Human cytomegalovirus UL23 inhibits transcription of interferon-γ stimulated genes and blocks antiviral interferon-γ responses by interacting with human N-myc interactor protein
(A) U251, U251-C, and U251-UL23 cells were transfected with reporter plasmid pGL3-Promoter-3×GAS and the internal control reporter pRL-TK. After 24 hours, cells were cultured in the absence (-IFN-γ) and presence (+IFN-γ) of IFN-γ (1000 U/ml). At 12 hours post-treatment, cells were mock-infected (mock) or infected with TowneBAC, UL23, UL23stop, R-UL23, or R-stop (MOI = 1) for 24 hours. Luciferase activity was determined luminometrically as relative light units (RLU). Only the results from experiments in the presence of IFN-γ are shown. (B-D) U251, U251-C, and U251-UL23 cells were cultured in the absence (-IFN-γ) and presence (+IFN-γ) of IFN-γ (1000 U/ml) for 12 hours, and then mock-infected or infected with different viruses. At 24 hours postinfection, total RNAs were extracted from cells. The levels of the IRF1 (B), IFP35 (C), and IFI44 mRNAs (D) were determined by qRT-PCR using those of actin as the internal control. Only the results from experiments in the presence of IFN-γ are shown. The values of the relative luciferase and mRNA level represent the ratios of the levels of luciferase and host mRNAs in different cells treated with IFN-γ to those in parental U251 cells in the absence of IFN-γ, respectively. The experiments were repeated three times. The standard deviation is indicated by the error bar.