FIG 1.

HIV-1 Vpr interacts with REAF and overcomes restriction. (A) REAF protein in HeLa-CD4 cells (Ø) and HeLa-CD4 shRNA-REAF cells (shREAF). GAPDH was a loading control. (B) Infectivity of HIV-1 89.6^WT and HIV-1 89.6^Δvpr in HeLa-CD4 cells (Ø) and HeLa-CD4 shRNA-REAF cells (shREAF). Viral inputs were equivalent at approximately 50 FFU/ml, measured on the HeLa-CD4 cells. The error bars indicate the standard deviations of the means derived from a range of duplicate titrations. Fold changes in FFU are indicated. (C) Foci of infection resulting from equal p24 inputs (1 ng) of HIV-1 89.6^WT or HIV-1 89.6^Δvpr in HeLa-CD4 cells. The error bars indicate the standard deviations of the means derived from a range of duplicate titrations. (D) REAF protein in HeLa-CD4 cells 24 h postchallenge with HIV-1 89.6^WT or HIV-1 89.6^Δvpr. GAPDH was a loading control. (E) HEK-293T cells were transfected with Vpr-GFP expression plasmid or GFP control vector, expression was analyzed by Western blotting (left), and protein was immunoprecipitated (IP) with anti-GFP beads. Coimmunoprecipitated REAF was detected in the Vpr-GFP precipitation (right). (F) Nuclear (Nuc.) and cytoplasmic (Cyto.) REAF MFI in HeLa-CD4 cells measured by imaging flow cytometry. The error bars represent standard deviations of the means of replicates. (G) Percent change in REAF MFI from time 0 in the nucleus (left) and cytoplasm (right) of HeLa-CD4 cells over time after challenge with HIV-1 89.6^WT or HIV-1 89.6^Δvpr. The results are representative of three independent experiments. p.i., postinfection. (H) Nuclear enrichment scores of HeLa-CD4 cells over time postchallenge with HIV-1 89.6^WT or HIV-1 89.6^Δvpr. A lower nuclear enrichment score indicates that a lower proportion of overall REAF is located in the nucleus, as calculated with IDEAS software. The results are representative of three independent experiments. Where quantitative comparisons are made, the data were derived from the same blot or blots processed together.