Only group 3 Vif mutants interact with A3G. Cells were either mock transfected (lanes 1) or transfected with 2.5 μg of pcDNA-A3G-Myc (lanes 2 to 5, 8 to 9, 12 to 14, and 18 to 19), together with 2.5 μg of empty-vector DNA [Vif(−)] (lanes 2), pcDNA-hVif wt (lanes 3), or Vif mutants from groups 1 to 4 (lanes 4 and 5, 8 and 9, 12 and 14, and 18 and 19). Mutant Vif proteins were also expressed in the absence of A3G to control for nonspecific binding to the antibody matrix (lanes 6 and 7, 10 and 11, 15 and 17, and 20 and 21). To prevent possible degradation of A3G by Vif, 1 μg of a dominant negative mutant of Cul5, Cul5ΔRbx-HA (75), was cotransfected in all samples. Cells were harvested 24 h after transfection. Cell lysates were immunoprecipitated with anti-Myc antibody conjugated to agarose beads as described in Materials and Methods. Aliquots of total cell lysates (Input) and immunoprecipitated proteins (IP: α-myc) were separated by SDS-PAGE and subjected to immunoblot analysis using antibodies to A3G (I and II) or Vif (III and IV). Proteins are identified on the right. HC, immunoglobulin heavy chain.