three independent experiments.