Cell-cell fusion and mAb binding activities of NiV-F mutants.

Four mutants of NiV-F within the trimer-trimer interface were tested for their ability to promote cell-cell fusion when co-expressed with NiV-G in a β-Gal reporter cell-cell fusion assay using HEK293T cells as the target population. (A) The data shown are the mean percentage of WT fusion levels measured for each mutant calculated from four separate experiments. The data was normalized for cell surface expression of WT F measured by flow cytometry, using a F-specific mAb that recognizes total F surface expression. The bars represent the range from multiple experiments. WT: wild type F. Statistical analysis probes activity deviation relative to WT. *: pB) Expression of NiV-F mutants and NiV-G in HeLa USU cells from (A). Equal amount of remaining cells from fusion were lysed and clarified by centrifugation followed by immunoprecipitation by polyclonal rabbit anti F and G serum and protein G Sepharose. The precipitated products were analyzed on SDS PAGE followed by western blotting. F and G were probed by monoclonal mouse anti F (upper panel) and G (lower panel) specific antibody. (C) Cleared lysates of NiV-F mutants expressed in 293T cells were immunoprecipitated with 3 different competition groups of neutralizing anti F mAb as indicated. All 3 mAbs efficiently immunoprecipitated WT F. The precipitated complexes were analyzed on SDS-PAGE followed by western blotting and F was probed using polyclonal rabbit anti-F antisera.