Structure-Based Mutations in the Herpes Simplex Virus 1 Glycoprotein B Ectodomain Arm Impart a Slow-Entry Phenotype

Fan, Qing; Kopp, Sarah J.; Connolly, Sarah A.; Longnecker, Richard

Excess gH/gL does not restore the fusion activity of the gB 3A mutant protein. We previously demonstrated that the gB 3A mutant protein exhibited reduced fusion in a cell-cell fusion assay using the HSV receptors nectin-1 and HVEM (20) . A quantitative cell-cell fusion assay was used to confirm this reduced fusion capacity and to test gB 3A fusion by using the PILR␣ receptor, an entry receptor that binds to gB. Fusion was reduced with all of the HSV receptors tested (Fig. 9A) . If the gB 3A mutations favor the promoter along with a plasmid encoding the PILR␣, HVEM, or nectin-1 receptor. Transfected cells were cocultured with effector CHO cells that had been transfected with plasmids encoding T7 polymerase, gD, gH, gL, and either WT gB or gB 3A. After coincubation overnight, luciferase activity was measured as an indication of cell-cell fusion. Background signals (effector cells transfected with the vector only) were subtracted, and the data were normalized to the fusion activity observed with WT gB. Data were normalized separately for each receptor. Each bar shows the mean and standard deviation of three independent experiments. (B, C) Impact of gH/gL levels on fusion mediated by WT gB (B) or gB 3A (C). Target CHO cells were transfected with a reporter plasmid encoding luciferase under the control of the T7 promoter and a plasmid encoding either HVEM or nectin-1. Effector CHO cells were transfected separately with plasmids encoding T7 polymerase, gD, gH, gL, and gB (B) or gB 3A (C). Increasing amounts of gH and gL plasmid DNA were added to the effector cell transfections. Target cells were cocultured with effector cells overnight, and luciferase activity was measured as an indication of cell-cell fusion. Background signals (effector cells transfected with the vector only) were subtracted, and data were normalized to the level of fusion achieved by WT gB in cells transfected with 0.8 g each of the gH and gL plasmids (standard gH/gL levels). Fusion with each receptor was normalized independently. V denotes empty-vector DNA transfected into the effector cells. Each bar shows the mean and standard deviation of three independent determinations. prefusion conformation of gB as we hypothesize, reduced fusion with all HSV receptors is expected.