Figure 6

Reduced 14-3-3 θ association with centrosomal proteins, centrin and Plk1, during HIV-1 infection-induced G₂,M arrest. (A) Jurkat cells were mock-infected (-) or infected with NL4-3_e-n-GFP derivatives encoding wild-type Vif and Vpr (wt), deleted Vpr (r-), deleted Vif (f-), or double deletion of Vpr and Vif (fr-) at an MOI of 1.5. Lysates were harvested two days post-infection and immunoprecipitated as in Fig. 2 (IP: 14-3-3 θ). Whole cell lysates (input) and IP samples were blotted for Plk1, 14-3-3 θ, centrin, Vif, and Vpr. There appeared to be poor transfer of centrin protein at the left edge of the gel (input lanes). The reduced band intensity for this sample does not reflect decreased centrin abundance as it was well-represented in the IP and similar experiments showed no changes in centrin expression upon HIV-1 infection. (B) DNA content analysis of the samples in (A) by propidium iodide staining. HIV-infected samples were pre-gated on GFP+ cells for DNA analysis. (C) Viability (large plot) and GFP expression by viable cells (inset) for samples in (A) and (B) were measured by flow cytometric detection of propidium iodide (PI) negative, large (high forward scatter) cells and GFP fluorescence (inset histogram), respectively, at the time lysates were harvested. Plots correspond to samples directly above in (B). The gates demarcate viable and GFP-positive cell populations and the percentage of cells within each gate is indicated. (D) Jurkat cells were mock-infected (m), infected with Vpr_v as in (A), or infected with NL4-3_e-n-GFP (HIV) and harvested after 40 hours for immunoprecipitation with 14-3-3 θ and immunoblotting with importin β, CyclinB1, 14-3-3 θ, and Vpr. (E) DNA content analysis performed as in (B) is shown for the samples in (D). The inset histogram depicts the percentage of GFP+ cells expressing the NL4-3_e-n-GFP provirus. The DNA analysis for the HIV-infected sample was performed on the gated GFP population indicated. (F) Jurkat cells were mock-infected (m), infected with RT- NL4-3_e-n-GFP virions (Vpr_v) to deliver Vpr protein, or treated with adriamycin (adr). Cell lysates (input) were harvested after 40 hours for immunoprecipitation with importin β (IP) and immunoblotting with importin β, CyclinB1, Plk1, Cdk1, and Vpr as indicated. (G) Cell cycle analysis is shown for the samples in (F) at the time of lysis as measured by propidium iodide DNA staining as in (B).