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.