*2.5. Chemical Compounds*

100mM uridine or cytidine (both Sigma-Aldrich) stock solutions were prepared in dimethyl sulfoxide (DMSO) and further diluted in cell culture medium. Diluted pyrimidines or DMSO corresponding to 1% of the supernatant volume in 12-well plates was added to the cells at the time of transfection and after medium changes. All concentrations indicated in the figures are final concentrations.

### *2.6. siRNA Knockdown with EBOV Minigenomes and Pyrimidine Complementation*

For siRNA knockdown of endogenous CAD, 293T cells were reverse transfected (i.e., transfected in suspension and subsequently seeded into plates) with 12 pmol pre-designed silencer select siRNAs (CAD-siRNA#1: s2320 [5-GAG GGU CUC UUC UUA AGU A-3]; CAD-siRNA#2: 117891 [5-GCU AGC UGA GAA AAA CUU U-3]; Negative Control siRNA #2; all Thermo Fisher Scientific) or a self-designed EBOV-anti-L siRNA [5-UUU AUA UAC AGC UUC GUA CUU-3] ordered from Eurofins Genomics (Ebersberg, Germany). Transfection was performed in 12-well plates using Lipofectamine RNAiMax (Thermo Fisher Scientific) following the manufacturer's instructions. 48 h post-siRNA transfection, the cells were transfected using Transit-LT1 (Mirus Bio LLC, Madison, USA) with all minigenome assay components, i.e., pCAGGS-based expression plasmids for NP (62.5 ng), VP35 (62.5 ng), VP30 (37.5 ng), L (500 ng), codon-optimized T7-polymerase (125 ng), firefly luciferase (as a control, 125 ng), and the T7-driven monocistronic minigenome (pT7-EBOV-1cis-vRNA-nLuc; 125 ng). For analyses of vRNA and mRNA levels the control firefly luciferase was replaced with GFP (200 ng), and for the replication-deficient minigenome assay a Pol-II-driven replication-deficient minigenome (pCAGGS-EBOV-1cis-vRNA-nLuc-RdM) was used. Transfections were performed using Transit LT1 as previously described [32]. All samples were harvested 48 h post-transfection for either determination of reporter activity or RNA isolation (see below). For measuring the luciferase activity, cells were lysed for 10 min in 1x Lysis Juice (PJK, Kleinblittersdorf, Germany) at room temperature and lysates were cleared of cell debris by centrifugation for 3 min at 10,000× g. Then, 40 μL of the cleared lysates were added to either 40 μL of Beetle Juice (PJK) or NanoGlo Luciferase Assay Reagent (Promega, Madison, USA) in opaque 96-well plates and luminescence was measured using a Glomax Multi (Promega) microplate reader. NanoLuc luciferase activities were normalized to firefly luciferase activities.

### *2.7. RNA Isolation and RT-qPCR*

RNA isolation from minigenome cell lysates was performed following the manufacturer's instructions using a NucleoSpin RNA kit (Machery-Nagel, Düren, Germany). After RNA purification, all samples were treated with DNase (TURBO DNA-free kit; Thermo Fisher Scientific) following the

manufacturer's instructions to avoid plasmid contamination. For cDNA generation, RNA samples were incubated with an oligo(dT)-primer for mRNA quantification or with a strand-specific primer (5-AGT GTG AGC TTC TAA AGC AAC C-3) for vRNA quantification using the RevertAid Reverse Transcriptase (Thermo Fisher Scientific) following the manufacturer's instructions. The subsequent qPCR was performed using a PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) with 1 μL of cDNA and primers targeting either the reporter gene (5-TTC AGA ATC TCG GG GTG TCC-3 , 5-CGT AAC CCC GTC GAT TAC CA-3), or GFP as a control (5-CTT GTA CAG CTC GTC CAT GC-3 , 5-CGA CAA CCA CTA CCT GAG CAC-3). Values for vRNA and mRNA levels were normalized to control GFP mRNA levels.

