FIG 6.

C184 of FIV Vif is essential for Vif-CUL5, Vif-FcaA3, and Vif-ELOB/C interactions. (A) Closeup view of the homology model of the FIV Vif (orange)-CUL5 (green) complex in a cartoon representation, with residues that underwent mutational analysis shown in a stick representation. These residues are K181, K182, C184, C187, and C192 in the loop following helix 3 of FIV Vif; C138 that forms a disulfide bond with C192 is also shown. (B) Sequence representation of the FIV Vif C-terminal domain (CTD). (C) myc-CUL5 or the empty pcDNA3.1 plasmid was cotransfected with expression plasmids for wild-type FIV Vif-V5 or the indicated FIV Vif mutants. Immunoprecipitated complexes were analyzed by immunoblotting with anti-V5 for FIV Vif and with anti-myc for CUL5. (D) HEK293T cells were transfected with expression plasmids for wild-type FIV Vif-V5 or the indicated FIV Vif mutants. Immunoprecipitated complexes were analyzed by immunoblotting with anti-V5 for FIV Vif and with anti-CUL5 for CUL5. (E and F) The FIV Vif C184S mutant has lost the capability to bind to FcaA3s, ELOB, and ELOC. HEK293T cells were transfected with expression plasmids for FcaZ2bZ3-HA, wild-type FIV Vif-V5, or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid (E) or with wild-type FIV Vif-V5, the indicated FIV Vif mutants, T7-ELOC, or HA-ELOB and the empty pcDNA3.1 plasmid (F). Cells were harvested 48 h after transfection, and proteins of cell lysates (input) and immunoprecipitated complexes were analyzed by using Western blots stained with anti-V5 antibody for FIV Vif, anti-HA antibody for FcaZ2bZ3-HA and HA-Elongin B, and anti-T7 antibody for T7-Elongin C. (G) HEK293T cells were transfected with expression plasmids for FcaA3Z2bZ3-HA and wild-type FIV Vif-V5 or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid. Cells were harvested and analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies, respectively.