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

Complementation with hyperfusogenic gB rescues the small-plaque phenotype. To confirm that the small-plaque phenotype of the gB 3A virus maps to the gB 3A protein, we determined whether this phenotype could be rescued by complementing the virus with a hyperfusogenic form of gB (gB 876T) (20, 28) that has a truncation gB Mutations That Impact Fusion ® in its cytoplasmic tail. Vero cells were transfected overnight with a plasmid encoding gB 876T or gB 3A or with the vector. Transfected cells were infected with gB 3A-1 or gB 3A-2 virus, and plaques were imaged after 3 days. The plaque size on gB 876Texpressing cells was up to 10-fold larger than that on cells transfected with the vector (Fig. 7) . These results indicate that expression of gB 876T partially rescues the gB 3A virus small-plaque phenotype, consistent with this phenotype mapping to the gB 3A protein. Interestingly, cells expressing gB 3A showed somewhat larger plaques than cells transfected with the vector, suggesting that greater levels of gB 3A expression may enhance virus spread. (29, 30) . To determine whether an increase in temperature could promote the entry of the gB 3A viruses into cells, the penetration assay was performed at both 37°C ( Fig. 8A to C) and 40°C (Fig. 8D to F). Viruses were allowed to penetrate cells for 2, 4, or 6 h before treatment with low-pH citrate buffer to inactivate extracellular virus or with PBS as a positive control. As expected, the citrate buffer treatment did not affect the entry of the G3217 (WT) compared with the PBS control because maximal penetration has occurred by 2 hpi (Fig. 8C) . As previously seen, the gB 3A viruses failed to penetrate the cells after a 6-h incubation at 37°C (Fig. 8A and B) ; however, a small number of plaques were apparent when the gB 3A viruses were incubated with cells for 4 h at 40°C prior to citrate buffer treatment (Fig. 8D and E). These results suggest that the energy provided by the increased temperature may overcome the penetration defect in the gB 3A virus, potentially by promoting the prefusion-to-postfusion conformational change in gB.