Fig. 1

HSV-induced H2AX phosphorylation requires the presence of the viral polymerase and ATM kinase activity. a Human foreskin fibroblast (HFF) cells were infected with HSV-1 (MOI 5) and cell lysates were prepared at various times post-infection. The lysates were immunoblotted and probed with antibodies for total H2AX or its phosphorylated counterpart (γH2AX), with GAPDH as a loading control. b The transcription inhibitor actinomycin D (A, 1 μg/ml) and the translation inhibitor cycloheximide (C, 100 μg/ml) were added to HFFs 1 h before HSV-1 infection or 1 μM doxorubicin (Doxo) treatment. The DNA synthesis inhibitor phosphonoacetic acid (P, 400 μg/ml) was added at 1 h p.i. All compounds remained in the media until lysis at 24 h p.i. c HFFs were infected with HSV-1 and/or HSV-2 with or without phosphonoacetic acid (PAA, 400 μg/ml). Cells were lysed at 24 h p.i., immunoblotted, and probed for total H2AX, γH2AX, or VP16, with actin loading control. d Vero or HFF cells were either mock-infected, infected with HSV-1 KOS strain (K), or infected with a mutant virus lacking UL30, either strain ΔS1 (Δ) or HP66 (H) at MOI 5. Cell lysates were prepared at 16 h.p.i. and analyzed by immunoblot. e and f HFFs were infected with HSV-1 (panel e) or HSV-2 (panel f), immediately overlaid with an ATM inhibitor (KU-55933, KU), an ATR inhibitor (VE-821, VE), or the two in combination (KV), and lysed at 24 h p.i. prior to immunoblot analysis