Fig 4. Effects of ectopic IE1 expression on the IFNβ-mediated ISG54 induction.

(A) HF cells transduced by control MIN retroviral vector (HF-MIN) or MIN-IE1 (HF-MIN-IE1) were untreated or treated with IFNβ (1 x 10³ units/ml) for 8 h. Total RNAs were prepared and the mRNA levels of ISG54 and β-actin were determined by qRT-PCR. The IE1 protein levels in cells were determined by immunoblotting with anti-IE1 antibody (6E1). (B) HF cells were uninfected or infected with Ad-Trans alone or with Ad-Trans plus Ad-IE1 at a total MOI of 10 plaque forming units (PFU) per cell for 48 h. Cells were then incubated for 8 h in the absence or presence of IFNβ (1 x 10³ units/ml). qRT-PCR and immunoblot assays were performed as described in (A). (C) Reporter assays using the ISG54 ISRE-luciferase (Luc) reporter construct. 293T cells were cotransfected with 0.5 μg of ISG54 ISRE-Luc reporter plasmid and 0.5 μg of plasmid encoding intact HA-IE1, HA-IE1(1–475), or HA-IE1(Δ421–475) or 1.5 μg of plasmid encoding HA-IE1(Δ290–320), HA-IE1(1–420), or HA-IE1(Δ290–320/Δ421–475) mutants as indicated. All samples for transfection were made up to the same total amount of DNA using empty vectors. At 24 h, cells were untreated or treated with 1 x 10³ units/ml (left) or 1 x 10⁴ units/ml of IFNβ in absence or presence of 1 μM tricostatin A (TSA) (right) for 8 h, and luciferase reporter assays were performed. The results shown are the mean values and standard errors of three independent experiments. The expression levels of IE1 proteins were determined by immunoblotting with anti-HA antibody. (D) HF cells stably expressing wild-type or mutant IE1 (produced using retroviral vectors) were untreated or treated with IFNβ (1 x 10³ units/ml) for 8 h. Total RNAs were prepared and the amounts of ISG54 mRNA were determined by qRT-PCR. The results shown are the mean values and standard errors of three independent experiments. The amounts of mRNA in cells treated with IFNβ compared to that in untreated cells are shown as fold inductions. The expression levels of IE1 were determined by immunoblotting using anti-IE1/IE2 antibody (810R).