### *2.8. Immunofluorescence Analysis*

Huh7 cells, which are more suitable for IFA than 293T cells, were seeded on coverslips in 12-well plates and transfected 24 h later with 500 ng pCAGGS-EBOV-NP and 500 ng pCAGGS-FLAG-HA-CAD (or CAD mutants) and, in selected experiments, additionally with 500 ng pCAGGS-myc-VP35 as indicated. For a mock control, cells were transfected with pCAGGS. Transfection was performed using polyethylenimine (Sigma-Aldrich) following the manufacturer's instructions. 48 h post-transfection, cells were fixed using 4% paraformaldehyde (Roth, Karlsruhe, Germany) in DMEM for 20 min and then treated with 1 M glycine (in phosphate-bu ffered saline++ (PBS with 0.9M Ca2+ and 0.5M Mg<sup>2</sup>+)) for 10 min. Then, cells were permeabilized with 0.1% Triton X-100 in PBS for another 10 min and incubated with 10% fetal calf serum (FCS) in PBS for 45 min. Primary antibodies (rabbit anti-EBOV-NP 1:500; mouse anti-FLAG 1:2500; chicken anti-myc 1:1200) were diluted in PBS with 10% FCS and cells were incubated for 1 h at room temperature with the prepared antibody solutions. Secondary antibodies (Alexa Fluor 488 anti-mouse 1:1200; Alexa Fluor 568 anti-rabbit 1:500; Alexa Fluor 647 anti-chicken 1:1200) were prepared as described for the primary antibodies. After 45 min of staining, cells were washed with PBS and water before mounting with ProLong Diamond Antifade mountant with <sup>4</sup>,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific). Slides were analyzed by confocal laser scanning microscopy using a Leica SP5.

### *2.9. Infection of Transfected Huh7 Cells*

To investigate the localization of CAD during EBOV infection, Huh7 cells were seeded in 8-well chambered slides (ibidi, Martinsried, Germany) and transfected as described above (immunofluorescence analysis) with 500 ng pCAGGS-FLAG/HA-CAD. At 48 h post-transfection, the transfected cells were infected with EBOV at an MOI of 1, and the samples were fixed 16 h post-infection in 10% formalin twice overnight prior to removal from the BSL4 facility and immunofluorescence analysis.

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

CoIPs were performed as previously described [19]. Briefly, 293T cells were seeded in 6-well plates and transfected with expression plasmids encoding FLAG/HA-tagged CAD and EBOV-NP using Transit LT-1 (Mirus Bio LLC) following the manufacturer's instructions. The medium was changed after 24 h and the cells were harvested 48 h post-transfection. For coIP, cells were lysed in 1 mL coIP lysis bu ffer (1% NP-40; 50 mM Tris pH 7.4; 167 mM NaCl in water) with protease inhibitors (cOmplete; Roche, Mannheim, Germany). To investigate a possible RNA dependency of the interaction between CAD and NP, 100 μg/mL RNase A (Machery-Nagel) were added to the samples. Subsequently, the samples were incubated rotating at 15 RPM for 2 h at 4 ◦C. Then, 150 μL of the cleared lysates were taken as an input control (representing a sixth of the complete pre-immune lysate and 20% of the sample used for immunoprecipitation) and subjected to acetone precipitation. The remaining 750 μL of cell lysate were mixed with the prepared bead-antibody solution (Dynabeads Protein G, Thermo Fisher Scientific; 1 μL anti-FLAG M2 antibody per 10 μL beads). Immunoprecipitation was performed for 10 min, as recommended by the manufacturer, at room temperature and rotation at 15 RPM. Then, samples were transferred to new tubes and boiled for 10 min at 99 ◦C. Input and coIP samples were analyzed by SDS-PAGE and Western blotting.

### *2.11. Western Blotting*

For validation of CAD knockdown efficiency and analyses of coIP input and lysates, samples were subjected to SDS-PAGE and Western blotting as previously described [34]. FLAG-tagged CAD was detected using a monoclonal anti-FLAG antibody (1:2000), while NP, wild type CAD, and GAPDH were detected using anti-NP (1:1000), anti-CAD (1:250), and anti-GAPDH (1:1000) antibodies. As secondary antibodies, 680RD-coupled goat-anti-mouse, goat-anti-mouse-Alexa Fluor 680, and 800CW-coupled goat-anti-rabbit antibodies (1:14000) were used. Fluorescent signals were detected and quantified using an Odyssey CLx infrared imaging system (Li-Cor Biosciences). For knockdown quantification, CAD signals were normalized to GAPDH signals.

### *2.12. Statistical Analyses*

One-way ANOVA with a Dunnett's multiple comparisons test was performed using the GraphPad Prism 8.1.0 software.
