Vif causes prominent G2 arrest in the absence of Vpr. (A) Schematic of the NL4-3 HIV-1 molecular clones used. The NL4-3e-n-HSA (e-f+r+) lacks a functional env gene, due to a frameshift mutation, and the nef gene was replaced with HSA . The NL4-3e-n-GFP has the same env frameshift, but the nef gene was replaced with EGFP [4, 6]. The e-f+r- and e-f-r- mutants of NL4-3e-n-HAS and NL4-3e-n-GFP have been previously described [3, 4]. (B) Jurkat cells were synchronized with a G1/S-phase blocker, aphidicolin, for 16 hours and then released for 10 hours prior to infection. The cells were blocked again at the time of infection with the following HIV-1 NL4-3e-n-HSA strains at an MOI of 5: e-f+r+, e-f+r-, or e-f-r-. DNA content was examined by flow cytometry using the cell permeable dye DRAQ5 (Biostatus) every 3 hours after release from the second aphidicolin blockade as previously described . Infected cells highly expressing HSA and mock-infected cells are shown. These data are representative of three experiments using either the HSA- or GFP-expressing viruses. (C) The percentage of cells in the G2 phase of the cell cycle was graphed over the course of the experiment represented in panel B. Data are represented as the mean ± the standard deviation (SD) of quadruplicates and are representative of three experiments.