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

Western blot assays. Western blot assays were performed to examine the expression of HSV-1 gB (50). Viruses harvested from Vero cells infected at an MOI of 0.01 were lysed in 200 l of lysis buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1 mM Na 3 VO 3 , 1% Nonidet P-40) containing protease inhibitors (Roche Diagnostics, Indianapolis, IN). Proteins were boiled for 5 min under reducing conditions, separated by SDS-PAGE on 4 to 20% gels, and transferred to nitrocellulose. Blots were probed with anti-gB PAb R74 at a 1:10,000 dilution for 1 h at room temperature, followed by IRDye 800CW goat anti-rabbit IgG (LI-COR, Lincoln, NE) at 1:10,000. The bands were visualized by Odyssey imaging (LI-COR). Incorporation of gB 3A into the virion was examined as previously reported (52) . Briefly, medium from infected Vero cells containing extracellular virus was subjected to low-speed centrifugation to pellet cells and debris. The supernatant was centrifuged at 48,000 ϫ g for 1 h at 4°C over a 10% sucrose cushion. The pellet was dissolved in sample buffer and separated by SDS-PAGE on a 4 to 20% gel under reducing conditions. Western blot analysis was performed with R74 at a 1:10,000 dilution and anti-VP5 MAb (East Coast Bio) at a 1:2,000 dilution, followed by the addition of IRDye 800CW goat anti-rabbit IgG (LI-COR, Lincoln, NE) and IRDye 680LT donkey anti-mouse antibody at 1:10,000 (LI-COR, Lincoln, NE).