FIGURE 9.

Vpx interferes with IRF5 DNA binding activity but does not affect IRF5 phosphorylation or nuclear translocation. A, 293T cells were transfected with plasmids expressing IRF5, IKKϵ, TBK1, and Vpx as indicated. After 48 h, cells were lysed, and aliquots of each cell lysate were either untreated or treated with calf intestinal phosphatase (CIP) for 30 min at 37 °C before subjected to SDS-PAGE. IRF5 and Vpx were immunoblotted separately with anti-FLAG or anti-Vpx antibody. Each phosphorylated and nonphosphorylated IRF5 band was quantified by densitometry using Image lab software 3.0 (Bio-Rad). Data are presented as the ratio of phosphorylated to nonphosphorylated IRF5. Values are mean ± S.D. of three experiments. B, THP-1 cells transduced with either an empty lentiviral vector or the vector encoding HIV-2 Vpx were treated with 100 nm PMA and then mock-stimulated (control) or stimulated with 10 μm R848 for 12 h. The cells were lysed and separated into cytoplasmic and nuclear fractions using a nuclear (N) and cytoplasmic (C) kit (Thermo Scientific). Equal amounts of protein from each fraction were loaded onto the gel and blotted with antibodies as indicated. Immunoblot data are representative of three independent experiments. Each IRF5 band was quantified by densitometry using Image lab software 3.0 (Bio-Rad) and presented as the ratio of nuclear to cytoplasmic IRF5. Values are mean ± S.D. of three experiments. C–E, THP-1 cells transduced with an empty lentiviral vector or the vector encoding HIV-2 Vpx were differentiated with 100 nm PMA for 24 h and then either stimulated with 10 μm R848 or not (E). After 6–12 h, cells were collected for ChIP assay as described under “Experimental Procedures.” Each PCR was run in triplicate, and data are representative of three independent experiments. Values are mean ± S.D. of the triplicate experiments.