Role of E2F-Rb family proteins in the inhibition of E1A expression by IFNs.
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(A) EMSA binding reactions were performed using nuclear extract from HDF-TERT cells and a 32P-labeled probe corresponding to the IFN-regulated E1A enhancer repressor element (E1A-ENH, Ad5 nt 271–288, lane 1). Lanes 2–10 show EMSA binding reactions that contained specific antibodies directed E2F, DP, and Rb family members (E2Fs 1, 3 and 4, Rb, p107, p130, and DP1) or control antibodies (normal mouse and rabbit). The compositions of DNA-protein EMSA complexes are indicated on the left. Lanes 11–13 show binding reactions with HDF-TERT nuclear extracts prepared from untreated cells or cells treated with IFNα or IFNγ for 24 hr. (B and C) ChIP assays were performed to examine the binding of Rb and p107 to the E1A enhancer region in vivo with or without IFN treatment. HDF-TERT cells were treated with IFNs or left untreated for 24 hr and then infected with 200 virus particles/cell dl309 or Ad5-mut1. ChIP assays were performed at 18 hr post-infection using antibodies against Rb (B), p107 (C), with isotype-matched controls. Immunoprecipitated DNAs were quantified by qPCR (primer pairs are listed in S1 Table in S1 Materials and Methods). Enrichment is represented as percentage of input DNA and the values represent the mean ± sd, n = 3. (* P ≤ 0.05, ** P ≤ 0.01). (D–F) The effect of the cdk4 inhibitor PD0332991 on Ad5 replication via the E2F binding site in the E1A enhancer region was evaluated. HDF-TERT cells were incubated with 2 μM PD0332991 or DMSO for 6 hr prior to virus infection. (D, left) Cell extracts were prepared at 6, 12 and 30 hr following PD0332991 pretreatment and Rb, p107 and p130 phosphorylation was examined by Western blot. (D, right) PD0332991 pretreated cells were infected with Ad5-WT and Ad5-mut1 at 200 virus particles/cell and Ad early gene expression (E1A, DBP) was analyzed by Western blot at 24 hr post-infection. γ-tubulin was used as a loading control for the samples. (E) Cell numbers were determined after 6 hr PD0332991 pretreatment (0), and in virus-infected cells at 6 and 24 hr post-infection. (F) Cells were pretreated with PD0332991 for 6 hr and infected as described in (D) and viral DNA replication was quantified at 24 hr post-infection by qPCR. The values were normalized to the input at 6 hr post-infection and are plotted as mean ± sd. n = 3. (G and H) HDF-TERT cells were infected Ad-CMV-12S-E1A or the empty control virus, Ad-CMV, at 5,000 virus particles/cell for 1 hr and the cells were left untreated or treated with IFNs for 24 hr. The cells were super-infected with dl309 at 200 virus particles/cell. (G) Cells were harvested at 48 hr post-infection and E1A expression was analyzed by Western blot. α-tubulin was used as a loading control for the samples. (H) Cells were harvested at 48 hr post-infection and viral DNA replication was quantified by qPCR using a dl309-specific primer pair (see S1 Table in S1 Materials and Methods). The values were normalized to 1.0 in untreated cells and are plotted as mean ± sd, n = 3. (** P ≤ 0.01, *** P ≤ 0.001).