Figure 5

The 34-amino acid C-terminal tail of EloB is required for regulation of Vif function. (A) Schematic representations of functional domains of EloB based on existing evidence and of the EloB C-terminal deletion mutant used in this study. (B) Structural alignment of EloB (red) and ubiquitin (green). The tertiary structure of the EloB UbL domain bears striking similarity to that of ubiquitin, whereas the tertiary structure of the EloB C-terminal tail was not found in that of ubiquitin. (C) 293 T cells grown in 25 cm² flasks to about 80% confluency were co-transfected with 450 ng of VR-A3G-HA, 3.5 μg of NL4-3ΔVif, and 1.2 μg of VR-Vif or VR1012 in the presence of 750 ng of wild-type EB (lanes 1–3) or 1.5 μg of EBΔC34 (lanes 4–6). Cells were harvested 48 h after transfection and analyzed by Western blot. A3G expression was calculated relative to that detected in the absence of both Vif and EBΔC34 (set to 100%). (D) 293 T cells (10⁶) were co-transfected with 300 ng of VR-A3G-HA, 600 ng of VR-Vif or VR1012 and increasing amounts of vectors expressing EB or VR1012 (150, 250 and 350 ng). Cells were harvested 48 h after transfection and analyzed by Western blot. (E) 293 T cells (10⁶) were co-transfected with 300 ng VR-A3G-HA, 600 ng VR-Vif or VR1012 and increasing amounts of vectors expressing EBΔC34 or VR1012 (300, 500 and 700 ng). Cells were harvested 48 h after transfection and analyzed by Western blot. (F) 293 T cells (10⁶) were co-transfected with 7.5 ng VR-P53-HA and 1 μg VR-E4orf6-Myc or VR1012 in the presence of 750 ng of wild-type EB (lanes 1–2) or 1.5 μg of EBΔC34 (lanes 3–4). The cells were harvested 48 h post-transfection and analyzed by Western blotting. All experiments were repeated three times.