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

Cell-cell fusion assay. The cell-cell fusion assay was performed as previously described (49) . Briefly, CHO-K1 cells were seeded into six-well plates and incubated overnight. One set of cells (effectors) was transfected with plasmids encoding T7 RNA polymerase, gD, gH, and gL, as well as WT gB, mutant gB, or empty vector, using 5 l of Lipofectamine 2000 (Invitrogen) and 400 ng of each plasmid. In some experiments, the total amount of gH and gL plasmid DNA transfected was titrated from 200 ng/well to 2 g/well. A second set of cells (targets) was transfected with 400 ng of a plasmid carrying the firefly luciferase-encoding gene under the control of the T7 promoter and 1.5 g of the empty vector or a plasmid encoding PILR␣, HVEM, or nectin-1 with 5 l of Lipofectamine 2000. After 6 h of transfection, the cells were detached with Versene and resuspended in 1.5 ml of F12 medium supplemented with 10%