Figure 4.

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.).