Fig 1.
Identification of Flap endonuclease 1 as novel interactor of HCMV IE1.
Yeast cells Y153 were transformed with two separate vectors, one of which encoded a GAL4 activation domain (AD) in fusion with the indicated cellular protein, the second plasmid encoded a GAL4 DNA-binding domain (DBD) in fusion with the respective IE1 versions. Thereafter, yeast colonies were selected for the presence of both plasmids on dropout medium lacking tryptophan and leucine and subsequently analyzed for the expression of β-galactosidase. (A) Interaction analysis between HCMV IE1 14–382, which serves as yeast two-hybrid screen bait, fused to the GAL4 DBD and the putative novel interactors as fusions with the GAL4 AD. (B) Interaction analysis between Flap endonuclease 1 in fusion to the GAL4 (AD) and HCMV IE1 as full length (FL) or truncated (14–382 and 1–359) versions as fusions with the GAL4 DBD.
Fig 2.
Characterization of the IE1-FEN1 interaction via coimmunoprecipitation (CoIP) experiments.
(A, B, and E) HEK293T cells were cotransfected with the indicated Myc (M)- and FLAG (F)-tagged constructs. Upper two panels: Western blot detection of FEN1- and IE1 variants after immunoprecipitation (IP) with an anti-FLAG antibody. Lower panel/panels: detection of FEN1 and IE1 variants in cell lysates before precipitation (input). (A) IP of F-IE1 and F-IE1 1–382, CoIP of M-FEN1 176–380. (B) IP of F-FEN1 and F-FEN1 176–380, CoIP of M-IE1 1–382. (E) IP of F-FEN1 176–380, CoIP of different wildtype (wt) and mutant versions of M-IE1 1–382. (C) HFFs stably expressing mCherry or mCherryFEN1 were infected with AD169 at an MOI of 3 and lysed at the indicated times post infection. Upper two panels: Western blot detection of mCherryFEN1 and IE1 after immunoprecipitation (IP) with an anti-mCherry antibody. Lower panel: detection of IE1 in cells lysates before precipitation (input). (D) Schematic of the IE1core structure. Regions that were mutated in order to generate Y206A, Loop mut, Helix 8 mut, and 5x mut are depicted in green, blue, orange, and red, respectively.
Fig 3.
IE1-mediated upregulation of FEN1 during HCMV infection.
(A, B, and D) HFF cells were either mock infected or infected with AD169 at an MOI of 3 (A), with TB40/E at an MOI of 1 (B), or with AD169 (wt) at an MOI of 7 and equivalent genome copy numbers of a recombinant strain lacking IE1 (ΔIE1) (D). At the indicated times post infection, cells were harvested and analyzed by Western blotting for FEN1, IE1, IE2 (for D), UL44, MCP, and β-actin. (C) HFF FLAG (F)-IE1 cells with doxycycline-inducible expression of FLAG (F)-IE1 were either not induced or induced with 0.5 μg/ml doxycycline for the indicated times. Cells were harvested analyzed by Western blotting for FEN1, IE1, and β-actin. (E) HFFs, which were treated for 24 h with 50 nM of a control siRNA (siC) or a siRNA targeting E2F1 (siE2F1), were infected with AD169 at an MOI of 3. At the indicated times post infection, cells were harvested and analyzed by Western blotting for E2F1, FEN1, IE1, UL44, and β-actin.
Fig 4.
Stabilization of FEN1 by IE1 binding.
(A, B, C, and D) 293T cells were transfected with FLAG (F)-FEN1 alone (A) or in combination with either IE1 wildtype (wt) (B) or IE1 5x mut (C). 18 hours post transfection, cells were treated with 10 μg/ml protein synthesis inhibitor cycloheximide (CHX) and harvested at the indicated times post addition. Western blot analyses were performed to detect FLAG (F)-FEN1, IE1, and β-actin. (D) Decline of FEN1 obtained by three independent experiments is represented by mean values ± SD. The p-values were calculated using two-tailed Student’s t-tests. n.s., not significant; **, p ≤ 0.01, *, p ≤ 0.05.
Fig 5.
Promotion of viral DNA replication by FEN1.
(A) Detection of FEN1 protein levels in vector-, siC-, and siFEN1-transduced HFFs by Western blotting. β-actin levels served as loading control. (B and C) siC- and siFEN1-transduced HFFs were infected at an MOI of 0.01 (B) or at indicated MOIs (C) and multistep growth curve analysis (B) or virus release assay (C) was performed. Cell supernatants were harvested at the indicated times after infection and analyzed for viral genome equivalents by HCMV IE1-specific quantitative real-time PCR. For C, siC-transduced HFFs were set to 1. Values are derived from biological triplicates and represent mean values ± SD. (D) HFF cells were either transfected with two control siRNAs (siC A and siC J) or siRNA directed against FEN1 or left untreated. 24 h later, cells were either directly harvested (Mock) or infected with AD169 at an MOI of 0.1 and harvested 96 hpi. Cells were analyzed by Western blotting for FEN1, IE1, MCP, and β-actin. (E and F) siC A- and siFEN1-transfected HFFs were infected with AD169 (E) or TB40/E (F) at an MOI of 0.1, and total DNA was extracted at 8 hpi (left) and 96 hpi (right) using the DNeasy blood and tissue kit (Qiagen). Viral genomes were quantified by TaqMan real-time PCR specific for IE1, and genome copy numbers were calculated. siC A-transfected HFFs were set to 1. Values are derived from biological triplicates and represent mean values ± SD. For panels B, E, and F, the p-values were calculated using two-tailed Student’s t-tests. n.s., not significant; ****, p ≤ 0.0001; ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05.
