FIG. 4.

IE86 attenuates NFκB DNA binding activity. (A) HFF cells were mock infected or infected with WT-HCMV or UV-HCMV at a multiplicity of 5 PFU/cell. Nuclear extracts were prepared 6 h postinfection and assayed by EMSA for binding of NFκB to the PRDII region of the IFN-β promoter. NS indicates a nonspecific shift. (B) HFF cells were infected with WT-HCMV or UV-HCMV. Six hours postinfection, nuclear lysates were isolated and assayed for NFκB binding. A competition analysis was performed on UV-HCMV extracts by adding unlabeled specific-competitor oligonucleotide probe (PRDII) or a mutated probe sequence (mPRDII) in increasing concentrations to the binding mixture to confirm the specificity of the NFκB DNA binding. (C) HFF cells were infected with HCMV, UV-HCMV, UL83Stop virus, or IE2ΔSX virus at a multiplicity of 5 PFU/cell. Nuclear extracts were prepared 6 h postinfection and assayed for NFκB binding by EMSA. A Western blot is also included to confirm the expression of the various viral proteins. (D) HFF cells were transduced with adenoviruses expressing IE86, pp65, GFP, or IκBαSR and then infected at a multiplicity of 5 PFU/cell with UV-HCMV. Nuclear lysates were prepared 6 h postinfection and assayed for NFκB binding by EMSA. A Western blot is included to confirm the expression of IE86, pp65, IκBα, GFP, adenovirus hexon, and tubulin.