Vif N-terminal mutants localize to cytoplasm, but are inefficient at restoring HIV infectivity. HA-tagged Vif wild-type and mutant proteins along with CBF-β were overexpressed in HEK 293 T cells. Two days post-transfection, cells were lysed and cleared lysate was mixed with anti-HA matrix affinity beads for 4-8 hrs. Incubated beads were washed several times followed by elution of bound proteins. A) Select Vif N-terminal mutants that do not efficiently degrade A3G and A3F have a reduced ability to co-precipitate Cul5; however, CBF-β and Elo B/C can still bind Vif. B) Plasmids (Vif-YFP 2 ug and CBF-β 0.5 ug) were transfected into 293 T cells using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. Cells were visualized at 25°C using a Zeiss LSM510-Meta confocal imaging system. Imaging demonstrates that the Vif double mutant V25/H27A and single mutant H108A localize to the cytoplasm of the cell similar to wild-type. C) Vif wild-type and mutant containing virus were produced and used to infect MAGI cells. Infected cells were stained using X-gal. The histogram demonstrates that Cul5-binding deficient Vif mutants were inefficient at restoring HIV infectivity in the presence of A3G. Error bars represent the standard error from triplicate experiments. Capsid p24 levels are shown in the western blot.