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 Mutations That Impact Fusion ® to generate GS3217 (WT) and gB 3A virus stocks. Two distinct gB 3A virus stocks (gB 3A-1 and gB 3A-2) were created by independent transfections of BAC pQF297 into Vero-Cre cells. To confirm the expression of the mutant gB proteins and gB incorporation into the virions, Western blot assays were performed (Fig. 1G ). Vero cells were infected with the gB 3A viruses and the GS3217 (WT) virus at 0.01 PFU/cell. Because of the growth defects of gB 3A viruses (described below), infected cells and supernatants were harvested 7 days postinfection with the gB 3A viruses and 3 days postinfection with the G3217 (WT) virus. A Western blot assay of infected cell lysates probed with anti-gB serum R74 shows a band at the expected size for WT gB for all three viruses (Fig. 1G , top). To confirm that gB was incorporated into the gB 3A virions, virus was harvested from culture supernatants by centrifugation through a 10% sucrose cushion. Western blot assays of virus released into the medium demonstrated that mutant gB was incorporated into virions (Fig. 1G , middle and bottom).