FIG 5.

The ΔUL46 virus failed to block innate immunity triggered by the ligand of STING, 2′,3′-cGAMP. (A) HEL cells were infected with HSV-1(F) or the ΔUL46 virus (0.1 PFU/cell). The cells were harvested at 3, 6, 9, and 24 h after infection, and equal amounts of proteins were analyzed with antibodies against STING and β-actin. (B) HEL cells were infected with HSV-1(F) or the ΔUL46 virus (0.1 PFU/cell). The cells were harvested at 3, 6, and 9 h after infection, and total RNA was extracted and used for quantification of the IFN-β and ISG56 transcripts by real-time PCR. The experiment was repeated three independent times. 18S rRNA served as a loading control. (C) HEL cells were either mock infected (lanes 2 to 4 and 11) or exposed to HSV-1(F) (lanes 5 to 7 and 12 to 14) or to the ΔUL46 virus (lanes 8 to 10 and 15 to 17) (0.5, 2.5, or 5 PFU/cell). Two hours after infection, the cells were treated with 2′,3′-cGAMP (3 μM) that was either added to the cultures (cGAMP) or transfected to the cells (tcGAMP). The cells were harvested at 8 h after infection, and total RNA was extracted and used for semiquantification of the ISG56 transcripts by PCR. 18S rRNA was used as a control. (D) HEL cells were either mock infected or exposed to HSV-1(F) or to the ΔUL46 virus (5 PFU/cell). The cells were harvested at 6, 9, or 24 h after infection, and equal amounts of proteins were analyzed by immunoblot analysis using antibodies against ICP0, VP22, and β-actin. (E) HEL, HEp-2, or STING-depleted HEL cells were infected with either HSV-1(F) or the ΔUL46 virus (0.01 PFU/cell). The cells were harvested at 3, 24, 48, or 72 h after infection, and titrations were done in Vero cells. (F) HEL or HEp-2 cells or their derivatives expressing UL46 were infected with either HSV-1(F) or the ΔUL46 virus (0.01 PFU/cell). The cells were harvested at 3, 24, and 48 h after infection, and titrations were done in Vero cells.