Release of soluble EBOV GP_1,2Δ complexes. (A) RK-13 cells were infected with vSCGP8, pulse labeled 10 h p.i. and chased for 6 h. Culture medium (m) and cells (c) were collected separately. Soluble (s) and membrane-bound (p) proteins were separated by ultracentrifugation (25 000 rpm, 1 h, 4°C). Labeled proteins were immunoprecipitated using rabbit anti-GP₂ Igs or human monoclonal antibody KZ52. (B) Western blot analysis of culture medium from EBOV-infected Vero E6, HeLa, and RK-13 cells.

Kinetics of release and oligomeric structure of soluble GP. (A) RK-13 cells infected with vSCGP8 were pulse-chase labeled and proteins were immunoprecipitated from cells (left panel) and medium (right panel). (B) Surface biotinylation of RK-13 cells expressing EBOV GP. Release of biotin-labeled GP from the cell surface into culture supernatant was observed at indicated time intervals. (C) Analysis of the oligomeric structure of shed GP. Vero E6 cells were infected with EBOV. At 5 days p.i., virus from culture medium was pelleted through a 20% sucrose cushion, and subsequently the supernatant and virus were analyzed by ultracentrifugation at linear sucrose gradients in the presence of 1% NP-40 and 0.4% DOC. Fractions 1–15 were collected from the bottom and analyzed by Western blot. Due to the effect of several detergents present in the samples such as NP-40, DOC, and finally SDS, the bands appeared slightly more diffused compared to other analyses.

Involvement of TACE in EBOV GP shedding. (A) 293 cells were transfected with pcDNA3.1-GP or co-transfected with pcDNA3.1-GP and pcDNA3Mu TACE FL. At 36 h p.t., cells were pulse labeled and chased for 8 h. Labeled proteins were analyzed by immunoprecipitation. (B) 293 cells were infected with vSCGP8 and subsequently transfected with ASOs. Mismatch ASO2M and ASO4M were used as negative controls. At 7 h p.t., cells were labeled and chased for 8 h. Proteins from culture medium were analyzed by immunoprecipitation. (C) EC-2 cells (TACE^ΔZn/ΔZn) lacking functional TACE were transfected with pcDNA3.1-GP or co-transfected with pcDNA3Mu TACE FL and pcDNA3.1-GP. Cells were pulse-chase labeled 18 h p.t., and medium and cells were analyzed by immunoprecipitation. For all three panels (A–C) statistical analysis using at least three independent experiments has been performed. Mean values of GP release (% ±s.d.) are shown in bars. Quantification of the released GP_2Δ was performed using Fuji BAS 1000 Bio-Imaging Analyzer.

Analysis of glycosylation pattern of GP₂ and GP_2Δ. (A) RK-13 cells were infected with vSCGP8 (wtGP) or vaccinia viruses expressing glycosylation mutants (GP618N/T and GP563N/T) and subjected to pulse-chase labeling and immunoprecipitation with horse anti-EBOV Igs followed by endoglycosidase treatment with Endo H or PNGase F. Forms of GP₂ differing in N-glycosylation pattern are indicated as follows: A—containing only complex type oligosaccharides, B—containing only high-mannose type oligosaccharides, C—completely deglycosylated molecules. (B) Culture medium from Vero E6 cells infected with EBOV was ultracentrifuged, and pellet and supernatant were collected separately. Samples were either treated with Endo H or PNGase F or used untreated. Proteins were analyzed by Western blot.

Determination of the carboxy-terminus of GP_2Δ. (A) Schematic illustration of EBOV GP₂. Subtilisin-like cleavage site (RTRR), shedding cleavage site (D₆₃₇–Q₆₃₈), position of carboxy-terminal deletion mutants (ΔTM, Δ630, Δ640), cysteine residues (C), N-glycosylation sites N₅₆₃ and N₆₁₈ (white stars), transmembrane anchor (TM), cytoplasmic tail (CT), and fusion peptide (FD) are indicated. (B) Western blot analysis of GP deletion mutants. Culture medium from HeLa cells infected with vTF7-3 and transfected with pGEMmGP8/ΔTM, pGEMmGP8/Δ630, or pGEMmGP8/Δ640 was treated with PNGase F and subjected to Western blot analysis. GP₂ from virus particles (p) and shed GP_2Δ from EBOV-infected HeLa cells (s) were used as controls. (C) SDS–PAGE analysis of purified soluble GP under nonreducing conditions. The left panel shows silver staining and the right panel immunodetection with anti-GP₂ Igs. (D) Analysis of purified GP preparation by MALDI-TOF MS. GP_2Δ and PNGase F peaks are indicated. Upper box: GP_2Δ mass after subtraction of carboxyamidoyl group mass (CAM); lower box: theoretical masses of GP_2Δ with indicated carboxy-terminal amino acid. (E) Influence of amino-acid exchanges on GP shedding. RK-13 cells infected with vSCGP8 (wtGP) or virus mutants with indicated exchanges were labeled and analyzed by immunoprecipitation. Quantification of released GP_2Δ was performed using Bio-imaging. Representative results from individual experiments are presented as mean values (±s.d.).

GP_1,2Δ is present in the blood of infected guinea pigs and is able to inhibit the virus-neutralizing activity of human monoclonal antibody KZ52. (A) Virus neutralization inhibition assays were performed using different EBOV dilutions in the presence of KZ52 antibody or shed GP or both at indicated concentrations. Total number of infectious units obtained for each reaction is indicated in bars. (B) Vero E6 cells were infected with EBOV and culture medium collected 5 days p.i. (EBOV-m). Guinea pigs (strain Hartley) were infected intraperitoneally with 1 × 10³ PFU/animal of guinea pig-adapted EBOV (Volchkov et al, 2000). At days 6 and 9 p.i. (EBOV-6d, EBOV-9d), GP₂, GP_2Δ, and VP40 were identified in sera (sr) and in culture medium (m) by Western blot analysis using specific antibodies. Samples from blood (at day 9 p.i.) and culture medium were either treated with Endo H or PNGase F or used untreated.