Oct-2 DNA-binding activity is not required for Oct-2 inhibition of BZLF1 function.

<p>(A) (Left panel) An EMSA was performed using <i>in vitro</i>-translated wild-type or mutant Oct-2 (Q221A) protein and a ³²P-labelled oligonucleotide probe containing the consensus Oct-2 binding site. Protein-DNA complexes are indicated by arrows. (Right panel) Immunoblot analysis was performed to compare the levels of wild-type and mutant Oct-2 (Q221A) in the <i>in vitro</i>-translated samples. (B) HONE-Akata cells were transfected with 5 ng BZLF1, 500 ng wild-type Oct-2, 500 ng mutant Oct-2 (Q221A), or control vectors as indicated. Immunoblot analysis was performed two days after transfection to compare levels of transfected BZLF1 and Oct-2, as well as the levels of BMRF1 and BRLF1 protein derived from the endogenous viral genome. β-actin served as a loading control. (C) EBV-negative BJAB cells were transfected with a BMRF1p-luciferase construct in the presence or absence of co-transfected BZLF1 (30 ng), wild-type Oct-2 (1470 ng), two different mutant Oct-2 proteins (Oct-2 Q221A or Oct-2 Δ262–302) (1470 ng), or control vectors as indicated. The fold luciferase activity for each condition is shown relative to control vector; the value for the activity of the promoter construct plus the vector control is set at 1. Values are given as means ± standard deviations of results from two replicates. (D) A ChIP assay was performed using HONE-Akata cells transfected with Flag-tagged-BZLF1, wild-type Oct-2, mutant Oct-2 (Q221A), or control vectors as indicated. Cross-linked DNA-protein complexes were immunoprecipitated using antibodies against Flag (BZLF1) or an IgG control. Antibody-bound DNA sequences were then PCR-amplified using primers spanning the EBV BRLF1, BMRF1, or SM promoters, the viral FR repeats, cellular Gadd45a promoter, or the GAPDH gene (negative control). Binding bands were quantified using ImageJ software and represented as numerical values in bar diagrams in the lower panel. The amount of BZLF1 binding to each promoter in the presence or absence of wild-type or mutant Oct-2 (Q221A) was compared to input. (E) A ChIP assay was performed using HONE-Akata cells transfected with 3 µg wild-type Oct-2 or mutant Oct-2 (Q221A), with or without 3 µg co-transfected BZLF1, or control vectors as indicated. Cross-linked DNA-protein complexes were immunoprecipitated using antibodies against Oct-2 or an IgG control. Antibody-bound DNA sequences were then PCR-amplified using primers spanning the cellular Gadd45a promoter, viral FR repeats, viral latency Qp, or the GAPDH gene (negative control). Binding bands were quantified using ImageJ software and represented as numerical values in bar diagrams in the lower panel. The amount of Oct-2 binding to each DNA region in the presence or absence of BZLF1 was compared to input.