FIG 8.

An infection-associated DDB2-ATM negative feedback loop. (A and B) DDB2 contributes to the kinetics of ATM accumulation. HEL fibroblasts were transiently transfected with siCON or siDDB2 and subsequently infected with HCMV at an MOI of 0.1 24 h later. Samples were harvested at the indicated times p.i. (A) ATM and DDB2 levels were assessed by immunoblot analyses. (B) ATM transcript levels were measured by qRT-PCR. The fold change in ATM transcript levels in siDDB2-treated fibroblasts is shown relative to ATM transcript levels measured in siCON-treated fibroblasts. Mean values are shown, with bars denoting 1 standard deviation, for three independent experiments. (C and D) Influence of ATM on DDB2 transcript levels. HEL fibroblasts were treated with siCON or siATM and infected with HCMV at an MOI of 0.1 24 h later. Cells were harvested at the indicated times p.i. For panel C, DDB2 transcript levels were measured by qRT-PCR. The fold change in DDB2 transcript levels in siATM-treated fibroblasts is plotted relative to DDB2 transcript levels measured in siCON-treated fibroblasts. Mean values are shown, with bars denoting 1 standard deviation, for three independent experiments. For panel D, the levels of ATM protein expression in siCON- and siATM-treated fibroblasts were assessed by immunoblot analysis. (E) DDB2-dependent ATM accumulation in response to HCMV infection is independent of viral DNA replication. HEL fibroblasts were treated with siCON or siDDB2 and infected with HCMV at an MOI of 0.1 24 h later in the presence (+) or absence (−) of the viral DNA synthesis inhibitor phosphonoacetic acid (PAA) (100 μg/ml). The levels of ATM protein expression in siCON- and siDDB2-treated cells with or without PAA were assessed by immunoblot analyses.