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

Virus cell penetration kinetics. To assay the cell entry of the HSV-1 gB 3A viruses, a cell penetration experiment was performed as previously described (56) . GS3217 (WT) virus or HSV-1 gB 3A viruses were diluted to 150 PFU/ml in PBS and allowed to adsorb to Vero cell monolayers in six-well plates for 1 h on ice, Cells were washed three times with cold PBS, and then 1 ml/well of medium prewarmed to 37°C or 40°C was added (time zero). Cells were incubated at 37°C or 40°C for up to 12 h. At the time points indicated, cells were treated for 1 min with citrate buffer (135 mM NaCl, 10 mM KCl, 40 mM citric acid, pH 3.0) or with PBS as a control. The cells were carefully rinsed five times with PBS. The monolayers were overlaid with methylcellulose medium (0.5% methylcellulose in DMEM with 1% heat-inactivated serum [Sigma-Aldrich]) and incubated at 37°C for 3 days for HSV-1 3A viruses and WT virus. Plaques were visualized by Giemsa staining and counted under light microcopy.