Figure 1.

Decreased expression of 14-3-3 θ, β, γ, and Chk1 protects against Vpr_v- and HIV-1 (vif-)-induced cell cycle arrest. (A) Diagram of the basic HIV-1 genomes used throughout the study. Env-deficient NL4-3, NL4-3_e-n-GFP, (top) also lacks nef, which was replaced with EGFP. Vpr protein associated with virions (Vpr_v) was delivered into cells using an RT point mutant, D186N, derivative of NL4-3_e-n-GFP (bottom). (B) Jurkat T cells were transfected with 500 pmol of nonspecific (ns), 14-3-3 θ, or Chk1 siRNA and four days later mock-infected or infected with either NL4-3_e-n-GFP (NL4-3), NL4-3_e-n-GFP Vif- (NL4-3 vif-), or RT- NL4-3_e-n-GFP (Vpr_v). Cell cycle arrest by was measured by flow cytometric detection of DRAQ5 DNA staining 44 hours post-infection. DNA analysis of cultures infected with RT+ NL4-3_e-n-GFP was restricted to actively infected GFP+ cells. Data is representative of six independent experiments. (C) The Dean-Jett-Fox cell cycle model was used for determination of G1 and G₂,M populations and the ratio is plotted for the FACS data in (B). The transfected siRNA is indicated in the legend. (D) Western blot detection of 14-3-3 θ and Chk1 expression four days post-transfection for the samples in (B) and (C). β-actin was probed as a loading control. (E) Live cell counts were measured for mock-infected and Vpr_v-infected samples in (B-D) at the indicated times after infection by flow cytometric constant time acquisition. (F) Jurkat cells were transfected as in (B) with the addition of a co-transfected sample that received both 14-3-3 θ and Chk1 siRNA (θ + Chk1). Western blot analysis of 14-3-3 θ, Chk1, and β-actin, as a loading control, is shown. (G) Cell cycle analysis as in (C) for the samples in (F).