Nontargeting control

Ambion Silencer siRNAs (S2 Table) and nontargeting controls (Thermo Fisher Scientific) were reverse transfected with DharmaFECT 1 Transfection Reagent (Horizon Discovery, Cambridge, United Kingdom) according to manufacturer protocols. Briefly, siRNAs were mixed with DharmaFECT 1 Transfection Reagent (1:100 dilution in HBSS) and 50 μL of siRNA mix were added to each well in a 24 well plate, or 100 μL in a 12 well plate. 1.2 x 10⁵ cells were added per well in 250 μL in a 24 well plate or 2.4 x 10⁶ in 500 μL in a 12 well plate, for a final concentration of 25 nM siRNA. Plates that would be subjected to SINV infection were first poly-L-lysine treated. Cells were induced for TRIM25 expression using a final concentration of 1 μg/mL dox one day post-transfection, as applicable. Cells were harvested for RNA extraction for RT-qPCR to quantify gene knockdown or subjected to SINV infection 48 h post-transfection. To assess ISG induction in TRIM25 inducible cells upon poly(I:C) treatment, cells were treated with 1 μg poly(I:C) HMW (InvivoGen, San Diego, CA) in the presence or absence of 1 μg/mL dox per well, and harvested for RNA extraction for RT-qPCR.

The HEK293T cell line was a gift from Dr. Thomas Klonisch; the MCF-7 and MDA-MB-231 cell lines were a gift from Dr Spencer Gibson and the MEF cell line was a gift from Dr Peter Pelka. Cell culture conditions were as previously published (14 (link)). DNA primers and RNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). LNA GapmeRs were purchased from Exiqon (Woburn, MA, USA). siRNAs and non-targeting controls were purchased from Thermo Fisher Scientific (Ottawa, Canada). The plasmid for overexpression of BC200 under control of the U6 snRNA promoter was synthesized by Genscript Inc. (Piscataway, NJ, USA). The plasmid for BC200 expression from the endogenous promoter was generating by amplifying and cloning a 3788 nt fragment of the genomic sequence of the BC200 gene (–2314 to +1474) into the pJET1.2 vector. All standard laboratory chemicals and reagents were purchased from Thermo Fisher Scientific.