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.