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

gB 3A viruses display a small-plaque phenotype. Compared to the GS3217 (WT) virus, both gB 3A viruses formed smaller plaques on Vero cells after 3 days of incubation (Fig. 3A) . Relative to the GS3217 (WT) gB plaques, the gB 3A plaques were approximately 200-fold smaller, with a 50-to 300-fold range of reduction (Fig. 3B ). The plaque size was partially restored when VgB cells (Vero-24 cells expressing WT gB) were infected with the gB 3A viruses (Fig. 3A) , with gB 3A plaques sizes only two to three times smaller than those of the WT (Fig. 3B ). This partial restoration of plaque size suggests that the mutations in the gB-encoding gene of gB 3A are responsible for gB 3A viruses have slow growth kinetics. To investigate the growth of the gB 3A viruses, we performed single-step growth curve assays (Fig. 4A ). Vero cells were infected with gB 3A-1, gB 3A-2, or GS3217 (WT) virus at 5 PFU/cell, and virus titers were determined at 6, 12, 18, and 24 hpi (Fig. 4A) . A defect in gB 3A mutant virus replication was detected at 12, 18, and 24 hpi. By 24 hpi, the mutant virus titers were 3 log units lower than those of the GS3217 (WT) virus. To examine the replication of the gB 3A viruses at a lower multiplicity of infection (MOI), we performed a multistep growth curve assay (Fig. 4B ). Vero cells were infected with the viruses at 0.01 PFU/cell, and virus titers were determined every 12 h until 48 hpi. At 12 hpi, the GS3217 (WT) virus titer had increased but the gB 3A virus titers had not. At 24 hpi, the gB 3A titers lagged behind the GS3217 (WT) virus titer by more than 5 log units (Fig. 4B ). At 48 hpi, the gB 3A virus titers were increasing but had not reached the titer achieved by the GS3217 (WT) virus. Consistent with their small-plaque phenotype (Fig. 3) and delayed development of a CPE (Fig. 2) , the gB 3A viruses demonstrated significant growth defects in both the single-step and multistep growth curves (Fig. 4) . gB Mutations That Impact Fusion ® gB 3A viruses show slow penetration of cells. The entry of HSV-1 into the cell begins with viral gB and/or gC attachment to cell surface HS proteoglycans. After binding, the virus is resistant to removal from the cell surface with phosphate-buffered saline (PBS) rinses but bound virions are sensitive to inactivation by a low-pH citrate buffer (21) (22) (23) (24) (25) , Once fusion has occurred, the virions are no longer sensitive to inactivation by citrate buffer (21, 26, 27) . This ability to differentiate between "bound viruses" and "fused viruses" by using low-pH inactivation allows penetration assays to be performed. GS3217 (WT) or gB 3A HSV-1 (150 PFU) was allowed to adsorb to Vero cell monolayers for 1 h on ice. Cells were then incubated at 37°C for 0 to 4 h. At specific time points, cells were treated with citrate buffer to inactivate extracellular virus or with PBS as a control. After treatment, the cells were overlaid with methylcellulose medium and plaques were allowed to form. For G3217 (WT) virus, incubation at 37°C for 2 h prior to the citrate buffer treatment was sufficient to reach nearly maximal titers (Fig. 5A) . Indeed, penetration of the G3217 (WT) virus was detectable after only 20 min at 37°C. In contrast, when the gB 3A viruses were allowed to adsorb to Vero cells for 1 h at 4°C and then allowed to enter the cells for 2 h at 37°C before citrate buffer inactivation, very few plaques formed (Fig. 5B ). When the gB 3A viruses were rinsed with PBS instead of citrate buffer at 2 hp