Functional regulation of the structure-specific endonuclease FEN1 by the human cytomegalovirus protein IE1 suggests a role for the re-initiation of stalled viral replication forks
Fig 6
Requirement of FEN1’s enzymatic activity for viral DNA replication.
(A) HFFs were infected with AD169 at an MOI of 0.1 or left uninfected (mock) and, at 2 hpi, treated either with DMSO or with different concentrations PTPD. At 96 hpi, cells were harvested and analyzed by Western blotting for FEN1, IE1, MCP, and β-actin. (B) HFFs were infected with AD169 at an MOI of 0.1 and, at 2 hpi, treated with the FEN1 inhibitor PTPD (25 μM), ganciclovir (GCV) (20 μM) or left untreated. At 96 hpi, total DNA was extracted, viral genomes were quantified by TaqMan real-time PCR specific for IE1, and genome copy numbers were calculated. Untreated cells were set to 1. Values are derived from biological triplicates and represent mean values ± SD. (C) Cell viability measured after a 96 h treatment of HFFs with the positive control Staurosporin (STP) (5 μM), ganciclovir (GCV) (20 μM) and PTPD (concentrations as indicated). Untreated cells were set to 100%. Values are derived from biological triplicates and represent mean values ± SD. For panels B and C, the p-values were calculated using two-tailed Student’s t-tests. n.s., not significant; ****, p ≤ 0.0001.