FIG. 3.

The chimeric PIV5 F protein harboring the SV41 F-derived domains M1 and M2 mediates cell-cell fusion with the coexpressed SV41 HN protein. (A) Schematic diagram of chimeric F proteins. Dark boxes represent amino acid regions derived from the PIV5 F protein, while light boxes represent those derived from the SV41 F protein. (B) Western blot of chimeric F proteins. Subconfluent HeLa cell monolayers in six-well culture plates were transfected with the recombinant plasmid encoding each F protein and lysed at 24 h posttransfection. The cell lysates were subjected to SDS-PAGE under reducing conditions, followed by Western blot analysis with MAb 1D1 that is specific for the PIV5 F₁ subunit. (C) Detection of cell surface-expressed chimeric F proteins by cell surface biotinylation and comparison of their fusion activity. For detection of the F proteins on the cell surface, subconfluent HeLa cell monolayers in six-well culture plates were transfected with the recombinant plasmid encoding each F protein, biotinylated with thiocleavable sulfo-NHS-SS-biotin at 24 h posttransfection, and the biotinylated proteins in the cell lysates were immunoprecipitated with rabbit antiserum specific for the PIV5 F₂ subunit. The immunoprecipitates were subjected to SDS-PAGE under nonreducing conditions, followed by blotting onto a PVDF membrane. The biotinylated proteins on the membrane were detected by the avidin-biotin-peroxidase complex and visualized by enhanced chemiluminescence (ECL). The F protein bands were then quantified, normalized by the value given by the PIV5 F protein, and expressed as cell surface expression (CSE) levels. The average of results from three independent experiments was determined; error bars indicate standard deviations. For analyzing fusion activity of the F proteins, subconfluent HeLa cell monolayers in six-well culture plates were transfected with the recombinant plasmid encoding each F protein together with recombinant plasmid encoding the PIV5 (W3A) HN protein, SV41 HN protein, or CH5-41. After 24 h, the cells were fixed with 4% paraformaldehyde, and the average fusion index was determined as described in Materials and Methods; error bars indicate standard deviations.