*2.1. Cells*

HEK 293T (human embryonic kidney) cells (Collection of Cell Lines in Veterinary Medicine CCLV-RIE 1018) and Huh7 (human hepatocarcinoma) cells (kindly provided by Stephan Becker, Philipps University Marburg, Marburg, Germany) were maintained in Dulbecco's modified Eagle's medium (DMEM; ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (PS; ThermoFisher Scientific) and 1× GlutaMAX (ThermoFisher Scientific). All cells were grown at 37 ◦C with 5% CO2.

### *2.2. Plasmids and Antibodies*

Expression plasmids for the RNP proteins, the T7 polymerase and the T7-driven monocistronic minigenomes (pT7-1cis-vRNA-rLuc, pT7-1cis-vRNA-nLuc) have been previously described [19,41]. Plasmids for the expression of N-terminally flag/HA-tagged constructs were generated by insertion of the respective genes (NXF1, p15, NP) into a pCAGGS-flag/HA vector. All mutant versions and individual domains of NXF1 were first inserted into pCAGGS and subsequently subcloned into the pCAGGS-flag/HA plasmid. EBOV VP40 and VP24 were also cloned into pCAGGS. The PolII-driven replication-deficient minigenome expressing NanoLuc luciferase as its reporter was generated by flanking the minigenome in vRNA-orientation with hammerhead and Hepatitis Delta Virus ribozymes and insertion of the minigenome into pCAGGS. Deletion of 55 nt in the antigenomic replication promotor to generate a replication-deficient version of the PolII-driven minigenome was performed as previously described [41]. Detailed cloning strategies are available on request.

The anti-flag (mouse anti-flag mAb, clone M2, cat. no. F1804) antibody used for co-immunoprecipitation assays (coIPs) and Western blot analyses was purchased from Sigma-Aldrich. Primary antibodies against NP (rabbit anti-EBOV NP pAb, cat. no. 0301-012), VP35 (rabbit anti-ZEBOV VP35 a ffinity purified polyclonal antibody, cat. no. 0301-040) and VP40 (mouse anti-EBOV VP40 mAb, clone 3G5, cat. no. 0201-016) were obtained from IBT Bioservices, and the antibody against NXF1 (mouse anti-NXF1 mAb, clone 53H8, cat. no. ab50609) was obtained from Abcam. For VP30, a mouse monoclonal antibody (clone 5F11 A1) was produced by the FLI biobank as previously described [42] and following approved protocols using a ffinity-purified flag/HA-tagged VP30 expressed in mammalian cells as the immunogen. Secondary antibodies against mouse (Peroxidase A ffiniPure Goat Anti-Mouse IgG, light chain specific, cat. no. 115-035-174, used for Western blots with exception of that shown in Figure 4) and rabbit (Peroxidase A ffiniPure Goat Anti-Rabbit IgG H+L, cat. no. 111-035-003) were obtained from Jackson Immunoresearch or Abcam (anti-mouse IgG for IP, HRP, cat. no. ab131368, used for the Western blot shown in Figure 4).

### *2.3. Co-Immunoprecipitation of Viral Proteins*

For coIPs, 293T cells were seeded into 6-well plates and transfected at 30% confluency with expression plasmids encoding the flag/HA-tagged host proteins (NXF1, p15, mutated versions or individual domains of NXF1) and for their putative interaction partners (p15, NXF1 or EBOV proteins) using Transit LT-1 (Mirus Bio LLC, Madison, WI, USA) following the manufacturer's instructions. Supernatant was exchanged after 24 h and coIP was performed 48 h post transfection (p.t.). To this end, cells were washed in PBS and pelleted before being lysed in 1 mL coIP lysis buffer (1% NP-40; 50 mM Tris; 150 mM NaCl; pH 7.4) with protease inhibitor (cOmplete; Roche, Basel, Switzerland) and incubated with rotation at 15 rpm for 2 h at 4 ◦C. Initial experiments investigating an interaction between viral proteins and NXF1 (Figure 1A) where performed in the absence of RNase treatment. However, since RNase treatment increases the interaction between NXF1 and NP (see Figure 2), in all further experiments 100 μg/mL RNase A (Machery-Nagel, Düren, Germany) was added to the samples prior to incubation. Lysates were separated from cell debris by centrifugation (11,000× *g*, 10 min, 4 ◦C) and 150 μL was used for the input control (representing a sixth of the whole lysate and 20% of the sample subjected to IP) and subjected to acetone precipitation. To this end 750 μL acetone pre-chilled to −20 ◦C was added to the samples, and they were incubated for at least 30 min at −20 ◦C. Proteins were then pelleted by centrifugation (20,000× *g*, 15 min, 4 ◦C), resuspended in 60 μL 1× SDS sample buffer and boiled for 10 min at 99 ◦C. The remaining 750 μL of lysate was added to the prepared bead-antibody solution (Dynabeads Protein G, ThermoFisher Scientific; 1 μL anti-flag M2 antibody per 10 μL beads), resuspended, and the IP was performed for 10 min (as recommended by the manufacturer to minimize non-specific binding) at room temperature with rotation at 15 rpm. Beads were washed three times with PBS containing 0.02% Tween-20 and transferred to new tubes. The beads were resuspended in 60 μL 1× SDS sample buffer and boiled for 10 min at 99 ◦C. Input and coIP samples were analyzed via SDS-PAGE and semi-dry Western blot, using an ethanol-containing blotting buffer (5.8 g Tris(hydroxymethyl)aminomethan; 2.9 g Glycin; 200 mL Ethanol; ad 1 mL H2O) together with Nitrocellulose membranes and a blotting current of 0.8 mA/cm<sup>2</sup> for 1 to 2 h, and 7% skim milk powder in PBS for blocking.
