Fig. 2.

FIV Vif interacts with Cul5, ElonginB, and ElonginC and induces the degradation of fA3s in a proteasome-dependent manner. 293T cells (A) and CrFK cells (B) (2 × 10⁶) were transfected with 4.0 μg of VR-HIV Vif-myc, VR-FIV Vif-myc, or VR-FIV Vif-HA. After 48 h, the cell lysates were immunoprecipitated with anti-myc antibody, followed by Western blot analysis with anti-HA, anti-myc, anti-hCul5, anti-hElonginB, and anti-hElonginC antibodies. (C) CrFK cells (10⁶) were cotransfected with 100 ng of VR-fA3Z3-HA, VR-fA3Z2-Z3-HA, or VR-fA3Z2a-HA and 400 ng of VR-FIV Vif-myc or empty plasmid (VR1012). The transfected cells were treated with the proteasome inhibitor MG132 at 10 mM (lanes 3, 4, 7, 8, 11, and 12) or DMSO (lanes 1, 2, 5, 6, 9, and 10) at 36 h after transfection. Cells were harvested 12 h later (48 h after transfection) and then analyzed by Western blotting using anti-HA, anti-myc, and anti-tubulin antibodies. The percentages of fA3s in the presence of FIV Vif with DMSO or MG132 treatment were calculated relative to that in the absence of FIV Vif (set to 100%).