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

To determine the time required for gB 3A virus penetration, the gB 3A viruses were allowed to bind to cells for 1 h on ice and then incubated at 37°C for up to 12 h prior to the citrate buffer treatment. Both gB 3A viruses did not show appreciable penetra- gB 3A viruses do not exhibit a cell binding defect. To examine whether poor binding might contribute to the slow growth kinetics of the gB 3A viruses, the cell binding kinetics of the gB 3A viruses and G3217 (WT) were compared. The gB 3A and G3217 (WT) viruses (200 PFU) were added to Vero cell monolayers, and the cells were incubated at 4°C or 37°C for up to 120 min. At 4°C, viruses can bind to but not fuse with the cells. At distinct time points, the cells were rinsed with PBS twice to remove unbound virus and methylcellulose medium was added. Plaques were counted after 3 days of incubation to determine the level of resistance to the PBS washes. The gB 3A and G3217 (WT) viruses showed similar plaque counts when incubated at 4°C (Fig. 6A ) or 37°C (Fig. 6B) prior to the PBS washes. The plaque counts of all viruses increased as the incubation time prior to the PBS washes increased. These results suggest that the growth defect seen in the gB 3A viruses is not due to a severe cell binding defect in the gB 3A viruses.