Fig 5.

HSV-1 DNA replication triggers the accumulation of DNA breaks. (A) Images showing the phosphorylation levels of histone H2AX (γH2AX) over the course of HSV-1 infection. Serum-starved fibroblasts were infected (1 IU/cell) and fixed at various times. Protein levels and localization were examined by immunofluorescence using antibodies specific to γH2AX and HSV-1 ICP4. (B and C) Analysis of H2AX phosphorylation after inhibition of the viral DNA polymerase using 400 μg/ml PAA. Serum-starved fibroblasts were infected (0.1 IU/cell for immunofluorescence assay; 3 IU/cell for Western blotting), and drug was applied at 1 hpi. Cells were fixed at 8 hpi (immunofluorescence assay) or harvested at 18 hpi (Western blotting). (D) Production of infectious HSV-1 virions in cells treated with 400 μg/ml PAA as described above. Values are representative of virus yield at 18 hpi and are expressed relative to mock-treated cells. (E) Analysis of poly(ADP-ribosyl)ation after inhibition of HSV-1 DNA replication. Serum-starved fibroblasts were infected with HSV-1 (3 IU/cell) or mock infected and treated with PAA as described above. H₂O₂-treated cells were pretreated with PAA for 1 h before H₂O₂ application. Whole-cell lysates were analyzed by Western blotting. (F) PARP-1 localization during HSV-1 infection. Serum-starved fibroblasts were infected with HSV (0.5 IU/cell) and either 400 μg/ml PAA or vehicle was applied at 1 hpi. Cells were fixed at 12 hpi and examined by immunofluorescence using antibodies specific to PARP-1 and HSV-1 UL30. *, P < 0.05.