FIG. 2.

HVif_IIIB affects the intracellular distributions of A3A, A3B, and A3C and colocalizes in P-bodies with A3F. (A and B) 293T cells were transiently transfected with either A3A-HA, A3B-Myc, A3C-HA, or A3F-HA and an HVif_IIIB expression vector or the negative-control plasmid pcDNA3.1. Cells were fixed and probed with mouse anti-HA or mouse anti-Myc and rabbit anti-Vif antibodies. (A) In the absence of HVif_IIIB, A3A and A3C are present in both the nucleus and the cytoplasm, whereas A3B is predominantly intranuclear (control panels). In the presence of HVif_IIIB, A3A, A3B, and A3C migrate from the nuclei to the cytoplasm (HVif_IIIB panels). (B) Control panel shows A3F staining throughout the cytoplasm as well as in discrete cytoplasmic granules. HVif_IIIB colocalizes in the cytoplasmic granules with A3F (HVif_IIIB panel). (C) 293T cells transiently expressing A3F-HA were stained for A3F and Rck/p54. Cycloheximide (CHX) treatment did not disperse the A3F-Rck/p54-containing P-bodies. In contrast, CHX substantially dispersed the Rck/p54-containing P-bodies in cells that lacked A3F. (D) Western blot analyses of cytosolic extracts from 293T cells transiently cotransfected with A3B-Myc (left) or A3G-Myc (right) and either wt HIV-1_IIIB, Δvif HIV-1_IIIB, or codon-optimized Vif_IIIB (HVif_IIIB). α-Tubulin (α-tub), loading control.