Inhibition of Ad replication by IFNs is blocked by E1A expression.

(A) HDF-TERT cells treated with IFNs or left untreated for 24 hr and then infected with dl309 or the replication-defective mutant virus ΔTP-GFP at 25 virus particles/cell. Nuclear DNA was isolated at the indicated time points and viral DNA levels were quantified by qPCR. (B) HDF-TERT cells treated with IFNs or left untreated for 24 hr and then infected with ΔTP-GFP at 25 virus particles/cell. E1A mRNA levels were quantified by RT-qPCR. The results were normalized to cellular GAPDH mRNA levels. The data are plotted as mean ± sd, n = 3. (C) HDF-TERT-E1A cells treated with IFNs or left untreated for 24 hr and Western blot analyses were performed at 24 hr post-infection to detect expression and phosphorylation of STAT1, and the expression of ISG54 and ISG15. E1A 12S and 13S products are shown in the top panel. γ-tubulin is shown in the bottom panel as a loading control for the samples. (D) HDF-TERT-E1A cells treated with IFNs or left untreated for 24 hr and then infected with dl309 at 25, 200 or 1000 virus particles/cell. Nuclear DNA was isolated at 48 hr post-infection and viral DNA levels were quantified by qPCR. The values were normalized to 1.0 in untreated cells and are plotted as mean ± sd, n = 3. (E) HDF-TERT-E1A cells treated with IFNs or left untreated for 24 hr and then infected with dl309 at 25 virus particles/cell. E1A mRNA levels were quantified at 24 hr post-infection by RT-qPCR. The values were normalized to 1.0 in untreated cells and are plotted as mean ± sd, n = 3. (F) HDF-TERT were infected with in340-Δ2-CMV (Ad-CMV) or in340-Δ2-CMV-E1A (Ad-CMV-E1A) viruses at 5,000 virus particles/cell for 1 hr and subsequently treated with IFNs or left untreated. Cellular extracts were prepared at 24 hr post-treatment and analyzed by Western blot using antibodies against E1A, STAT1 and STAT1 (pY701). α-tubulin is shown in the bottom panel as a loading control for the samples. (G) HDF-TERT cells were infected and treated as in (F) and super-infected with dl309 at 200 virus particles/cell 24 hr after the addition of IFNs. dl309 DNA replication was quantified 48 hr post-infection by qPCR using a dl309-specific primer pair. The values were normalized to 1.0 in untreated cells and are plotted as mean ± sd, n = 3. (** P ≤ 0.01, *** P ≤ 0.001).