FIG. 2.

An F-Edm-452C/460C double mutant is proteolytically activated and intracellular transport competent but lacks fusion activity. (A) Immunoprecipitation (IP) of F-Edm, F-Edm-452C/460C, and F-Edm-307C/448C from cleared Vero cell lysates obtained 36 h posttransfection. An antiserum directed against residues 127 to 193 in the F HR-A domain was employed. Precipitated material was subsequently subjected to immunostaining using antibodies directed against the cytosolic F tail as previously described (19). Both the F₀ precursor and the proteolytically matured F₁ fraction of F-Edm and F-Edm-452C/460C are present, while for F-Edm-307C/448C, only the uncleaved F₀ precursor is visible. Samples were fractionated under reducing conditions, mock-transfected cells did not express any MV F antigenic material, and immunoblots were decorated with rabbit im- munoglobulin G light-chain-specific horseradish peroxidase conjugate. (B) The F-Edm-452C/460C mutant but not F-Edm-307C/448C is expressed at the cell surface. Surface biotinylation to assess plasma membrane steady-state levels of MV F was carried out as described previously (16). Values above the blot are based on densitometric quantification of signal intensities using a VersaDoc imaging station. Averages of the results of two independent experiments and standard error of the mean values are presented. (C) Quantitative cell-to-cell fusion assays reveal that both F-Edm variants lack fusion activity. Effector Vero cells were infected with MVA-T7 and cotransfected with 3 μg plasmid DNA, each encoding H-Edm or the F construct specified. Target cells harbored a luciferase reporter construct under the control of the T7 promoter, and luciferase activities as an indicator of cell-to-cell fusion activity were assessed 200 min postmixing of both populations. Mock effector cells received only MVA-T7 and H-Edm encoding plasmid. Bar graphs and numbers show averages and standard deviation values of the results of three independent experiments.