FIG. 9.

A27-dependent cell fusion was partially inhibited by A26 protein. (A) Live imaging recording of cell fusion from within of virus-infected BSC40 cells by time-lapse immunofluorescence microscopy. BSC40 cells were infected with Wt-VV, IA27L, and IA27L-A26WR at an MOI of 5 PFU per cell and incubated at 37°C in the presence of 5 mM IPTG for 34 h, and cell images showing cell fusion development were collected at 30 min, 10 h, 20 h, 27 h, and 34 h p.i. The bottom color figures show confocal images of the infected cells fixed at 34 h p.i. and stained with anti-VV (1:5,000) Ab (green), fluorescein isothiocyanate-conjugated goat anti-rabbit Igs, and DAPI (blue). (B) Parental L or sog9 cells were infected with purified IMV virions for 1 h at 37°C, treated with PBS (pH 4.7) at 37°C for 2 min, incubated in normal medium for 1 h to develop cell fusion from without, and subsequently fixed. Plasma membrane was stained with PKH26 (red) and nuclei were stained with Hoechst 33258 (blue), and cells were visualized by confocal microscopy. White arrows refer to fused cells. (C) Quantification of cell fusion from without of L and sog9 cells in medium containing 5 mM IPTG as shown in panel B. The percentage of cell fusion was quantified based on equations described in Materials and Methods. (D) The fused cell population of L cells in panel C were subdivided into three subpopulation for quantification as described in Materials and Methods: small fused cells (2 to 5 nuclei per cell), medium fused cells (6 to 10 nuclei per cell), and large fused cells (>10 nuclei per cell). A total of >300 cells were counted for each virus, and the experiments were independently repeated twice.