Fig 1.
E4orf4 reduces the phosphorylation of DDR proteins during mutant Ad infection.
(A) HeLa cells were either mock-infected or infected with the Ad mutant dl366*, lacking the E4 region, or with dl366*+E4orf4, lacking all E4 orfs except E4orf4. Infections were done with 20 ffu per cell. Proteins were harvested at the indicated times post infection and Western blot analysis was carried out with the specified antibodies. Protein bands were quantified by densitometry, the levels of phosphorylated proteins were normalized to the levels of the corresponding total proteins, and the ratios between normalized phosphorylation in dl366*-infected cells (defined as 1) and parallel values in dl366*+E4orf4-infected cells are shown below the bands. (B) HUVEC cells were either mock-infected or infected with the Ad mutants dl366* and dl366*+E4orf4 at 30 ffu per cell. Proteins were harvested 24 hrs post infection and Western blot analysis was carried out with the specified antibodies. (C) The intensities of protein bands in Western blots from two independent experiments performed as described in B were quantified by densitometry. Relative phosphorylation levels were determined as described in A and the average of the two experiments is shown in the graph. Error bars represent standard error. *: p<0.005. (D) HeLa cells were infected as in A, fixed 24 hrs post infection, and stained with the indicated antibodies and with DAPI. Representative pictures taken with a Zeiss Axioskop at a magnification of 100 are shown. (E) A representative picture of a dl366*-infected HeLa cell stained with antibodies to p53BP1-S1778 and the Ad DNA binding protein (DBP) and with DAPI is shown at a magnification of 640.
Fig 2.
DDR inhibition by E4orf4 is PP2A-dependent.
(A) L11 cells in which a PP2A-B55 shRNA can be transiently expressed following Dox induction were induced with Dox (+) or left untreated (-). 48 hrs later, the cells were transfected with an empty vector (-), or with a vector expressing shRNA-resistant PP2A-B55-HA (+). One day later, dl366* and dl366*+E4orf4 mutant viruses were used to infect the transfected cells, and some of the cells were left uninfected (Mock). Protein extracts were prepared 24 hrs post-infection and subjected to Western blot analysis with the indicated antibodies. Quantitation and normalization were performed as described in the legend to Fig 1. (B) HEK293-derived clone 13 cells containing Dox-inducible WT E4orf4 or clone 3 cells expressing an E4orf4 mutant unable to bind PP2A (R81F84A: E4orf4-mut) were induced with Dox for three hrs (+E4orf4)) or were left uninduced (-E4orf4), and were then treated with 4 μM hydroxyurea (HU) or with 25 ng per ml neocarzinostatin (NCS), or were left untreated (Control). Protein extracts were prepared after 3 hrs incubation with the drugs and subjected to Western blot analysis, using antibodies to phosphorylated and non-phosphorylated Chk1 and to E4orf4. Quantitation and normalization were performed as described above.
Fig 3.
E4orf4 inhibits DNA damage repair.
(A) Clone 13 cells were induced with Dox for 2.5 hrs to stimulate E4orf4 expression (+E4orf4), or were left uninduced (-E4orf4), and were then treated with 100 μM H2O2 for 30 minutes or were left untreated. Cell samples were then subjected to an alkaline comet assay. Representative microscopic images of the assay taken at a magnification of 200 are shown. (B). Quantitation of the comet data from A is shown. The length and intensity of DNA tails relative to heads is represented by the comet tail moment which was determined using the TriTek Comet Score software (n ≥ 100). Bars represent standard error of the mean. The statistical differences between control and E4orf4-expressing cells or between drug-treated and untreated cells are indicated with p values: *: p<0.001.
Fig 4.
ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4.
(A) ATM-deficient A-T fibroblasts and matching WT fibroblasts were either mock-infected or infected with the Ad mutants dl366* and dl366*+E4orf4. Proteins were harvested 24 hrs later and Western blot analysis was carried out with the indicated antibodies. Quantitation and normalization were performed as described in the legend to Fig 1. (B) Clone 13 cells expressing E4orf4 under Dox regulation were induced for 3 hrs (+) or were left uninduced (-). They were then treated with NCS for one hr in the presence or absence of an ATM inhibitor (ATMi: KU60019). Protein extracts were subjected to Western blot analysis with the indicated antibodies. Quantitation and normalization were performed as described in the legend to Fig 1. (C) HeLa cells were either mock-infected or infected with the Ad mutants dl366* and dl366*+E4orf4. An ATR inhibitor (ATRi: ETP46464) was added to the infected cells and another group of infected cells was left untreated (-). Proteins were harvested 24 hrs post infection and Western blot analysis was carried out with the indicated antibodies. (D) The intensities of protein bands in Western blots from two independent experiments performed as described in C were quantified by densitometry. Relative pATM-S1981 levels were calculated as described in the legend to Fig 1, and the average of the two experiments is shown in the graph. Error bars represent standard error of the mean and statistical significance was determined using a paired student t test. *: p<0.003; NS: non-significant (p = 0.09).
