FIG 3.

Expression and fusion mediated by mutant gB constructs in a gB3A background. (A) Cell surface expression and fusion function. Mutations identified in the revertant viruses were cloned into a gB3A expression construct. One set of CHO-K1 cells (effector cells) was transfected with plasmids encoding T7 polymerase, gD, gH, gL, and either a version of gB or an empty vector. Another set of CHO-K1 cells (target cells) was transfected with plasmids carrying the luciferase gene under the control of a T7 promoter and either the nectin-1 or HVEM receptor. The expression of gB on the surface of effector cells was detected by a CELISA using anti-gB PAb R74. To assess fusion function, effector cells were cocultured with target cells for 18 h, and luciferase expression was assayed. Fusion with target cells expressing nectin-1 or HVEM is shown. Results are expressed as a percentage of the expression of fusion mediated by WT gB, after subtracting background values measured in the absence of gB (vector control). The means and standard deviations from at least three independent experiments are shown. Asterisks indicate that the difference in fusion levels between the mutants and WT gB was statistically significant (P < 0.05 using a t test). Differences in cell surface expression were not statistically significant. (B) Western blots. CHO-K1 cells were transfected with plasmids encoding gB constructs or an empty vector. Cell lysates were subjected to SDS-PAGE followed by Western blotting using anti-gB PAb R74. The location of the 100-kDa molecular weight marker is shown.