*2.6. Colocalization Studies*

For colocalization studies of CedV F and mutants with early endosomes, antibody uptake assays were performed as described above with modifications. Briefly, at 24 h p.t., CedV F-expressing HeLa cells were incubated with the polyclonal rabbit anti-CedV F serum (1:200) for 1 h at 4 ◦C. After washing, cells were shifted to 37 ◦C for 5 or 30 min to allow endocytosis to occur. Then, surface-bound primary antibodies were blocked with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:50; LifeTechnologies, Darmstadt, Germany). After fixation with 2% PFA for 20 min and permeabilization with 0.2% Triton X-100 in PBS, a mouse anti-human early endosomal antigen 1 (EEA-1) antibody (1:50; BD Biosciences, Heidelberg, Germany) was added for 1 h at 4 ◦C for staining of early endosomes. Internalized primary rabbit antibodies were stained with AF 488-conjugated secondary antibodies (1:500; LifeTechnologies, Darmstadt, Germany). Primary mouse antibodies bound to EEA-1 were detected with AF 568-conjugated secondary antibodies (1:500; LifeTechnologies, Darmstadt, Germany). Cell nuclei were counterstained with <sup>4</sup>,6-Diamidin-2-phenylindol (DAPI). Representative images were recorded with a confocal laser scanning microscope (Leica SP5) and processed with the ImageJ software version 1.45 s [47].

### *2.7. Surface Biotinylation and Western Blot Analysis*

MDCK-2 cells were transfected with plasmid DNA encoding either CedV F or mutant CedV F protein genes. At 24 h p.t., cells were washed and incubated twice with 2 mg/mL EZ-Link® Sulfo-NHS-LC-Biotin (ThermoFisher Scientific, Waltham, MA, USA) for 20 min at 4 ◦C. Following cell lysis, F proteins were immunoprecipitated overnight using NeutrAvidin Agarose Resin (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Proteins were separated on a 10% SDS-gel under reducing or non-reducing conditions and then transferred onto nitrocellulose. Biotinylated CedV F proteins were detected by incubation with the HA-tag specific rabbit antibody H6908 (dilution 1:2,000 in PBS-Tween (0.05%)) followed by labeling with anti-rabbit HRP-conjugated secondary antibodies (1:5.000). Under non-reducing conditions, HRP-labeled Concanavalin A (ConA; Sigma-Aldrich, Darmstadt, Germany, 1:1,000 in PBS containing 0.05% (v/v) TWEEN 20, 1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2 for 16 h at 20 ◦C) was used to stain NeutrAvidin-precipitated surface glycoproteins, which served as a loading control and as a reference to compare CedV F0 protein intensities. Proteins were visualized using a chemiluminescent substrate (Clarity Western ECL substrate, Bio-Rad, Feldkirchen, Deutschland) and the Bio-Rad Molecular Imager Chemi DocTM XRS+ in combination with the Image Lab software (Bio-Rad, Feldkirchen, Deutschland, Version 6.0.1).

### *2.8. Quantification of Endocytosis Rate by MESNA Reduction*

MDCK-2 cells transiently expressing either CedV F or mutant F proteins were surface labeled with cleavable EZ-Link sulfo-NHS-SS-biotin (ThermoFisher Scientific, Waltham, MA, USA) at 4 ◦C. Then, wells were flooded with pre-warmed DMEM (37 ◦C) and shifted to 37 ◦C for either 5, 15, 30, or 90 min to allow endocytosis to occur. After rapid cooling to 4 ◦C, biotin still bound to the cell surface was cleaved by a membrane-impermeable reducing agent, 2-mercaptoethane-sulfonic acid sodium salt (MESNA; 50 mM in MESNA buffer (50 mM Tris, pH 8.5, 100 mM NaCl, 2.5 mM CaCl2)) a total of three times for 20 min. One sample was kept on ice and was neither incubated at 37 ◦C nor cleaved with MESNA and thus served as the surface biotinylation control to determine the total amount of biotinylated protein (control). After cell lysis in RIPA buffer, CedV F and CedV mutant F proteins were immunoprecipitated using the H6908 antibody as described above and separated by SDS-PAGE under non-reducing conditions. Transfer to nitrocellulose was followed by the detection of biotinylated proteins with HRP- labeled streptavidin and a chemiluminescent substrate. Protein bands were quantified using ImageLab software (version 6.0.1). Mean internalization rates per min were calculated as the ratio of band intensities from internalized, intracellular biotinylated protein after 5, 15, 30, and 90 min and total surface-labeled F protein (control, 50%) divided by 5, 15, 30, and 90 min, respectively.

### *2.9. Fusion Assay*

A total of 4 × 10<sup>5</sup> Vero76 cells or 3 × 10<sup>5</sup> MDCK-2 cells were co-transfected with expression plasmids encoding for CedV G (0.5 μg/well) and either F or mutant F proteins (0.5 μg/well). At 48 h p.t., cells were fixed with ethanol and stained with 1:10-diluted Giemsa staining solution. Representative images (20× magnification) were recorded with an inverted microscope.

### *2.10. Luciferase Reporter Gene-Based Fusion Assay*

Vero76 cells were reverse transfected with expression plasmids encoding for CedV G and either F or mutant F proteins as described above and seeded in 24-well plates. In addition, pCITE Renilla (200 ng/well), a vector containing the renilla luciferase gene under the control of the T7 promoter, was co-transfected. In parallel, Vero76 cells were reverse transfected with the pCAGGS T7 polymerase vector. At 27 h p.t., Vero76 cells expressing the T7 polymerase were washed twice with EDTA-containing PBS (5 mM), detached and added to the cells pre-transfected with pCAGGS CedV F and G and pCITE Renilla to allow fusion to proceed. After 3 h, cells were lysed using Lysis Buffer (pjk, GmbH, Kleinblittersdorf, Germany) and luciferase activity was measured by adding Renilla Glow substrate (pjk GmbH, Kleinblittersdorf, Germany) according to the manufacturer's instructions. Samples were tested in duplicate in three independent experiments. Reporter activity detected upon co-transfection of the parental CedV F with CedV G protein was set to one, which served as a reference point for fusion activity. Reporter activities measured for the fusion of mutant CedV F proteins were used to calculate their fusion activity in relation to the parental protein. Background activity of the luciferase reporter was assessed with cells transfected with pCAGGS CedV G and pCITE Renilla only overlaid with T7 polymerase expressing cells.
