Fig. 1: A protein proximity screen for Ebola virus proteins identifies an expanded viral interactome.

a Left: Schematic of structural protein arrangement in the virion. Right: EBOV genome denoting virally encoded genes and screening constructs showing BioID2 ligase (green blocks) at the N or C-termini of each protein. b Expression of viral protein-BioID2 fusion constructs and GFP-BioID2 non-specific control in stable cell lines. Blots were probed with biotin ligase-specific antibody, and loading levels were controlled by detection of β-actin. Blots were performed twice with similar results. c Tagged proteins were evaluated for trVLP activity by replacing constructs encoding WT proteins with the tagged protein at the first step to generate trVLPs, and the resulting NanoLuc activity was measured. NanoLuc activity was compared to an RdRp L active site mutant using one-way ANOVA with multiple comparisons correction. Data are presented as mean values from three biological replicates +/- standard deviation (SD). * p = 0.01, *** p < 0.0001. Statistical significance relative to the inactive L mutant sample was determined using ordinary one-way ANOVA with Dunnett’s multiple comparison correction. d N- and C-terminal BioID2 tagged constructs were transfected into HEK293T cells, challenged with EBOV at an MOI of 1, and stained for BioID2 protein (green) and EBOV VP30 as a marker of infection and inclusion bodies (red) at 20 hpi with cell nuclei stained with Hoechst 33342 (blue). Overlap is indicated by yellow. Individual channels for each image are shown in Supplementary Fig. 2a. One technical replicate of one representative biological replicate is shown. Scale bar = 25 μm. e BioID2-tagged VP40 incorporation into filamentous viral particles was evaluated by transfecting cells with each construct, which were then infected and stained as described above. Insets show magnification with BioID2 staining apparent at the plasma membrane, with budding filamentous and torus-shaped structures indicative of virus particles. Scale bar = 10 μm. One technical replicate of one representative biological replicate is shown. f Biotinylation of cellular proteins was measured in cell lysates after inducing expression of each indicated protein by tetracycline and adding biotin to cell medium. GFP-BioID2 and Flp-In TREx 293 cells not expressing any BioID2 construct were used as non-specific biotinylation controls (last two lanes). Blots were probed with fluorescently labeled streptavidin. One representative experimental replicate of each construct is shown. Biotinylation of all four replicates is shown in Supplementary Fig. 2b. Source data are provided as a Source Data File.