Proliferation of PBMCs and activation of ERK1/2 and NF-κB upon infection with PBj-wt or PBj-Nef202/203GG virus. (A) Analysis of virus gene expression by in situ immunostaining of PBj-wt or PBj-Nef202/203GG virus infected cell cultures with bar chart showing the percentage of infected cells at day 8 post infection as determined by cell counting. Magnification, 200 × (ns, P = 0.31). (B) Macaque PBMCs from 3 animals were infected with PBj-wt or PBj-Nef202/203GG virus. At day 10 p.i., cell proliferation was assessed by 3H-thymidine incorporation. PHA/IL-2 stimulated as well as non-stimulated uninfected PBMCs served as controls. Error bars, SD (**, P < 0.04 compared to control; ***, P = 0.036; ns, P = 0.21). Numbers represent stimulation index compared to non-stimulated uninfected cells. (C) In vitro ERK1/2 kinase activity. Non-stimulated macaque PBMCs were left untreated or infected with PBj-wt or PBj-Nef202/203GG virus. γ-32P-phosphorylation of ELK-1 quantified ERK1/2-activity. Western blot detection of ERK-2 served as loading control. (D) EMSA of NF-κB activation. Non-stimulated macaque PBMCs were left untreated, stimulated by PHA/IL-2 or infected with PBj-wt or PBj-Nef202/203GG virus. On day 10 p.i., NF-κB activity was assessed using a specific 32P-labelled oligonucleotide. The specificity of NF-κB binding complexes was confirmed by using NF-κB-p50 and NF-κB-p65 specific antibodies in supershift experiments. (E) Differential binding of PBj-wt and PBj-Nef202/203GG Nef to cellular signaling proteins. GST-PBj-Nef fusion proteins were used to precipitate potential binding partners from lysates of non-stimulated T cells. Precipitates were analyzed for Raf-1, ERK-2 and p56lck by Western Blot. Precipitations with GST, Protein A-coupled anti-Raf-1, and total cell lysates (TCL) served as controls.