Mutant Vpr fails to stimulate the association of CyclinB1, Cdk1, and Plk1 with 14-3-3 θ but still binds 14-3-3 θ itself

(A) Jurkat cells were infected as in Fig. 2 with RT- NL4-3virions (Vpr) lacking Vpr (Δ) or containing either wild-type (wt), R80A mutant (80A), or I70S mutant (70S) Vpr. Cells lysates were harvested two days post-infection. for immunoprecipitation with 14-3-3 θ antibody (IP: 14-3-3 θ) and the lysates (input) and IP were blotted as indicated with antibodies recognizing Plk1, CyclinB1, Cdk7, Cdk1, 14-3-3 θ, and Vpr. (B) DNA content analysis of the samples in (A), top row (Vpr), and of aphidicolin-synchronized Jurkat cells released from the G1 block for the indicated number of hours (bottom row; sync.). (C) Immunoprecipitation and western blot were performed as in (A) of lysates from the cell cycle synchronized cells shown in (B, bottom row). (D) Jurkat cells were infected with NL4-3(RT+) derivatives containing either wild-type Vpr and Vif (lane 1), Vpr but no Vif (lane 2), neither Vpr nor Vif (lane 3), or R80A mutant Vpr and no Vif (lane 4). Two days post-infection cells were lysed, immunoprecipitated, and immunoblotted as in (A) (top). Flow cytometric DNA content analysis was performed at the time of harvest and shown for the GFP+ (HIV-infected) population of each sample (bottom, numbering corresponds to lane numbers of blots). GFP expression is shown as an inset with the percentage of cells in the GFP-positive gate indicated within the plot.Copyright information:Taken from "14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr"http://www.biology-direct.com/content/3/1/17Biology Direct 2008;3():17-17.Published online 29 Apr 2008PMCID:PMC2390528.