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.
Fig 7.
IE1-mediated nucleolar exclusion of FEN1 during HCMV infection.
(A) mCherryFEN1 cells were infected with AD169 at an MOI of 1 and, at 24 hpi, analyzed by detecting the red fluorescent protein mCherry as well as UL44 as marker for infected cells by utilizing an antibody directed against UL44. Nucleoli are marked by arrowheads in the inset images. (B) mCherryFEN1 cells were infected with AD169 at an MOI of 1 and, at indicated time points, analyzed by detecting the red fluorescent protein mCherry as well as IE1 by utilizing an antibody directed against IE1. (C and D) Doxycycline-inducible HFF IE1 wildtype (wt) (C) and HFF IE1 5x mut (D) were transduced with mCherryFEN1. Cells treated (+ Dox) or untreated (- Dox) with doxycycline (0.5 μg/ml) were analyzed by detecting the red fluorescent protein mCherry as well as IE1 by utilizing an antibody directed against IE1.
Fig 8.
Accumulation of phosphorylated FEN1 species upon IE1 expression.
(A, B, C, D, G and H) 293T cells were transfected with the indicated constructs and, at 42 h post transfection, treated with MG132 (5 μM) when indicated. 48 h post transfection, cells were harvested and analyzed by Western blotting for IE1, FLAG, pFEN1 (Ser187), and β-actin. (A, B, D, G and H) Protein levels of FEN1 and pFEN1 were quantified via densitometric analyses utilizing the Image Lab Software. Protein levels without IE1/MG132 were set to 1. (B and H) Ratios between pFEN1 and FEN1 obtained by three independent experiments are represented by mean values ± SD. The p-values were calculated using two-tailed Student’s t-test. n.s., not significant; ***, p ≤ 0.001. (E and F) HFF cells were either mock infected or infected with AD169 at an MOI of 3 (E) or with AD169 (wt) at an MOI of 7 and equivalent genome copy numbers of a recombinant strain lacking IE1 (ΔIE1) (F). At the indicated times post infection, cells were harvested and analyzed by Western blotting for FEN1, pFEN1, IE1, IE2 (for F), UL44, MCP, and β-actin.
Fig 9.
Requirement of FEN1’s enzymatic activity for the induction of γH2AX.
(A, B, D, and E) HFF cells were either mock infected or infected with AD169 at an MOI of 3 (A, E), 1 (B), or with AD169 (wt) at an MOI of 7 and equivalent genome copy numbers of a recombinant strain lacking IE1 (ΔIE1) (D). Cells were treated at 2 hpi with 400 μM phosphonoformic acid (PFA) (B) or with the indicated concentration of the FEN1 inhibitor PTPD (E). At the indicated times post infection, cells were harvested and analyzed by Western blotting for the indicated proteins. (C and F) HFF IE1 cells with doxycycline-inducible expression of IE1 were treated with control siRNA (siC) or siRNA targeting FEN1 (siFEN1) when indicated (F) and either not induced or induced with 0.5 μg/ml doxycycline and analyzed by Western blotting for γH2AX, IE1, and β-actin.
Fig 10.
Requirement of FEN1’s enzymatic activity for the re-initiation of stalled viral replication fork.
(A, B, and D) HFF cells were either mock infected (A, B, and D) or infected with AD169 at an MOI of 3 (B and D). When indicated, cells were treated with DMSO or PTPD (25 μM) at 2 hpi (D). DMSO or 1 μM camptothecin (CPT) were applied for 1 h unless otherwise indicated. Subsequently, cells were harvested and analyzed by Western blotting for the indicated proteins. (C) HFFs were infected with AD169 at an MOI of 0.1, and total DNA was extracted at 48 hpi (first bar) or at 51 hpi after a 1 h treatment with DMSO (second bar) or 1 μM CPT (third bar) using the DNeasy blood and tissue kit (Qiagen). Viral genomes were quantified by TaqMan real-time PCR specific for IE1, and genome copy numbers were calculated. HFFs infected for 48 h were set to 1. Values are derived from biological triplicates and represent mean values ± SD. (E) Visualization of viral DNA labeled with two synthetic nucleotides: EdC (second panel), which was incorporated before the induction of replication fork stalling with 1 μM CPT, and BrdU (third panel), which was incorporated after release from the CPT block. Time points reflect times after release. (F and G) Mean fluorescence intensities of incorporated EdC (F) or BrdU (G) quantified in 50 cells treated with DMSO (left) or 25 μM PTPD (right) 6 h after CPT release. For panels C, F, and G, the p-values were calculated using two-tailed Student’s t-tests. n.s., not significant; ****, p ≤ 0.0001; *, p ≤ 0.05.
Table 1.
Oligonucleotides.