siRNA knockdown in the EBOV minigenome assay.

For siRNA knockdown, 293T cells were reverse transfected with 12 pmol anti-L siRNA (5′-UUU AUA UAC AGC UUC GUA CUU-3′) or predesigned silencer select siRNAs (NXF1: s20532, Negative Control siRNA#2; Thermo Fisher Scientific) using Lipofectamine RNAiMax (Thermo Fisher Scientific) according to the instructions of the manufacturer in 12-well plates. To assess the impact of NXF1 on viral translation, the cells were transfected with 500 ng of either minigenome-derived mRNA (nluc) or Firefly mRNA (RiboPro) using Lipofectamine MessengerMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions 2 days after the siRNA transfection. As a negative control, no mRNA was added (–mRNA). Further, as a control for the successful knockdown of NXF1, siRNA-treated cells were also transfected with the plasmids encoding the minigenome (pT7-EBOV-1cis-vRNA-hrluc), the T7 polymerase, and the RNPs (NP, L, VP35, and VP30), as well as a firefly luciferase transfection control. For the complementation of siRNA-mediated knockdown of NXF1, the NXF1-depleted cells were transfected with all components required for the replication-deficient minigenome as previously described (49), together with different NXF1 constructs harboring mutations in the siRNA binding site. The plasmid amounts for the NXF1 mutants used in this assay were adjusted to ensure equal expression levels (corresponding to 250 ng of pCAGGS-flag/HA-NXF1-ΔsiRNA), as judged using the same workflow and Western blot analysis 2 days posttransfection with an anti-flag antibody. Two days after the second transfection, cells were lysed for 10 min in 1× Lysis Juice (PJK), and the lysates were cleared by centrifugation (3 min, 10,000 × g, room temperature). Then, 40 μL of the cleared lysate was added to 40 μL of either Renilla Glo Juice or Beetle Juice (both PJK) or NanoGlo luciferase assay reagent (Promega) in opaque 96-well plates. For measurement of luminescence, a GloMax Multi (Promega) multiplate reader was used. Minigenome reporter activity or viral mRNA reporter activity was normalized to firefly luciferase activities.