Properties of Replication-Competent Vesicular Stomatitis Virus Vectors Expressing Glycoproteins of Filoviruses and Arenaviruses

Characterization of rescued rVSVs. Rescued rVSVs were used to infect VeroE6 cells at an MOI of 0.1 PFU/cell. (A) Cytopathogenic effect of infected VeroE6 cells is shown by phase-contrast microscopy 24 h postinfection with VSVΔG/MARVGP (lower panel) in comparison with a mock-infected culture (upper panels). (B) Particle morphology. Electron micrographs show wild-type VSV (upper panel) and VSVΔG/MARVGP (lower panel). (C) Immunofluorescence staining of VeroE6 cells infected with VSVΔG/MARVGP with the GP-specific monoclonal antibody 5EII (dilution, 1:1,000) (lower panel). The upper panel shows the same cells in bright-field microscopy. (D) Growth curves. VeroE6 cells were infected with wild-type VSV (VSVwt), VSV/ZEBOVsGP, VSV/MARVGP₁, VSVΔG/ZEBOVGP, VSVΔG/LASVGP, or VSVΔG/MARVGP at an MOI of 10. Supernatants were collected at the indicated times and titrated by defining the TCID₅₀.

Biosynthesis of the foreign glycoproteins expressed after infection with rVSVs. VeroE6 cells were infected with rVSVs at an MOI of 10. (A) For cells infected with VSVΔG/MARVGP, proteins were pulse labeled at 24 h postinfection for 30 min with 20 μCi of [³⁵S]cysteine per ml and chased for 240 min. GP-specific proteins were immunoprecipitated from cell lysates with mouse anti-Marburg virus GP immunoglobulin (II9G4) (dilution, 1:800) and analyzed on SDS-10% PAGE under reducing conditions. The presence of decRVKR (25 μM) during labeling and chase abolished cleavage of pre-GP (lane 2). (B) For cells infected with VSVΔG/EBOVGP, cells were lysed 24 h postinfection and analyzed by Western blotting with a GP₁-specific antibody at a dilution of 1:4,000 (lane 1) and GP₂-specific rabbit antiserum at a dilution of 1:2,000 (lane 2). (C) For cells infected with VSVΔG/LASVGPC, cells were lysed 24 h postinfection and analyzed by Western blotting with a GP2-specific antiserum (dilution, 1:2,000). (D) For cells infected with wild-type VSV (VSVwt) (lane 1), VSV/ZEBOVsGP (lane 2), and VSV/MARVGP₁ (lane 3), supernatants were analyzed 12 h postinfection by Western blotting with a VSV G-specific antibody (dilution, 1:1,000), a Zaire Ebola virus GP-specific antibody (12/1.1; dilution, 1:4,000), and a Marburg virus GP₁-specific antibody (5EII; dilution, 1:4,000).