Figure 2

Vif-induced dysfunction of Cdk1. Jurkat cells were synchronized and infected as in Figure 1 with the GFP-expressing viruses. These data are representative of three experiments with infection efficiencies ranging from 85-95% based on GFP expression. (A) Nuclear translocation of Cdk1 was barely detectable in Vif-expressing cells. Vif and Cdk1 localization patterns were visualized by immunofluorescent confocal microscopy using the following antibodies: rabbit anti-Vif (AIDS Research and Reference Reagent Program [ARRRP]) [53], mouse anti-Cdk1 (anti-cdc2, Santa Cruz Biotechnology), goat anti-rabbit-Alexa565 (Molecular Probes), and goat anti-mouse-Alexa647 (Molecular Probes). GFP, expressed by infected cells, was measured by direct fluorescence. Nuclei were counterstained with Hoechst 33342 (Molecular Probes). (B) At least 350 cells were counted from representative fields, and the percentage of cells showing the indicated phenotypes for Cdk1 were plotted at each time point. (C) Cdk1 is phosphorylated (inactivated) in Vif-expressing cells. Bulk lysates were prepared at the indicated time points and analyzed for inhibitory Cdk1 Tyr15 phosphorylation by immunoblotting using a rabbit anti-phospho-Cdk1 Tyr15 antibody (anti-phospho-cdc2 Tyr15, Cell Signaling Technology). Total Cdk1 expression was examined using a mouse anti-Cdk1 antibody (anti-cdc2, Santa Cruz Biotechnology) on the same blot after stripping off the phospho-Cdk1 antibody. An immunoblot using a mouse-anti β-actin antibody (anti-β-actin, Sigma-Aldrich) is provided as a loading control. (D) Densitometry of the bands in panel C was performed using ImageJ (NIH), and the intensity of each band was normalized to β-actin. The normalized ratio of phospho-Cdk1 to total Cdk1 was plotted for each time point.