FIG. 1.

Construction and expression of Vif mutants. (A) A series of in-frame deletions mapping to different regions in Vif was created by PCR-based site-directed mutagenesis in the backbone of pcDNA-hVif (46). The positions of the deleted amino acid residues are shown for each mutant. The Vif-M1 mutant carries four point mutations affecting the conserved HCCH motif (H108L, C114S, C133S, and H139L) and was constructed by PCR-based mutagenesis. Mutant Vif-M2 carries the same four amino acid changes as Vif-M1, as well as an in-frame deletion of residues 144 to 149 affecting the conserved BC (SOCS) box of Vif. Mutant M3 carries three amino acid changes, resulting in the change of P₁₆₁PLP₁₆₄ to AALA. (B) Cells (5 × 10⁶) were transfected with 5 μg each of the individual Vif mutants. Cells were harvested 24 h posttransfection, and whole-cell lysates were analyzed by immunoblotting them with a monoclonal antibody to Vif. A mock-transfected sample was included as a background control.