Fig 5.
ATM and ATR inhibit Ad replication whereas E4orf4 enhances it.
(A) ATM-deficient A-T cells, reconstituted with WT ATM or with an empty vector, were infected with the Ad mutants dl366* and dl366*+E4orf4 (30 ffu/cell), or were mock infected. An ATR inhibitor (ATRi) was added to parallel cell samples as described in the methods section. The cells were visualized 24 hrs post infection using a Zeiss Axioskop at a magnification of 100. Representative pictures of two independent experiments are shown. The percent of area covered with cells out of the total field area (ce) was determined using Image-Pro Premier 9.1 and the percent of area covered with cell clusters larger than 6000 pixels2 out of total cell area (cl), was calculated using FIJI. (B) Viruses from two independent experiments performed as described in A were collected from infected cells 24 hrs post infection and their titer was determined. Data is displayed in box plots as previously described [72] and black dots represent outliers. Statistical significance was calculated using unpaired t-test and p values of significant differences between dl366* and dl366*+E4orf4 viruses are shown.
Fig 6.
Loss of ATM and expression of E4orf4 accelerate Ad infection.
(A) A-T cells and matching WT control cells were infected with the Ad mutants dl366* and dl366*+E4orf4 (100 ffu/cell) or were mock infected. The cells were visualized at the indicated times post infection (p.i.) using a Zeiss Axioskop at a magnification of 100. Mock infected samples are from the 24 hrs time point. Representative pictures of two independent experiments are shown. The percent of area covered with cells out of the total field area (ce) was determined using Image-Pro Premier 9.1 and the percent of area covered with cell clusters larger than 1000 pixels2 out of total cell area (cl), was calculated using FIJI. (B) Production of viruses in cells infected as described in A was determined by a titer assay. p.i.: post infection. Data is displayed in box plots as previously described [72]. Statistical significance was calculated using unpaired t-test and p values of significant differences between dl366* and dl366*+E4orf4 viruses are shown.
Fig 7.
The effect of ATM and ATR inactivation and E4orf4 expression on various stages of Ad replication.
(A) ATM-deficient A-T cells, reconstituted with WT ATM or with an empty vector, were infected with the Ad mutants dl366* and dl366*+E4orf4, or were mock infected. An ATR inhibitor (ATRi) was added to parallel cell samples as described in the methods section. Proteins were harvested 24 hrs later and Western blot analysis was carried out with the indicated antibodies. Three different exposures of the blot stained with a capsid-specific antibody are shown (long, short, very short). Protein levels were quantified by densitometry. Values of virus protein levels obtained by densitometry were normalized to the Tubulin loading control. The value for dl366* infection of A-T + VECTOR cells was defined as 1 and relative protein levels are shown beneath the relevant protein bands. (B) Virus DNA levels were quantified by quantitative PCR and normalized to cellular DNA represented by the PRPH2 gene. The viral DNA level in a dl366* infection of A-T + WT ATM cells was defined as 1 and relative DNA levels are shown.
Fig 8.
E4orf4 sensitizes cells to killing by drugs inducing DNA damage or replication stress.
Clone 13 cells were induced with Dox for two hrs to stimulate E4orf4 expression (+E4orf4), or were left uninduced (-E4orf4), and were then treated with 4 μM HU or 2.5 ng/ml NCS for three hrs or left untreated (Control). Cell survival was measured by a clonogenic assay using various cell dilutions, without further Dox addition. The number of colonies was counted two weeks later, and relative survival was calculated separately for–E4orf4 and +E4orf4 samples by defining both control samples as 100%. E4orf4 itself decreased cell viability to 40% of control cells. Two independent experiments, each with triplicate samples were performed. Error bars represent pooled standard deviation and statistical significance was determined using a paired student t test. *: p<0.05.